CN101848924A - 基因敲除的嗜温和嗜热生物以及其使用方法 - Google Patents
基因敲除的嗜温和嗜热生物以及其使用方法 Download PDFInfo
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Abstract
本发明的一个方面涉及经遗传修饰的嗜热或嗜温微生物,其中第一天然基因被部分、基本上或完全缺失、沉默、失活或下调,该第一天然基因编码参与有机酸或其盐的代谢产生的第一天然酶,从而提高所述嗜热或嗜温微生物以发酵产物的形式产生乙醇的天然能力。在某些实施方案中,上述微生物还包含第一非天然基因,该第一非天然基因编码参与乙醇的代谢产生的第一非天然酶。本发明的另一个方面涉及用于将木质纤维生物质转化成乙醇的方法,该方法包括将木质纤维生物质与经遗传修饰的嗜热或嗜温微生物接触。
Description
相关申请
本申请要求2007年5月9日提交的美国临时专利申请系列号60/916,978的权益;其全部内容通过引用合并入本文。
发明背景
能量转换、利用和获得是形成我们这个时代的许多巨大挑战,包括与持续性、环境质量、安全性和贫困相关的挑战的原因。需要应用新的新兴技术来应对这些挑战。生物技术,最强有力的新兴技术之一,可产生重要的新型能量转换方法。植物生物质(Plant biomass)和其衍生物是用于将能量生物转换成对人类有用的形式的资源。
在植物生物质的形式中,木质纤维生物质(‘生物质’)因为其大规模可获得性、低成本和环境友好生产而特别适合于能源应用。特别地,许多基于纤维质生物质的能源生产和利用循环在生命循环(life-cycle)基础上具有几乎零温室气体排放。阻碍从生物质原料更广泛地产生能源的主要障碍是用于使此类材料顺利转化成有用燃料的低成本技术的普遍不存在。木质纤维生物质包含可转化成乙醇的碳水化合物级分(例如,纤维素和半纤维素)。为了转化此类级分,必须将纤维素和半纤维素最终转化或水解成单糖;历史已证明水解是存在问题的。
生物学介导的方法对于能量转换,特别地对于将木质纤维生物质转化成燃料是很有前景的。牵涉酶促或微生物水解的生物质处理方案通常包括4个生物学介导的转化:(1)糖解酶(saccharolytic enzyme)(纤维素酶和半纤维素酶)的产生;(2)存在于经预处理的生物质中的碳水化合物组分的水解为糖;(3)己糖(例如,葡萄糖、甘露糖和半乳糖)的发酵;和(4)戊糖(例如,木糖和***糖)的发酵。这4个转化在称为联合生物加工(consolidated bioprocessing)(CBP)的流程配置(processconfiguration)的单个步骤中发生,所述流程配置与其他整合度不太高的配置不同在于其不包括用于纤维素酶和/或半纤维素酶产生的专门处理步骤。
CBP提供了比以专门的纤维素酶产生为特征的加工更低的成本和更高的效率的潜能。该益处部分地因避免的资金成本、底物和其他原料,以及与纤维素酶的产生相关的功用而产生。此外,几个因素支持通过使用CBP,包括使用酶-微生物协作以及嗜热生物和/或复合的纤维素酶***来实现更高的水解速率,从而减少反应器体积和资金投资。此外,粘附纤维素的纤维溶解微生物(cellulolytic microorganism)可能成功地与非粘附的微生物例如污染物竞争纤维素水解的产物,这可提高基于微生物纤维素利用的工业流程的稳定性。开发促成CBP的微生物的进展将通过两个策略来实现:对天然存在的纤维素分解微生物进行工程改造以提高产品相关性质例如收率(yield)和滴度;和对展示高产品收率(productyield)和滴度的非纤维素分解微生物进行工程改造以表达异源纤维素酶和半纤维素酶***,从而使得能够利用纤维素和半纤维素。
许多细菌具有通过糖酵解过程将简单的己糖发酵成酸性和pH-中性产物的混合物的能力。糖酵解途径是丰富的并且包括一系列酶促步骤,通过所述酶促步骤六碳糖分子经过多个中间体被分解成2个分子的三碳化合物丙酮酸。该过程导致净产生ATP(生物能量供应)和还原性辅因子NADH。
丙酮酸是代谢的重要中间化合物。例如,在好氧条件下,丙酮酸可被氧化成乙酰辅酶A(乙酰CoA),然后乙酰CoA进入三羧酸循环(TCA),转而产生合成前体、CO2和还原性辅因子。然后通过经由一系列酶促步骤将氢等价物提供给氧来氧化所述辅助因子,从而导致水和ATP的形成。该能量形成过程称为氧化磷酸化。
在厌氧条件(无可利用的氧)下,发生其中有机化合物的降解产物用作氢供体和受体的发酵。来自糖酵解的过量NADH在牵涉将有机底物还原成产物例如乳酸和乙醇的反应中被氧化。此外,在称为底物水平磷酸化的过程中通过有机酸例如乙酸的产生中再生ATP。因此,糖酵解和丙酮酸代谢的发酵产物包括多种有机酸、醇和CO2。
大多数兼性厌氧菌在好氧或厌氧条件下都不产生高收率的乙醇。大多数兼性厌氧菌通过丙酮酸脱氢酶(PDH)和三羧酸循环(TCA)有氧代谢丙酮酸。在厌氧条件下,用于代谢丙酮酸的主要能量途径是通过丙酮酸甲酸裂解酶(PFL)途径产生甲酸和乙酰-CoA。然后乙酰-CoA通过磷酸转乙酰酶(PTA)和乙酸激酶(ACK)被转化成乙酸(共产物(co-production)为ATP),或通过乙醛脱氢酶(AcDH)和乙醇脱氢酶(ADH)被还原成乙醇。为了保持还原当量(reducing equivalent)的平衡,在丙酮酸还原成乳酸的过程中利用乳酸脱氢酶(LDH)将从糖酵解产生的过量NADH再氧化成NAD+。NADH还可在乙酰-CoA还原成乙醇的过程中通过AcDH和ADH而被再氧化,但这在具有功能性LDH的细胞中是次要反应。因此未获得乙醇的理论收率,因为大部分乙酰CoA被转化成乙酸,从而再生ATP,并且糖酵解中产生的过量NADH被LDH氧化。
微生物的代谢工程还可导致编码酶例如乳酸脱氢酶的产生的基因的被靶向敲除的产生。在该情况下,基因的“敲除”是指部分、基本上或完全缺失、沉默、失活或下调。如果在糖酵解途径的早期阶段不能利用通过LDH的作用进行的丙酮酸至乳酸盐(乳酸的盐形式)的转化,那么丙酮酸可通过丙酮酸脱氢酶或丙酮酸-铁氧环蛋白氧化还源酶(pyruvate-ferredoxin oxidoreductase)的作用来更有效地转化成乙酰CoA。如果乙酰CoA通过磷酸转乙酰酶进一步被转化成乙酸盐(乙酸的盐形式)也不可利用,即,如果编码PTA和ACK的产生的基因被敲除,那么乙酰CoA可通过AcDH和ADH更有效地转化成乙醇。因此,具有此类被靶向的基因敲除的经遗传修饰的微生物菌株(其消除了有机酸的产生)将可具有提高的以发酵产物的形式产生乙醇的能力。
产乙醇微生物例如运动发酵单孢菌(Zymomonas mobilis)、棕榈发酵细菌(Zymobacter palmae)、巴氏醋杆菌(Acetobacter pasteurianus)或八叠球菌(Sarcina ventriculi)以及一些酵母(例如,酿酒酵母(Saccharomyces cerevisiae))能够进行第二类型的厌氧发酵,通常称为乙醇发酵,其中丙酮酸通过丙酮酸脱羧酶(PDC)被代谢成乙醛和CO2。然后乙醛通过ADH再生NAD+而被还原成乙醇。乙醇发酵导致1个分子的葡萄糖代谢成2个分子的乙醇和2个分子的CO2。如果可如上所述避免丙酮酸转化成不期望的有机酸,那么这样的经遗传修饰的微生物将可具有提高的以发酵产物的形式产生乙醇的能力。
发明概述
本发明的一个方面涉及包含SEQ ID NOS:1-5、30-31和47-61的任一个的核苷酸序列或其互补序列的分离的核酸分子。本发明的另一个方面涉及分离的核酸分子,所述核酸分子包含与SEQ ID NOS:1-5、30-31和47-61的任一个的核苷酸序列或其互补序列共有至少80%的同一性的核苷酸序列。在某些实施方案中,本发明涉及与SEQ ID NOS:1-5、30-31和47-61的任一个的核苷酸序列或其互补序列共有至少大约95%的序列同一性的上述核酸分子。
本发明的另一个方面涉及基因构建体(genetic construct),其包含与可在嗜热或嗜温细菌中表达的启动子可操作连接的SEQ ID NOS:1-5、30-31和47-61的任一个序列。本发明还涉及包含上述基因构建体的重组嗜热或嗜温细菌。
本发明还包括包含上述核酸分子的任一个的载体。本发明还包括包含上述核酸分子的任一个的宿主细胞。在某些实施方案中,本发明涉及上述宿主细胞,其中所述宿主细胞是嗜热或嗜温细菌细胞。
本发明的另一个方面涉及经遗传修饰的嗜热或嗜温微生物,其中第一天然基因被部分、基本上或完全缺失、沉默、失活或下调,该第一天然基因编码参与有机酸或其盐的代谢产生的第一天然酶,从而提高了所述嗜热或嗜温微生物以发酵产物的形式产生乙醇的天然能力。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述微生物是革兰氏阴性细菌或***。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述微生物是好热厌氧杆菌属(Thermoanaerobacterium)、热厌氧杆菌属(Thermoanaerobacter)、梭菌属(Clostridium)、地芽孢杆菌属(Geobacillus)、糖球菌属(Saccharococcus)、类芽孢杆菌属(Paenibacillus)、杆菌属(Bacillus)、热解纤维素菌属(Caldicellulosiruptor)、厌氧细菌属(Anaerocellum)或厌氧芽孢杆菌属(Anoxybacillus)的种。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述微生物是选自下列的细菌:热硫化氢好热厌氧杆菌(Thermoanaerobacterium thermosulfurigenes)、新西兰好热厌氧杆菌(Thermoanaerobacterium aotearoense)、解多糖好热厌氧杆菌(Thermoanaerobacterium polysaccharolyticum)、玉米好热厌氧杆菌(Thermoanaerobacterium zeae)、解木聚糖好热厌氧杆菌(Thermoanaerobacterium xylanolyticum)、解糖好热厌氧杆菌(Thermoanaerobacterium saccharolyticum)、布氏好热厌氧杆菌(Thermoanaerobium brockii)、热解糖嗜热厌氧杆菌(Thermoanaerobacterium thermosaccharolyticum)、热硫化氢嗜热厌氧杆菌(Thermoanaerobacter thermohydrosulfuricus)、嗜热厌氧乙醇杆菌(Thermoanaerobacter ethanolicus)、布氏嗜热厌氧杆菌(Thermoanaerobacter brocki)、热解纤维梭菌(Clostridiumthermocellum)、解纤维梭菌(Clostridium cellulolyticum)、Clostridiumphytofermentans、Clostridium straminosolvens、热葡糖苷酶地芽孢杆菌(Geobacillus thermoglucosidasius)、嗜热脂肪地芽孢杆菌(Geobacillusstearothermophilus)、Saccharococcus caldoxylosilyticus、喜温链球菌(Saccharoccus thermophilus)、坎皮纳斯类芽孢杆菌(Paenibacilluscampinasensis)、黄热芽孢杆菌(Bacillus flavothermus)、勘察加无氧芽孢杆菌(Anoxybacillus kamchatkensis)、Anoxybacillus gonensis、产乙酸热解纤维素菌(Caldicellulosiruptor acetigenus)、极度嗜热菌(Caldicellulosiruptor saccharolyticus)、克里斯托热解纤维素菌(Caldicellulosiruptor kristjanssonii)、欧文湖热解纤维素菌(Caldicellulosiruptor owensensis)、产乳酸乙酸热解纤维素菌(Caldicellulosiruptor lactoaceticus)和Anaerocellum thermophilum。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述微生物是解糖好热厌氧杆菌。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述微生物选自:(a)具有代谢己糖的天然能力的嗜热或嗜温微生物;(b)具有代谢戊糖的天然能力的嗜热或嗜温微生物;和(c)具有代谢己糖和戊糖的天然能力的嗜热或嗜温微生物。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述微生物具有代谢己糖的天然能力。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述微生物是Clostridium straminisolvens或热解纤维梭菌。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述微生物具有代谢己糖和戊糖的天然能力。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述微生物是解纤维梭菌、Clostridiumkristjanssonii或粪堆梭菌(Clostridium stercorarium)leptosaprartum亚种。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中***了第一非天然基因,该第一非天然基因编码第一非天然酶,该酶提供代谢戊糖的能力,从而使得所述嗜热或嗜热微生物能够以发酵产物的形式从戊糖产生乙醇。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述微生物具有代谢戊糖的天然能力。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述微生物选自解糖好热厌氧杆菌,解木聚糖好热厌氧杆菌,解多糖好热厌氧杆菌,和解糖好热厌氧杆菌。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中***了第一非天然基因,该第一非天然基因编码第一非天然酶,该酶提供代谢己糖的能力,从而使得所述嗜热或嗜热微生物能够从己糖以发酵产物的形式产生乙醇。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述有机酸选自乳酸和乙酸。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述有机酸是乳酸。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述有机酸是乙酸。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述第一天然酶选自乳酸脱氢酶、乙酸激酶和磷酸转乙酰酶。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述第一天然酶是乳酸脱氢酶。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述第一天然酶是乙酸激酶。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述第一天然酶是磷酸转乙酰酶。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中第二天然基因被部分、基本上或完全缺失、沉默、失活或下调,该第二天然基因编码参与有机酸或其盐的代谢产生的第二天然酶。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述第二天然酶是乙酸激酶或磷酸转乙酰酶。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述第二天然酶是乳酸脱氢酶。
本发明的另一个方面还涉及经遗传修饰的嗜热或嗜温微生物,其中(a)第一天然基因被部分、基本上或完全缺失、沉默、失活或下调,该第一天然基因编码参与有机酸或其盐的代谢产生的第一天然酶,和(b)***了第一非天然基因,该第一非天然基因编码第一非天然酶,该酶参与乙醇的代谢产生,从而使得所述嗜热或嗜温微生物能够以发酵产物的形式产生乙醇。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述第一非天然基因编码第一非天然酶,该酶提供代谢己糖的能力,从而使得所述嗜热或嗜温微生物能够代谢己糖。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述第一非天然基因编码第一非天然酶,该酶提供代谢戊糖的能力,从而使得所述嗜热或嗜温微生物能够代谢戊糖。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述第一非天然基因编码第一非天然酶,该酶提供代谢己糖的能力;并且其中***了第二非天然基因,该第二非天然基因编码第二非天然酶,该酶提供代谢戊糖的能力,从而使得所述嗜热或嗜温微生物能够代谢己糖和戊糖。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述有机酸是乳酸。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述有机酸是乳酸。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述第一非天然酶是丙酮酸脱羧酶(PDC)或乙醇脱氢酶(ADH)。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述第二非天然酶是木糖异构酶。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述第一非天然基因相应于SEQ ID NOS:6、10或14。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述非天然酶是木酮糖激酶。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述非天然基因相应于SEQ ID NOS:7、11或15。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述非天然酶是L-***糖异构酶。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述非天然基因相应于SEQ ID NOS:8或12。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述非天然酶是L-核酮糖-5-磷酸4-异构酶。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述非天然基因相应于SEQ ID NO:9或13。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述微生物能够将至少60%的来自被代谢的生物质的碳转化成乙醇。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述微生物选自:(a)具有水解纤维素的天然能力的嗜热或嗜温微生物;(b)具有水解木聚糖的天然能力的嗜热或嗜温微生物;和(c)具有水解纤维素和木聚糖的天然能力的嗜热或嗜温微生物。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述微生物具有水解纤维素的天然能力。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述微生物具有水解纤维素和木聚糖的天然能力。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中***了第一非天然基因,该第一非天然基因编码提供水解木聚糖的能力的第一非天然酶。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述微生物具有水解木聚糖的天然能力。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中***了第一非天然基因,该第一非天然基因编码提供水解纤维素的能力的第一非天然酶。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述有机酸选自乳酸和乙醇。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述有机酸是乳酸。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述有机酸是乙酸。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述第一天然酶选自乳酸脱氢酶、乙酸激酶和磷酸转乙酰酶。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述第一天然酶是乳酸脱氢酶。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述第一天然酶是乙酸激酶。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述第一天然酶是磷酸转乙酰酶。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中第二天然基因被部分、基本上或完全缺失、沉默、失活或下调,该第二天然基因编码参与有机酸或其盐的代谢产生的第二天然酶。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述第二天然酶是乙酸激酶或磷酸转乙酰酶。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述第二天然酶是乳酸脱氢酶。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中(a)第一天然基因被部分、基本上或完全缺失、沉默、失活或下调,该第一天然基因编码参与有机酸或其盐的代谢产生的第一天然酶,和(b)***了第一非天然基因,该第一非天然基因编码第一非天然酶,该酶参与多糖的水解,从而使得所述嗜热或嗜温微生物能够以发酵产物的形式产生乙醇。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述第一非天然基因编码第一非天然酶,该酶提供水解纤维素的能力,从而使得所述嗜热或嗜温微生物能够水解纤维素。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述第一非天然基因编码第一非天然酶,该酶提供水解木聚糖的能力,从而使得所述嗜热或嗜温微生物能够水解木聚糖。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述第一非天然基因编码第一非天然酶,该酶提供水解纤维素的能力;并且其中***了第二非天然基因,该第二非天然基因编码第二非天然酶,该酶提供水解木聚糖的能力,从而使得所述嗜热或嗜温微生物能够水解纤维素和木聚糖。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述有机酸是乳酸。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述有机酸是乙酸。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述第一非天然酶是丙酮酸脱羧酶(PDC)或乙醇脱氢酶(ADH)。在某些实施方案中,本发明涉及上述经遗传修饰的微生物,其中所述微生物能够将至少60%的来自被代谢的生物质的碳转化成乙醇。
在某些实施方案中,本发明涉及上述经遗传修饰的微生物的任一种微生物,其中所述微生物是嗜温微生物。在某些实施方案中,本发明涉及上述经遗传修饰的微生物的任一种,其中所述微生物是嗜热微生物。
本发明的另一个方面涉及用于将木质纤维生物质转化成乙醇的方法,其包括将木质纤维生物质与上述经遗传修饰的嗜热或嗜温微生物的任一种接触。在某些实施方案中,本发明涉及上述方法,其中所述木质纤维生物质选自:草、柳枝稷、互花米草、黑麦草、草芦、混合大网茅草(mixed prairie grass)、芒属植物、糖加工废渣、甘蔗渣、甘蔗秆、农业废料、稻草、稻壳、大麦稿秆、玉米芯、谷类稿秆、麦秸、油菜秆(canola straw)、燕麦秸秆、燕麦皮、玉米纤维、干草、大豆干草、玉米秸秆、森林废弃物、再循环木浆纤维、造纸污泥、锯屑、硬木材、软木材和其组合。在某些实施方案中,本发明涉及上述方法,其中所述木质纤维生物质选自玉米秸秆、甘蔗渣、柳枝稷和杨木材。在某些实施方案中,本发明涉及上述方法,其中所述木质纤维生物质是玉米秸秆。在某些实施方案中,本发明涉及上述方法,其中所述木质纤维生物质是甘蔗渣。在某些实施方案中,本发明涉及上述方法,其中所述木质纤维生物质是柳枝稷。在某些实施方案中,本发明涉及上述方法,其中所述木质纤维生物质是杨木材。在某些实施方案中,本发明涉及上述方法,其中所述木质纤维生物质是柳木。在某些实施方案中,本发明涉及上述方法,其中所述木质纤维生物质是造纸污泥。
附图概述
图1描述了糖酵解途径。
图2描述了戊糖和葡萄糖醛酸的互变以及突显了D-木糖至乙醇的途径中的酶木糖异构酶(XI或5.3.1.5)和木酮糖激酶(XK或2.7.1.17)。
图3描述了戊糖和葡萄糖醛酸的互变以及突显了L-***糖利用途径中的酶L-***糖异构酶(5.3.1.4)和L-核酮糖-5磷酸4-表异构酶(5.1.3.4)。
图4描述了戊糖和葡萄糖醛酸的互变并且显示木糖异构酶、木酮糖激酶、L-***糖异构酶和L-核酮糖-5-磷酸-4-表异构酶的基因存在于解纤维梭菌中。
图5描述了戊糖和葡萄糖醛酸的互变并且显示木糖异构酶和木酮糖激酶存在于C.phytofermentans中,而L-***糖异构酶和L-核酮糖-5-磷酸-4-表异构酶不存在于其中。
图6显示热解纤维梭菌(SEQ ID NO:77)、解纤维梭菌(SEQ ID NO:78)、解糖好热厌氧杆菌(SEQ ID NO:79)、粪堆梭菌(SEQ ID NO:80)、粪堆梭菌II(SEQ ID NO:81)、Caldiscellulosiruptorkristjanssonii(SEQ ID NO:82)、C.phytofermentans(SEQ ID NO:83)的比对,表明在16S rDNA基因的水平上存在大约73-89%的同源性。
图7显示基于质粒pIKM1的用于使热解纤维梭菌中的ack基因失活的双交换型敲除载体的构建。
图8显示基于复制型质粒pNW33N的用于使热解纤维梭菌中的ack基因失活的双交换型敲除载体的构建。
图9显示基于质粒pIKM1的用于使热解纤维梭菌中的ldh基因失活的双交换型敲除载体的构建。
图10显示基于复制型质粒pNW33N的用于使热解纤维梭菌中的ldh基因失活的双交换型敲除载体的构建。
图11显示基于复制型质粒pUC19的用于使热解纤维梭菌中的ldh基因失活的双交换型***载体的构建。
图13A和13B分别显示在纤维二糖和上培养的热解纤维梭菌的产物形成和OD600。
图16A和16B分别显示在纤维二糖和上培养的克里斯托热解纤维素菌的产物形成和OD600。
图18显示在48小时的2.5g/L木聚糖和2.5g/L纤维二糖的发酵后的总代谢副产品。
图19显示ack基因的图谱和通过PCR扩增的用于基因破坏的区域。
图20显示ldh 2262基因的图谱和通过PCR扩增的用于基因破坏的区域。
图21显示解纤维梭菌(C.cell.)ldh(2262)双交换型敲除片段的实例。
图22显示Clostridium phytofermentans的ack基因的图谱和通过PCR扩增的用于基因破坏的区域。
图23显示Clostridium phytofermentans中的使用mLs基因作为选择标记的假定的双交换型敲除构建体的实例。
图24显示ldh 1389基因的图谱和通过PCR扩增的用于基因破坏的区域。
图25显示使用mLs基因作为选择标记的假定的双交换型敲除构建体的实例。
图26是代表pMODTM-2<MCS>的bp 250-550的图示(SEQ ID NO: 84)。
图28显示用于将Ll.LtrB内含子再靶向(retarget)解纤维梭菌ACK基因(SEQ ID NO:21)中的***物的载体的实例。
图29显示将Ll.LtrB内含子再靶向解纤维梭菌LDH2744基因(SEQ ID NO:23)中的***物的载体的实例。
图30显示T.pseudoethanolicus 39E(SEQ ID NO:85)、T.sp菌株59(SEQ ID NO:86)、解糖梭菌B6A-RI(SEQ ID NO:87)、解糖梭菌YS485(SEQ ID NO:88)的比对和在16S rDNA基因水平上的共有序列(SEQ ID NO:89)。
图31显示T.sp.菌株59(SEQ ID NO:36)、T.pseudoethanolicus(SEQ ID NO:35)、解糖梭菌B6A-RI(SEQ ID NO:38)、解糖梭菌YS485(SEQ ID NO:32)的比对和在pta基因水平上的共有序列(SEQ ID NO:90)。
图32显示T.sp.菌株59(SEQ ID NO:37)、T.pseudoethanolicus(SEQ ID NO:34)、解糖梭菌B6A-RI(SEQ ID NO:39)、解糖梭菌YS485(SEQ ID NO:33)的比对和在ack基因的水平上的共有序列(SEQ ID NO:91)。
图33显示T.sp.菌株59(SEQ ID NO:41)、T.pseudoethanolicus 39E(SEQ ID NO:42)、解糖梭菌B6A-RI(SEQ ID NO:43)、解糖梭菌YS485(SEQ ID NO:40)的比对和在ldh基因的水平上的共有序列(SEQ ID NO:92)。
图34显示糖酵解/发酵途径的示意图。
图35显示pMU340质粒的实例。
图36显示pMU102运动发酵单孢菌(Z.mobilis)PDC-ADH质粒的实例。
图37显示pMU102棕榈发酵细菌(Z.palmae)PDC、运动发酵单孢菌ADH质粒的实例。
图38显示pMU360的质粒图谱。pMU360的DNA序列示于SEQ IDNO:61。
图39显示9个抗甲砜霉素转化株的集落的乳酸水平。
图40显示T.sacch.pfl KO单交换型质粒(SEQ ID NO:47)的实例。
图41显示T.sacch.pfl KO双交换型质粒(SEQ ID NO:48)的实例。
图42显示热解纤维梭菌pfl KO单交换型质粒(SEQ ID NO:49)的实例。
图43显示热解纤维梭菌pfl KO双交换型质粒(SEQ ID NO:50)的实例。
图44显示C.phyto.pfl KO单交换型质粒(SEQ ID NO:51)的实例。
图45显示C.phyto.pfl KO双交换型质粒(SEQ ID NO:52)的实例。
图46显示T.sacch.#59 L-ldh KO单交换型质粒(SEQ ID NO:53)的实例。
图47显示T.sacch.#59 L-ldh KO双交换型质粒(SEQ ID NO:54)的实例。
图48显示T.sacch.#59 pta/ack KO单交换型质粒(SEQ ID NO:55)的实例。
图49显示T.sacch.#59 pta/ack KO双交换型质粒(SEQ ID NO:56)的实例。
图50显示T.pseudo.L-ldh KO单交换型质粒(SEQ ID NO:57)的实例。
图51显示T.pseudo.L-ldh KO双交换型质粒(SEQ ID NO:58)的实例。
图52显示T.pseudo.ack KO单交换型质粒(SEQ ID NO:59)的实例。
图53显示T.pseudo.pta/ack KO双交换型质粒(SEQ ID NO:60)的实例。
附表概述
表1概述了代表性高度分解纤维素的生物。
表2概述了代表性天然纤维素分解和木聚糖分解的生物。
表3显示了基于其底物利用的细菌菌株的分类。
表4显示将内含子再靶向解纤维梭菌的乙酸激酶的***位置和引物。
表5显示将内含子再靶向解纤维梭菌的乳酸脱氢酶的***位置和引物。
表6显示经工程改造的嗜热厌氧杆菌(Thermoanaerobacter)和高温厌氧芽孢杆菌(Thermoanaerobacterium)菌株的发酵性能。
发明详述
本发明的方面涉及对嗜热或嗜温微生物进行工程改造以用于从木质纤维生物质产生乙醇。将嗜热细菌用于乙醇生产提供了优于基于嗜温乙醇产生者的常规方法的许多有利方面。例如,使用嗜热生物因更低的乙醇分离成本、对外加酶的加入的减少的需要和减少的加工时间而在经济上显著比常规处理方法更节省。
本发明的方面涉及方法,借助于该方法,可通过使用新流程配置来减少从包含纤维质生物质的材料生产乙醇的成本。特别地,本发明提供了许多用于在经遗传修饰的微生物中提高乙醇产量的方法。
在某些实施方案中,本发明涉及经遗传修饰的嗜热或嗜温微生物,其中基因或特别地多核苷酸序列(该基因或多核苷酸序列编码为所述微生物提供以发酵产物的形式产生有机酸的能力的酶)被部分、基本上或完全缺失、沉默、失活或下调,从而提高微生物产生乙醇作为主要发酵产物的能力。此外,由于加工步骤的新型整合(通常称为联合生物加工),本发明的方面提供了乙醇从包含纤维质-生物质的原料的更有效产生。在所述材料的加工中经遗传修饰的嗜热或嗜温微生物的加入使得发酵步骤能够在更高的温度下进行,从而提高了工艺经济。例如,反应动力学通常与温度成正比,因此更高的温度通常与总生产率(rate of production)的提高相关。此外,更高的温度促进挥发性产物从液体培养基清除并且减少在预处理后对冷却的需要。
在某些实施方案中,本发明涉及具有提高的产生酶的能力的经遗传修饰的或重组的嗜热或嗜温微生物,所述酶提供以发酵产物的形式产生乙醇的能力,该酶的存在改变了代谢木质纤维生物质材料的过程,从而以主要发酵产物的形式产生乙醇。在本发明的一个方面,将一个或多个非天然基因***经遗传修饰的嗜热或嗜温微生物,其中所述非天然基因编码参与乙醇的代谢产生的酶,例如,此类酶可提供代谢戊糖和/或己糖的能力。例如,在一个实施方案中,酶可参与D-木糖或L-***糖途径,从而使得微生物能够代谢戊糖,即,D-木糖或L-***糖。通过***(例如,导入或加入)编码参与D-木糖或L-***糖的代谢或利用的酶的非天然基因,微生物具有与所述天然生物相比较提高的产生乙醇的能力。
本发明还提供了可被整合入本发明的微生物的新型组合物。在一个实施方案中,本发明的分离的核酸分子包含作为SEQ ID NOS:1-76的任一个中显示的核苷酸序列的互补序列的核酸分子。在另一个实施方案中,本发明的分离的核酸分子包含作为SEQ ID NOS:1-76的任一个中所示的核苷酸序列或此类核苷酸序列的任一个的一部分的互补序列的核酸分子。与SEQ ID NOS:1-76的任一个中所示的核苷酸序列或其编码区域互补的核酸分子是与SEQ ID NOS:1-76的任一个中显示的核苷酸序列或其编码区充分互补的核酸分子,这样其可与SEQ ID NOS:1-76的任一个中显示的核苷酸序列或其编码区杂交,从而形成稳定的双链体。
在另一个优选实施方案中,本发明的分离的核酸分子包含核苷酸序列,所述核苷酸序列与SEQ ID NOS:1-76的任一个中显示的核苷酸序列(例如,与核苷酸序列的整个长度)或此类核苷酸序列的任一个的一部分至少大约50%、54%、55%、60%、62%、65%、70%、75%、78%、80%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更多的同源。
此外,本发明的核酸分子可以只包含SEQ ID NOS:1-76的任一个的核酸序列的一部分或其编码区;例如核酸分子可以是可用作探针或引物的片段或编码蛋白质的生物活性部分的片段。在另一个实施方案中,核酸分子可包含SEQ ID NOS:1-76的任一个的至少大约12或15,优选大约20或25,更优选大约30、35、40、45、50、55、60、65或75个连续核苷酸。
定义
术语“异源多核苷酸区段”意在包括编码一个或多个多肽或多肽的部分或片段的多核苷酸区段。异源多核苷酸区段可来源于任何来源,例如,真核生物、原核生物、病毒或合成的多核苷酸片段。
术语“启动子”或“替代启动子(surrogate promoter)”意欲包括多核苷酸区段,所述多核苷酸区段可在转录上控制其在天然状态下不转录控制的目的基因。在某些实施方案中,替代启动子的转录控制导致目的基因表达的提高。在某些实施方案中,替代启动子被置于目的基因的5’。替代启动子可用于置换天然启动子,或除了天然启动子之外还可使用其。替代启动子对于其中使用其的宿主细胞可以是内源的,或其可以是导入宿主细胞的异源多核苷酸序列,例如,对于其中使用其的宿主细胞是外源的。
术语“基因”或“多核苷酸区段”或“多核苷酸序列”意在包括核酸分子,例如,包括编码多肽的开放阅读框架,并且还可包括非编码调控序列和内含子的多核苷酸。此外,所述术语意在包括一个或多个被定位至功能基因座的基因。此外,所述术语意在包括用于选择目的的特殊基因。基因对于宿主细胞可以是内源的或可以被重组地导入宿主细胞,例如作为以附加体形式保持的质粒或被稳定地整合入基因组的质粒(或其片段)。除了质粒形式以外,基因可以例如以线性DNA的形式存在。在某些实施方案中,多核苷酸区段的基因参与碳水化合物至乙醇的生物转化中的至少一个步骤。因此,所述术语意在包括编码多肽,例如酶乙酸激酶(ACK)、磷酸转乙酰酶(PTA)和/或乳酸脱氢酶(LDH)、D-木糖途径中的酶例如木糖异构酶和木酮糖激酶、L-***糖途径中的酶例如L-***糖异构酶和L-核酮糖-5-磷酸4-表异构酶的任何基因。术语基因还意在包括所有拷贝的特定基因,例如,在细胞中编码特定基因产物的所有DNA序列。
术语“转录控制”意在包括在转录水平上调控基因表达的能力。在某些实施方案中,转录,从而基因表达可通过在目的基因的编码区的5’末端附近替换或添加替代启动子(从而导致改变的基因表达)来调控。在某些实施方案中,对一个或多个基因的转录控制进行工程改造来产生所述基因的最佳表达,例如以期望的比例表达。所述术语还包括本领域内公认的诱导型转录控制。
术语“表达”意在包括基因至少在mRNA产物水平上的表达。
术语“表达产物”意在包括表达的基因的所得产物,例如多肽。
术语“提高的表达”意在包括至少在提高的mRNA产量的水平上和优选在多肽表达的水平上的基因表达的变化。术语“提高的产量”意在包括表达的多肽的量上、多肽的酶促活性水平上或其组合上的提高。
术语“活性”、“活性”、“酶促活性”和“酶促活性”可互换使用并且意在包括通常归功于被选择的多肽(当在有利的条件下产生时)的任何功能活性。通常,被选择的多肽的活性包括与产生的多肽相关的总酶促活性。由宿主细胞产生的并且具有酶促活性的多肽可位于细胞的细胞内空间、与细胞结合、分泌入细胞外环境或其组合。用于测定与分泌活性相比较的总活性的技术在本文中进行了描述并且在本领域内是已知的。
术语“木聚糖分解活性”意在包括水解寡聚戊糖或多聚戊糖中的糖苷键的能力。
术语“纤维素分解活性”意在包括水解寡聚己糖和多聚己糖中的糖苷键的能力。纤维素水解活性还可包括解聚纤维素和半纤维素或对其去分支的能力。
如本文中所使用的,术语“乳酸脱氢酶”或“LDH”意在包括能够将丙酮酸转化成乳酸的酶。要理解,LDH还可催化羟基丁酸的氧化。
如本文中所使用的,“乙醇脱氢酶”或“ADH”意在包括能够将乙醛转化成醇,有利地,乙醇的酶。
术语“丙酮酸脱羧酶活性”意在包括多肽将丙酮酸酶促转化成乙醛的能力(例如,“丙酮酸脱羧酶”或“PDC”)。通常,被选择的多肽的活性包括与产生的多肽相关的总酶促活性,包括,例如,酶的优良的底物亲和力、热稳定性、在不同pH下的稳定性或此类属性的组合。
术语“产乙醇的”意在包括微生物从碳水化合物以发酵产物的形式产生乙醇的能力。该术语意在包括但不限于天然发生的产乙醇生物、具有天然发生的或诱导的突变的产乙醇生物以及已进行了遗传修饰的产乙醇生物。
术语“发酵”和“发酵作用”意在包括借以从碳水化合物产生乙醇(特别地作为发酵产物)的酶促过程(例如,细胞的或无细胞的,例如,裂解物或纯化的多肽混合物)。
术语“分泌的”意在包括多肽至周质空间或细胞外环境的转运。术语“提高的分泌”意在包括其中给定多肽以提高的水平(即,超过天然发生的分泌的量)分泌的状态。在某些实施方案中,术语“提高的分泌”是指当与天然发生的分泌水平相比较时,为至少大约10%或至少大约100%、200%、300%、400%、500%、600%、700%、800%、900%、1000%或更多的给定的多肽的分泌的提高。
术语“分泌多肽”意在包括单独地或与其他多肽组合在一起,促进另一种多肽从细胞的细胞内空间至细胞外环境的转运的任何多肽。在某些实施方案中,分泌多肽包括足以赋予革兰氏阴性或革兰氏阳性宿主细胞分泌活性的所有必需分泌多肽。通常,分泌蛋白在单个区域或基因座中被编码,可从一种宿主细胞分离所述区域或基因座并且使用基因工程将其转移至另一种宿主细胞。在某些实施方案中,分泌多肽来源于具有分泌活性的任何细菌细胞。在某些实施方案中,分泌多肽来源于具有II型分泌活性的宿主细胞。在某些实施方案中,宿主细胞是嗜热细菌细胞。
术语“来源于”意在包括多核苷酸区段从指定的来源的分离(完整地或部分地)或多肽从指定的来源的纯化。该术语意在包括例如直接克隆、PCR扩增或根据或基于与指定的多核苷酸来源相关的序列进行的人工合成。
“嗜热的”是指在大约45℃或更高的温度下旺盛生长的生物。
“嗜温的”是指在大约20-45℃的温度下旺盛生长的生物。
术语“有机酸”是本领域公认的。术语“乳酸”是指以游离酸或盐形式存在的有机酸2-羟基丙酸。无论使用何种中和剂即,碳酸钙或氢氧化铵,乳酸的盐形式称为“乳酸盐”。术语“乙酸”是指以游离酸或盐形式存在的也称为醋酸(ethanoic acid)的有机酸甲烷羧酸。乙酸的盐形式称为“乙酸盐”。
本发明的某些实施方案在嗜热或嗜温微生物内提供某些基因或特定多核苷酸序列的“***”,(例如,添加、整合、加入(incorporation)或导入),可以理解所述基因或特定多核苷酸序列的***包括“遗传修饰”或“转化”,这样所得的所述嗜热或嗜温微生物的菌株可被理解为是“经遗传修饰的”或“经转化的”。在某些实施方案中,菌株可以是细菌、真菌或酵母来源的。
本发明的某些实施方案在嗜热或嗜温微生物内提供某些基因或特定多核苷酸序列的“失活”或“缺失”,可以理解所述基因或特定多核苷酸序列的“失活”或“缺失”包括“遗传修饰”或“转化”,这样所得的所述嗜热或嗜温微生物的菌株可被理解为是“经遗传修饰的”或“经转化的”。在某些实施方案中,菌株可以是细菌、真菌或酵母来源。
术语“CBP生物”意在包括本发明的微生物,例如具有适合于CBP的特性的微生物。
在本发明的一个方面,***基因或特定的多核苷酸序列以激活它们编码的活性例如酶的表达。在某些实施方案中,可向嗜温或嗜热性生物中加入编码乙醇的代谢产生中的酶例如代谢戊糖和/或己糖的酶的基因。在本发明的某些实施方案中,酶可以提供代谢戊糖的能力和参与例如D-木糖途径和/或L-***糖途径。
在本发明的一个方面,使基因或特定核苷酸序列被部分、基本上或完全缺失、沉默、失活或下调,从而使它们编码的活性例如酶的表达失活。缺失提供最大的稳定性,因为不存在恢复功能的回复突变的机会。备选地,可通过破坏基因的功能和/或表达的核酸序列的***(例如,P1转导或本领域内已知的其他方法)来使基因部分、基本上或完全缺失、沉默、失活或下调。术语“消除”、“清除”和“敲除”可与术语“缺失”互换使用。在某些实施方案中,目的嗜热或嗜温微生物可通过定点同源重组进行工程改造来敲除有机酸的产生。在其他实施方案中,RNAi或反义DNA(asDNA)可用于部分、基本上或完全沉默、失活或下调特定的目的基因。
在某些实施方案中,本文所述的被靶向以进行缺失或失活的基因对于微生物的天然菌株可以是内源的,从而可被理解地称为“天然基因”或“内源基因”。如果生物未进行基因工程改造或未通过人的手以有意地改变生物的基因和/或表型组成的方式进行操作,那么其以“天然状态”的形式存在。例如,野生型生物可被认为处于天然状态。在其他实施方案中,被靶向以进行缺失或失活的基因对于生物可以是非天然的。
生物质
术语“木质纤维材料”、“木质纤维底物”和“木质纤维生物质”是指包含纤维素、半纤维素、木质素或其组合的任何类型的生物质,例如但不限于木质生物质、牧草、草本能源作物、非木质植物生物质、农业废料和/或农业废物、森林残留物和和/或森林废弃物、造纸污泥和/或废弃造纸污泥、污水处理污泥(waste-water-treatment sludge)、市政固体废物、来自采用湿法和干法工艺的玉米酒精厂(wet and dry mill cornethanol plant)的玉米纤维和糖加工废渣.
在非限定性实施例中,木质纤维材料可包括但不限于木质生物质例如再循环木浆纤维、锯屑、硬木、软木材和其组合;草,例如柳枝稷、互花米草、黑麦草、草芦、芒属植物或其组合;糖加工废渣,例如但不限于甘蔗渣;农业废料,例如但不限于稻草、稻壳、大麦稿秆、玉米芯、谷类稿秆、麦秸、油菜秆、燕麦秸秆,燕麦皮和玉米纤维;干草,例如但不限于大豆干草、玉米秸秆;和森林废弃物,例如但不限于再循环木浆纤维、锯屑、硬木(例如,白杨木、橡木、枫木(maple)、桦木、柳木),软木材或其任何组合。木质纤维材料可包括一种纤维;备选地,木质纤维材料可包括来源于不同木质纤维材料的纤维的混合物。特别有利的木质纤维材料是农业废料,例如谷类稿秆,包括麦秸、大麦稿秆,油菜秆和燕麦秸秆;玉米纤维;干草例如玉米秸秆和大豆干草;草例如柳枝稷、草芦、互花米草和芒属植物;或其组合。
造纸污泥也是用于乙醇生产的活原料(viable feedstock)。造纸污泥是从制浆和造纸产生的固体残渣,通常在初级澄清池(primaryclarifier)中将其从作业污水(process wastewater)中移出。以$30/湿吨的处理成本,污泥处理的成本相当于$5/吨生产用于销售的纸。湿污泥的处理成本极大地刺激了将所述材料转化为其他用途。例如转化成乙醇。本发明提供的加工方法可获得广泛应用。此外,糖化和/或发酵产物可用于产生乙醇或更高价值的另外的化学药品例如有机酸、芳香剂、酯、丙酮和聚合物中间体。
丙酮酸甲酸裂解酶(PFL)
丙酮酸甲酸裂解酶(PFL)是帮助调控无氧葡萄糖代谢的重要酶(于大肠杆菌(Escherichia coli)和其他生物中发现的)。通过使用自由基化学(radical chemistry),其催化丙酮酸和辅酶A至甲酸和乙酰-CoA(乙醇的前体)的可逆转化。丙酮酸甲酸裂解酶是由85kDa,759个残基的亚基组成的同二聚体。其具有其中***了包含主要催化残基的β指的10-链β/α桶形基元(barrel motif)。通过X射线衍射晶体分析法阐明的酶的活性位点具有3个进行催化作用的必需氨基酸(Gly734、Cys418和Cys419)、3个包围底物丙酮酸的主要残基(Arg435、Arg176和Ala272)以及2个侧翼疏水残基(Trp333和Phe432)。
研究已发现丙酮酸甲酸裂解酶的活性位点与I类和III类核糖核苷酸还原酶(RNR)的活性位点之间的结构相似性。3个催化残基的作用如下:Gly734(甘氨酰基)-通过Cys419将自由基转移至Cys418上和从其上转移出;Cys418(含硫基(thiyl radical))-对丙酮酸羰基的碳原子进行酰化化学作用;Cys419(含硫基)-进行氢原子转移。
提出的丙酮酸甲酸裂解酶的机制始于通过Cys419从Gly734至Cys418的自由基转移。Cys418含硫基共价地加至丙酮酸的C2(第2碳原子),从而产生乙酰基-酶中间体(其现可包含所述自由基)。乙酰基-酶中间体释放经历利用Cys419进行的氢原子转移的甲酰基。这产生了甲酸和Cys419基。辅酶-A经历利用Cys419基进行的氢原子转移,从而产生辅酶-A基。然后辅酶-A基从Cys418获得乙酰基来产生乙酰-CoA,从而留下Cys418基。丙酮酸甲酸裂解酶然后可经历自由基转移来将基转回Gly734。上述步骤的每一步也是可逆的。
两个另外的酶调控丙酮酸甲酸裂解酶的“开”和“关”状态,从而调控无氧葡萄糖代谢:PFL活化酶(activase)(AE)和PFL去活化酶(deactivase)(DA)。当丙酮酸可获得时,被激活的丙酮酸甲酸裂解酶使得能够形成乙酰-CoA,在能量的产生中非常重要的小分子。去活化的丙酮酸甲酸裂解酶,即使在底物存在的情况下,也不催化反应。PFL活化酶是自由基SAM(S-腺苷甲硫氨酸)超家族的一部分。
酶通过将Gly734(G-H)转化成(通过5′-脱氧腺苷基(deoxyadenosylradical)(自由基SAM)))Gly734基(G*)来“开启”丙酮酸甲酸裂解酶。PFL去活化酶(DA)通过淬灭Gly734基来“关闭”丙酮酸甲酸裂解酶。此外,丙酮酸甲酸裂解酶对分子氧(O2)敏感,分子氧的存在使酶关闭。
木糖的代谢
木糖是可被多种生物代谢成有用的产物的五碳单糖。存在两个主要的木糖代谢途径,各自在它们利用的特征酶上是独特的。一个途径称为“木糖还原酶-木糖醇脱氢酶”或XR-XDH途径。木糖还原酶(XR)和木糖醇脱氢酶(XDH)是用于该木糖降解方法的两个主要的酶。由XYL1基因编码的XR负责木糖至木糖醇的还原并且受助于辅因子NADH和NADPH。然后由通过XYL2基因表达的XDH将木糖醇氧化成木酮糖,并且只借助于辅助因子NAD+来完成。由于本途径中需要的不同的辅因子以及其可获得使用的程度,不平衡可导致木糖醇副产品的过量产生,从而不能产生足够的期望的乙醇。已在实验室中检测了XR和XDH酶水平的不同表达以试图最优化木糖代谢途径的效率。
木糖代谢的另一个途径称为“木糖异构酶”(XI)途径。酶XI负责木糖至木酮糖的直接转化,并且不通过木糖醇中间物进行加工。两个途径都产生木酮糖,虽然利用的酶不同。在产生木酮糖后,XR-XDH和XI途径都通过由基因XKS1编码的酶木酮糖激酶(XK)继续进行,从而进一步将木酮糖修饰成木酮糖-5-P,然后其由此进入戊糖磷酸途径以进行进一步分解代谢。
关于在木糖代谢过程中通过戊糖磷酸途径的流量(flux)的研究已揭示限制该步骤的速度可有益于发酵产生乙醇的效率。可提提高乙醇产量的对该流量的改变包括a)降低磷酸葡萄糖异构酶活性,b)缺失GND1基因,和c)缺失ZWF1基因(Jeppsson等人,2002)。因为戊糖磷酸途径在代谢过程中产生额外的NADPH,从而限制该步骤将帮助修正NAD(P)H和NAD+辅因子之间的已明显的不平衡,从而且减少木糖醇副产品。包括两个木糖代谢途径的另一个实验显示XI途径能够最有效地代谢木糖,从而产生最大的乙醇收率,尽管XR-XDH途径达到快得多的乙醇生产速度(Karhumaa等人,2007)。
微生物
本发明包括多个用于产生具有进行CBP所需的底物利用和产物形成性质的组合的微生物的策略。“天然分解纤维的策略”牵涉对天然发生的纤维素分解微生物进行工程改造以提高产物相关性质例如收率和滴度。“重组分解纤维的策略”牵涉对展示高成品收率和滴度的天然非纤维分解生物进行工程改造以表达使得能够进行纤维素利用或半纤维素利用或进行两者的异源纤维素酶***。
纤维素分解微生物
文献中报导的为分解纤维素的或具有纤维素分解活性的几种微生物已通过许多方法得到表征,包括它们在微晶纤维素以及多种其他糖上生长的能力。此外,所述生物可通过其他方法表征,包括但不限于它们解聚纤维素和半纤维素以及使其去分支的能力。热解纤维梭菌(菌株DSMZ 1237)用作基准来检查目的生物。如本文中所使用的,热解纤维梭菌可包括不同的菌株,包括但不限于DSMZ 1237、DSMZ 1313、DSMZ2360、DSMZ 4150、DSMZ 7072和ATCC 31924。在本发明的某些实施方案中,热解纤维梭菌的菌株可包括但不限于DSMZ 1313或DSMZ1237。在另一个实施方案中,用于本发明的特别适合的目的生物包括具有大于70%的与热解纤维梭菌的16S rDNA的同源性的纤维素分解生物。热解纤维梭菌、解纤维梭菌解糖好热厌氧杆菌、粪堆梭菌、粪堆梭菌II、Caldiscellulosiruptor kristjanssonii、C.phytofermentans的比对显示在16S rDNA基因水平上的73~85%的同源性(图6)。
表1
将生物培养在20g/L纤维二糖或20g/L中。于60℃下培养热解纤维梭菌和于55℃下培养C.straminisolvens。在含有50mM MOPS的M122中预培养来自-80℃冷藏库(来源DSMZ)的两者。在中对数生长期至晚对数生长期期间,将预培养物用于接种100mL血清瓶中的分批培养物,达到50mL的工作体积。定期取出液体样品用于代谢副产品和糖消耗的HPLC分析。在每一个这些时间点上获得OD600。图12A和12B分别显示C.straminisolvens在纤维二糖和中的产物形成和OD600。在OD下降和产物水平达到平衡前48小时,消耗了大量纤维二糖(37%)。图13A和13B分别显示热解纤维梭菌在纤维二糖和中的产物形成和OD600。在48小时内热解纤维梭菌消耗了大约60%的纤维二糖,在该点上产物形成达到平衡。由于有机酸的形成导致的抑制造成底物的不完全利用。
某些微生物包括例如热解纤维梭菌和C.straminisolvens不能代谢戊糖,例如D-木糖或L-***糖,但能够代谢己糖。D-木糖和L-***糖都是生物质中含量丰富的糖,D-木糖在软和硬木材中占据大约16至20%,L-***糖在玉米纤维占据大约25%。因此,本发明的一个目的是提供经遗传修饰的具有代谢戊糖例如D-木糖和L-***糖,从而增强它们用作用于生物质至乙醇工业(biomass-to-ethanol industry)中的发酵的生物催化剂的能力的纤维素分解微生物。
纤维素分解和木聚糖分解微生物
从文献中确定的为分解纤维素和分解木聚糖的几中微生物已就它们在微晶纤维素和桦木木聚糖以及多种其他糖类上生长的能力得到表征。热解纤维梭菌用于标准检查目的生物。在被选择用于表征的菌株中,解纤维梭菌、粪堆梭菌leptospartum亚种,克里斯托热解纤维素菌和Clostridium phytofermentans在上生长不良,但在桦木木聚糖上生长良好。表2概述了一些天然纤维素分解和木聚糖分解生物。
表2
将生物培养在20g/L维生二糖、20g/L或5g/L桦木木聚糖中。在37℃下培养解纤维梭菌,在60℃下培养粪堆梭菌leptospartum亚种,在75℃下培养克里斯托热解纤维素菌以及在37℃下培养Clostridiumphytofermentans。在补充有50mM MOPS的M122c中预培养来自-80℃冷藏库(来源DSMZ)的所有上述微生物。在中对数生长期至晚对数生长期期间,将预培养物用于接种100mL血清瓶中的分批培养物,达到50mL的工作体积。定期取出液体样品用于代谢副产品和糖消耗的HPLC分析。在每一个这些时间点上获得OD600。图14A和17B显示在纤维二糖和中培养的产物形成和OD600。
在分开的实验中,将生物培养在2.5g/L单种糖(包括纤维二糖、葡萄糖、木糖、半乳糖、***糖、甘露糖和乳糖)以及5g/L和桦木木聚糖中。在图18中,在2天后比较在纤维二糖和桦木木聚糖上的产物形成。表3概述了可基于细菌底物利用对其进行分类的方法。
表3
纤维二糖 | 葡萄糖 | 木糖 | 半乳糖 | ***糖 | 甘露糖 | 乳糖 | |
解纤维梭菌 | × | × | × | × | × | ||
粪堆梭菌亚种leptospartum | × | × | × | × | × | × | × |
C.kristjanssonii | × | × | × | × | × | × | |
C.phytofermentans | × | × | × | × | × |
微生物的转基因转变
本发明提供了用于某些微生物的转基因转变的组合物和方法。当将编码参与乙醇的代谢途径包括例如D-木糖和/或L-***糖的代谢的酶的基因导入缺乏一种或多种此类基因的细菌菌株例如热解纤维梭菌或C.straminisolvens时,可选择转化菌株培养在D-木糖或培养在L-***糖上。预期来自其他梭状芽胞杆菌种类的基因应当在热解纤维梭菌和C.straminisolvens中表达。靶基因供体可包括提供代谢己糖和戊糖的能力的微生物,例如解纤维梭菌、克里斯托热解纤维素菌、C.phytofermentans、粪堆梭菌和解糖好热厌氧杆菌。
解糖梭菌、解纤维梭菌和C.phytofermentans的基因组是可获得的。因此,本发明提供了相应于上面所示的3种宿主的每一种中的木糖异构酶和木酮糖激酶的序列。特别地,相应于来自解糖梭菌的木糖异构酶(SEQ ID NO:6)、木酮糖激酶(SEQ ID NO:7)、L-阿拉析糖异构酶(SEQ IDNO:8)和L-核酮糖-5-磷酸4-表异构酶(SEQ ID NO:9)的序列示于本文中。类似地,本文中提供了相应于来自解纤维梭菌的木糖异构酶(SEQ IDNO:10)、木酮糖激酶(SEQ ID NO:11)、L-阿拉析糖异构酶(SEQ ID NO:12)和L-核酮糖-5-磷酸4-表异构酶(SEQ ID NO:13)的序列。C.phytofermentans利用D-木糖途径但不利用L-***糖。因此,相应于来自C.phytofermentans的的木糖异构酶(SEQ ID NO:14)和木酮糖激酶(SEQ ID NO:15)的序列示于本文中。
C.kristjanssonii不代谢木糖。为此目的,已对C.kristjanssonii的木糖异构酶(SEQ ID NO:71)和木酮糖激酶(SEQ ID NO:70)基因进行了测序并且在本文中提供了所述序列。未曾显示C.straminisolvens在木糖上生长,然而其确实包含木糖异构酶(SEQ ID NO:73)和木酮糖激酶(SEQ IDNO:72)基因,所述基因在适应木糖作为碳源后可以是有功能的。
热解纤维梭菌和C.straminisolvens可缺乏D-木糖至乙醇途径和/或L-***糖利用途径中的一个或多个已知的基因或酶。图2和3描述了在热解纤维梭菌的此类途径的每一个途径中缺少的两个关键酶。C.straminisolvens具有木糖异构酶和木酮糖激酶,但此类酶的功能未知。基因组测序在C.straminosolvens中未显示L-***糖异构酶或L-核酮糖-5-磷酸4-表异构酶的拷贝。
热解纤维梭菌和C.straminisolvens不能代谢木酮糖,这可反映将D-木糖转化成D-木酮糖的木糖异构酶(在图2中称为“XI”或5.3.1.5)和将D-木酮糖转化成D-木酮糖-5磷酸的木酮糖激酶(在图2中也称为“XK”或2.7.1.1)基因的不存在(热解纤维梭菌)或活性和/或表达的缺乏(C.straminsolvens)。此外,对于C.straminsolvens,木糖的转运可以是限制。该潜在的限制可通过表达来自利用木糖的生物例如解糖梭菌和C.kristjanssonii的糖转运基因来克服。
热解纤维梭菌和C.straminisolvens也不能代谢L-***糖,这可反映了L-***糖异构酶(在图3中也称为5.3.1.4)和L-核酮糖-5-磷酸4-表异构酶(在图3中也称为5.1.3.4)的基因的不存在。
上述4个基因例如木糖异构酶、木酮糖激酶、L-***糖异构酶和L-核酮糖-5-磷酸4-表异构酶存在于几个梭状芽胞杆菌种类和解糖好热厌氧杆菌种类中,包括但不限于解纤维梭菌(参见图4)、解糖好热厌氧杆菌、粪堆梭菌、Caldiscellulosiruptor kristjanssonii和C.phytofermentans;这些菌株是此类糖的良好利用者。将要认识到,上述细菌菌株可用作本文中描述的基因的供体。
C.phytofermentans表达上述的2个木糖途径基因(木糖异构酶和木酮糖激酶),但缺乏或不表达上述***糖途径基因(L-***糖异构酶和L-核酮糖-5-磷酸4-表异构酶)(参见图5)。
因此,本发明的目的是修饰一些上述细菌菌株以通过例如导入从生物质来源的戊糖例如D-木糖或L-***糖的代谢产生乙醇所需的一个或多个酶的基因来最优化糖利用能力。启动子,包括热解纤维梭菌或C.straminisolvens的天然启动子例如磷酸丙糖异构酶(TPI)、GAPDH和LDH的启动子可用于表达此类基因。相应于热解纤维梭菌的天然启动子的序列包括(TPI)(SEQ ID NO:16)、GAPDH(SEQ ID NO:17)和LDH(SEQID NO:18)。当基因被克隆后,可在表达前进行密码子最优化。然后可将包含例如天然启动子、分解木聚糖的基因或分解***糖的基因和选择标记的盒用于转化热解纤维梭菌或C.straminisolvens和选择包含D-木糖或L-***糖作为唯一碳水化合物来源的培养基上的D-木糖和L-***糖生长。
转座子
为了选择已进入宿主的外源DNA,优选将DNA稳定地保持在目的生物中。关于质粒,存在两个通过其可使DNA稳定地保持在目的生物中的途径。一个途径是通过使用复制型质粒。此类质粒具有被宿主细胞识别并且允许质粒作为稳定的、自主的、染色体外元件(其在细胞***过程中被分配入子细胞)复制的复制起点。第二途径通过将质粒整合入染色体来发生。这主要通过同源重组来发生并且导致整合质粒或质粒的部分至宿主染色体的***。因此,质粒和选择标记作为染色体的整合片段而被复制并且被分离入子细胞。因而,为了确定质粒DNA在通过使用选择标记进行的转化事件过程中是否进入细胞,需要使用复制型质粒或将质粒重组至染色体上的能力。这些要求不能总是得到满足,特别是当操作不具有一套遗传工具(a suite of genetic tool)的生物时。
避免关于与质粒相关的标记的问题的一个方法是通过使用转座子。转座子是可移动的DNA元件,被定义为被称为转座酶的酶促机器识别的嵌合DNA序列(mosaic DNA sequence)。转座酶的功能是将转座子DNA随机***宿主或靶DNA。可通过标准基因工程将选择标记克隆入转座子。可在体内和体外反应中将所得的DNA片段偶联至转座酶机器,并且可通过电穿孔将所述复合物导入靶细胞。标记至染色体的稳定***只需要转座酶的功能并且减少对同源重组或复制型质粒的需要。
与转座子的整合相关的随机性质具有充当诱变形式的额外的有利方面。可建立包含转座子突变体的混合物的文库。此类文库可用于产生具有期望的表型的突变体的筛选或选择。例如,可就产生更多乙醇或更少乳酸和/或更少乙酸的能力筛选CBP生物的转座子文库。
天然分解纤维素的策略
天然发生的纤维素分解微生物是通过天然策略进行CBP生物开发的起始点。厌氧菌和兼性厌氧菌是特别吸引人的。主要目的是对成品收率和乙醇滴度进行工程改造以满足工业加工的需要。与此类目的相关的混合酸发酵的代谢工程改造在嗜温非纤维素分解肠细菌的情况下获得成功。适当的基因转移技术中的最新发展允许此类型的工作利用纤维素分解细菌来进行。
重组纤维素分解策略
具有期望的产物形成性质(例如,高乙醇收率和滴度)的非纤维素分解微生物是通过重组纤维素分解策略进行的CBP生物开发的起始点。此类开发的主要目的是对使得能够在经预处理的木质纤维素上生长和发酵的异源纤维素酶***进行工程改造。主要使用以高收率产生乙醇的细菌宿主(大肠杆菌、产酸克雷伯氏菌(Klebsiella oxytoca)和运动发酵单孢菌的经工程改造的菌株)和酵母酿酒酵母来进行纤维素酶的异源产生。产酸克雷伯氏菌的菌株中的纤维素酶的表达导致提高的水解收率(但对于微晶纤维素,在不加入纤维素酶的情况下不能生长),和在无定形纤维素上厌氧生长。尽管许多糖解酶已在酿酒酵母中功能性表达,但作为此表达的结果而发生的在纤维素上的厌氧生长还未得到最终的证明。
本发明的方面涉及嗜热或嗜温微生物作为用于通过天然纤维素分解策略进行修饰的宿主的用途。它们在生物技术的加工应用中的潜能源自它们在相对高的温度下生长(伴随高代谢速率)的能力、物理和化学上稳定的酶的产生以及提高的终产品收率。主要的嗜热细菌的种类包括真细菌类和古细菌。嗜热真细菌包括:向光细菌例如蓝细菌(cyanobacteria)、紫细菌和绿细菌;***例如杆菌(Bacillus)、梭状芽胞杆菌(Clostridium)、乳酸菌和放线菌(Actinomyces);以及其他真细菌例如硫化细菌(Thiobacillus)、Spirochete,Desulfotomaculum、革兰氏阴性需氧菌、革兰氏阴性厌氧菌和Thermotoga。产甲烷菌,极端嗜热生物(本领域内公认的术语)和Thermoplasma被认为属于古细菌。在某些实施方案中,本发明涉及嗜热菌(Thermus)属的革兰氏阴性有机营养嗜热菌、革兰氏阳性真细菌例如梭状芽孢杆菌(Clostridium),并且该属包括杆菌和球菌、真细菌组中的属例如嗜热厌氧菌属(Thermosipho)和Thermotoga、古细菌的属例如超嗜热古菌属(Thermococcus)、Thermoproteus(棒状的)、热丝菌属(Thermofilum)(棒状的),热网菌属(Pyrodictium)、Acidianus、硫化叶菌属(Sulfolobus)、Pyrobaculum、Pyrococcus、热盘菌属(Thermodiscus)、Staphylothermus、Desulfurococcus、Archaeoglobus和Methanopyrus。可适用于本发明的嗜热或嗜温生物(包括细菌、原核微生物和真菌)的一些实例包括但不限于:Clostridiumthermosulfurogenes、解纤维梭菌、热解纤维梭菌、Clostridiumthermohydrosulfuricum、Clostridium thermoaceticum、热解糖梭菌(Clostridium thermosaccharolyticum)、酒石酸梭菌(Clostridiumtartarivorum)、热解纤维素梭菌、Clostridium phytofermentans、Clostridiumstraminosolvens、Thermoanaerobacterium thermosaccarolyticum、解糖好热厌氧杆菌、乙酸乙基嗜热拟杆菌(Thermobacteroides acetoethylicus)、布氏好热厌氧杆菌、Methanobacterium thermoautotrophicum、Anaerocellum thermophilium、隐蔽热网菌(Pyrodictium occultum)、Thermoproteus neutrophilus、Thermofilum librum、Thermothrix thioparus、Desulfovibrio thermophilus、Thermoplasma acidophilum、Hydrogenomonasthermophilus、玫瑰红嗜热菌(Thermomicrobium roseum)、Thermus flavas、高温菌(Thermus ruber)、极端嗜热菌(Pyrococcus furiosus)、嗜热水生菌(Thermus aquaticus)、嗜热栖热菌(Thermus thermophilus)、Chloroflexus aurantiacus、Thermococcus litoralis、Pyrodictium abyssi、嗜热脂肪芽孢杆菌(Bacillus stearothermophilus)、Cyanidium caldarium、嗜热蓝藻层理鞭枝藻(Mastigocladus laminosus)、Chlamydothrixcalidissima、Chlamydothrix penicillata、Thiothrix carnea、微细席藻(Phormidium tenuissimum)、居热泉席蓝细菌(Phormidium geysericola)、Phormidium subterraneum、Phormidium bijahensi、Oscillatoria filiformis、铅色聚球藻(Synechococcus lividus)、Chloroflexus aurantiacus、布氏热网菌(Pyrodictium brockii)、氧化硫硫杆菌(Thiobacillus thiooxidans)、嗜酸热硫化叶菌(Sulfolobus acidocaldarius)、嗜热硫杆菌(Thiobacillusthermophilica)、嗜热脂肪芽孢杆菌、Cercosulcifer hamathensis、Vahlkampfia reichi、瓜形膜袋虫(Cyclidium citrullus)、Dactylariagallopava、铅色聚球藻、Synechococcus elongatus、Synechococcusminervae、Synechocystis aquatilus、温泉隐球蓝细菌(Aphanocapsathermalis)、Oscillatoria terebriformis、两栖颤藻(Oscillatoria amphibia)、Oscillatoria germinata、Oscillatoria okenii、蓝藻(Phormidiumlaminosum)、Phormidium parparasiens、墙生束藻(Symploca thermalis)、Bacillus acidocaldarias、凝结芽孢杆菌(Bacillus coagulans)、Bacillusthermocatenalatus、地衣芽孢杆菌(Bacillus licheniformis)、Bacilluspamilas、浸麻芽孢杆菌(Bacillus macerans)、芽孢杆菌(Bacilluscirculans)、侧胞芽孢杆菌(Bacillus laterosporus)、短芽孢杆菌(Bacillusbrevis)、枯草芽孢杆菌(Bacillus subtilis)、球形芽孢杆菌(Bacillussphaericus)、致黑脱硫肠状菌(Desulfotomaculum nigrificans)、嗜热链球菌(Streptococcus thermophilus)、嗜热乳酸杆菌(Lactobacillusthermophilus),保加利亚乳杆菌(Lactobacillus bulgaricus)、嗜热双歧杆菌(Bifidobacterium thermophilum)、Streptomyces thermonitrificans、Streptomyces thermonitrificans、链霉菌(Streptomyces thermovulgaris)、嗜热假诺卡氏菌(Pseudonocardia thermophila)、普通高温放线菌(Thermoactinomyces vulgaris)、糖高温放线菌(Thermoactinomycessacchari)、Thermoactinomyces candidas、Thermomonospora curvata、Thermomonospora viridis、柠檬高温单孢菌(Thermomonospora citrina)、热分解淀粉小双孢菌(Microbispora thermodiastatica)、充气小双孢菌(Microbispora aerata)、双孢小双孢菌(Microbispora bispora)、Actinobifida dichotomica、产色双岐放线菌(Actinobifida chromogena)、Micropolyspora caesia、干草小多孢菌(Micropolyspora faeni)、Micropolyspora cectivugida、Micropolyspora cabrobrunea、热绿小多孢菌(Micropolyspora thermovirida)、绿黑小多孢菌(Micropolysporaviridinigra)、Methanobacterium thermoautothropicum、Caldicellulosiruptoracetigenus、极度嗜热菌、克里斯托热解纤维素菌、Caldicellulosiruptorowensensis、Caldicellulosiruptor lactoaceticus、其变种和/或其后代。
在某些实施方案中,本发明涉及选自Fervidobacteriumgondwanense、Clostridium thermolacticum、Moorella sp.和海洋红嗜热盐菌(Rhodothermus marinus)的嗜热细菌。
在某些实施方案中,本发明涉及好热厌氧杆菌属或Thermoanaerobacter的嗜热细菌,包括但不限于选自下列的种:热硫化氢好热厌氧杆菌、Thermoanaerobacterium aotearoense、解多糖好热厌氧杆菌、玉米好热厌氧杆菌、解木聚糖好热厌氧杆菌、解糖好热厌氧杆菌、布氏好热厌氧杆菌、解糖好热厌氧杆菌、Thermoanaerobacterthermohydrosulfuricus、嗜热厌氧乙醇杆菌、Thermoanaerobacter brockii,其变种和其后代。
在某些实施方案中,本发明涉及地芽孢杆菌属(Geobacillus)、糖球菌属(Saccharococcus)、类芽孢杆菌属(Paenibacillus)、牙孢杆菌属(Bacillus)和厌氧芽孢杆菌属(Anoxybacillus)的微生物,包括但不限于选自下列的种:热葡糖苷酶地芽孢杆菌、嗜热脂肪地芽孢杆菌、Saccharococcus caldoxylosilyticus、喜温链球菌、坎皮纳斯类芽孢杆菌、黄热芽孢杆菌、Anoxybacillus kamchatkensis、Anoxybacillus gonensis、其变种和其后代。
在某些实施方案中,本发明涉及选自下列的嗜温细菌:Saccharophagus degradans、Flavobacterium johnsoniae、产琥珀酸拟杆菌(Fibrobacter succinogenes)、Clostridium hungatei、Clostridiumphytofermentans、解纤维梭菌、Clostridium aldrichii、Clostridiumtermitididis、解纤维素醋弧菌(Acetivibrio cellulolyticus)、产乙醇醋弧菌(Acetivibrio ethanolgignens)、多食醋弧菌(Acetivibrio multivorans)、Bacteroides cellulosolvens和Alkalibacter saccharofomentans、其变种和其后代。
本发明的方法
在糖酵解过程中,细胞将单糖例如葡萄糖转化成丙酮酸,净产生ATP和NADH。在用于氧化磷酸化的功能性电子传递***不存在的情况下,至少95%的丙酮酸在短途径中被消耗,该短途径再生NAD+,其进行持续的糖酵解和ATP产生的专性需要。此类NAD+再生***的废产物通常称为发酵产物。
微生物产生多种多样的发酵产物,包括有机酸例如乳酸盐(乳酸的盐形式)、乙酸盐(乙酸的盐形式)、琥珀酸盐和丁酸盐以及中性产物例如乙醇、丁醇、丙酮和丁二醇。发酵的终产物共有不同程度的几个基本特性,包括:它们在其中最初产生它们的条件下是相对无毒的,但在积累后毒性变得更大;和它们比丙酮酸减少得更多,因为它们的直接前体(immediate precursor)在糖酵解过程中已用作末端电子受体。本发明的方面涉及基因敲除技术用于提供用于从木质纤维素生物质底物产生乙醇的新型微生物的用途。通过使一个或多个基因缺失或失活,任选地然后就具有提高的以发酵产物的形式产生乙醇的性能的突变体进行基于生长的选择来制备经转化的生物,所述基因编码竞争性途径例如本文中描述的至期望的有机酸的非限定性途径。
在某些实施方案中,转化在天然状态中包含至少一个为微生物提供以发酵产物的形式产生乳酸的能力的基因的嗜热或嗜温微生物以减少或消除所述至少一个基因的表达。为所述微生物提供产生乳酸作为发酵产物的能力的基因可编码乳酸脱氢酶的表达。编码LDH的表达的基因或特定多核苷酸序列的缺失或抑制减少或消除了总的糖醇解途径中的籍以将丙酮酸转化成乳酸的反应方案(reaction scheme);来自此类糖酵解的第一阶段的所得的丙酮酸的相对丰度应当使得能够提高乙醇的产量。
在某些实施方案中,转化在天然状态中包含至少一个为微生物提供以发酵产物的形式产生乙酸的能力的基因的嗜热或嗜温微生物以消除所述至少一个基因的表达。为微生物提供产生乙酸作为发酵产物的能力的基因可编码乙酸激酶和/或磷酸转乙酰酶的表达。编码ACK和/或PTA的表达的基因或特定多核苷酸序列的缺失或抑制减少或消除了总的糖醇解途径中的籍以将乙酰CoA转化成乙酸的反应方案(图1);来自此类糖酵解的更后阶段的所得的乙酰CoA的相对丰度应当使得能够提高乙醇的产量。
在某些实施方案中,上述基因敲除方案可单独使用或协同使用。消除产生乳酸盐的机制(即,敲除编码LDH的表达的基因或特定多核苷酸序列)产生更多的乙酰CoA;其遵循,如果也消除产生乙酸盐的机制(即,敲除编码ACK和/或PTA的表达的基因或特定多核苷酸序列),乙酰CoA的丰度将进一步提高,这可导致提高的乙醇产生。
在某些实施方案中,嗜热或嗜温微生物不必具有天然的或内源的PDC或ADH。在某些实施方案中,可在经遗传修饰的本发明的微生物中重组表达编码PDC和/或ADH的基因。在某些实施方案中,可将本发明的基因敲除技术用于重组微生物,所述重组微生物可包含编码PDC和/或ADH的异源基因,其中所述异源基因以足以提高所述重组微生物(其可以是嗜热微生物)以发酵产物的形式产生乙醇或为所述重组微生物(其可以是嗜热微生物)提供以发酵产物的形式产生乙醇的能力的水平进行表达。
在某些实施方案中,本发明的方面涉及以为至少70%的理论收率(基于纤维素浓度或半纤维素浓度或两者)的浓度产生乙醇的木质纤维素底物的发酵。
在某些实施方案中,本发明的方面涉及以为至少80%的理论收率(基于纤维素浓度或半纤维素浓度或两者)的浓度产生乙醇的木质纤维素底物的发酵。
在某些实施方案中,本发明的方面涉及以为至少90%的理论收率(基于纤维素浓度或半纤维素浓度或两者)的浓度产生乙醇的木质纤维素底物的发酵。
在某些实施方案中,可使用一个或多个定点DNA同源重组事件来实现从处于天然状态的微生物中基本上或完全消除有机酸的产生。
在嗜热温度下操作同步糖化共发酵(SSCF)或CBP过程提供了几个优于30-37℃的常规嗜温发酵温度的重要益处。特别地,专门用于纤维素酶产生的处理步骤的成本大大减少(例如,2倍或更多)(对于嗜热SSCF)和消除(对于CBP)。与发酵罐冷却以及发酵前和发酵后的热交换相关的成本也预期得到减少(对于嗜热SSCF和CBP)。最后,表征嗜热生物催化剂的方法与表征常规嗜热生物催化剂的方法相比较,不太易于发生微生物污染。
依靠对碳流的改变而改变电子流方向的能力具有广泛的意义。例如,该方法可用于在除了解糖梭菌外的菌株中产生高乙醇收率和/或产生除了乙醇外的溶剂,例如高级醇(即,丁醇)。
通过反义寡核苷酸(asRNA)策略进行的代谢工程
发酵微生物例如酵母和厌氧菌将糖发酵成乙醇和其他还原性有机终产物。理论上,如果可抑制竞争性终产物例如乳酸盐和乙酸盐的形成,则可将碳流导向乙醇的产生。本发明提供了数个设计用于在本发明的CBP生物中除去竞争性途径的基因工程方法。此类方法的主要部分利用基因敲除构建体(对于单交换型重组)或等位基因交换构建体(对于双交换型重组)并且靶向ack和ldh的基因座。虽然此类工具使用“经过检验而可靠的”菌株开发技术,但仍存在几个可阻碍进程的潜在问题:(i)它们依赖于在所有情况下对于CBP生物来说是未知的宿主重组效率;(ii)它们可用于一次只敲除一个途径,因此连续的基因改变使得具有数个选择标记或重复利用标记成为必需;(iii)靶基因的缺失可以是有毒的或对下游基因表达具有极化效应。
本发明提供了导向不依赖于宿主重组效率的基因工程的另外的方法。一个此类备选工具称为反义RNA(asRNA)。虽然反义寡核苷酸25年来一直用于在体外和体内抑制基因的表达水平,但mRNA结构预测的最新进展已为asRNA分子的更灵巧设计提供了方便。这些进展已促进许多研究小组证明asRNA在细菌的代谢工程中的有用性。
在基因敲除和等位基因交换技术中使用asRNA的益处是多方面的:
(i)减少对多个选择标记的需要,因为可通过单个asRNA构建体靶向多个途径;(ii)可通过提高或降低asRNA之间的缔合速率来调节靶mRNA的衰减少水平;(iii)如果asRNA转录物由条件启动子驱动则可条件灭活途径。最近,该技术已用于提高革兰氏阳性嗜温菌丙酮丁醇梭菌(Clostridium acetobutylicum)(Tummala等人(2003))中的溶剂生成(solventogenesis)。虽然asRNA减弱基因表达的确切分子机制还不清楚,但当asRNA与靶mRNA杂交后触发了可能的机制。机制可包括一个或多个下列方面:(i)通过适当地与核糖体相互作用封闭核糖结合位点产生的对mRNA至蛋白质的翻译的抑制,(ii)通过快速降解双链体RNA的dsRNA-依赖的RNA酶例如RNA酶H减少mRNA的半衰期,和(iii)由于mRNA的早期转录终止而导致的转录的抑制。
反义序列的设计
asRNA的长度通常为18-25个核苷酸。存在几个可获得用于合理设计可用于选择asRNA序列的靶向RNA的核酸的计算工具(Sfold,Integrated DNA Technologies,STZ Nucleic Acid Design)。例如,可将热解纤维梭菌ack(乙酸激酶)的基因序列提交至合理设计服务器(rationaldesign server),然后可挑选出几个asRNA序列。简而言之,设计参数选择不包含预测的二级结构的mRNA靶序列。
递送载体的设计
复制型质粒将被用于把asRNA编码序列递送至靶生物。载体例如但不限于pNW33N、pJIR418、pJIR751和pCTC1将形成用于把asRNA编码序列递送至宿主细胞内部的asRNA构建体的主链。除了染色体外(基于质粒的)表达外,还可将asRNA在异源基因座上稳定地***微生物的基因组,从而获得asRNA的稳定表达。在某些实施方案中,可通过定点同源重组来对目的嗜热或嗜温微生物的菌株进行工程改造,从而敲除有机酸的产生,并且可通过asRNA使其他目的基因部分、基本上或完全缺失、沉默、失活或下调。
启动子的选择
为了确保asRNA转录物的表达,可将用于给定的宿主的相容性启动子融合至asRNA编码序列。可在单个PCR步骤中构建启动子-asRNA盒。可这样修饰被设计用于扩增启动子区域的有义和反义引物以使asRNA序列(通过合理设计方法挑选的)与反义引物的5’末端连接。此外,可向各引物的末端加入限制性位点例如EcoRI或BamHI,以使可用限制性内切酶直接降解终PCR扩增子,然后通过常规克隆技术将其***载体的主链。
关于本文中描述的不具有代谢戊糖的能力但能够代谢己糖的微生物,将认识到,可以根据根据本文中描述的方法使用反义RNA例如靶向热解纤维梭菌和Clostridium straminisolvens的ack和ldh基因以使之失活。
关于本文中描述的提供代谢戊糖和己糖的能力的微生物,将认识到,可以根据根据本文中描述的方法使用反义RNA例如靶向解纤维素梭菌、Clostridium phytofermentans和克里斯托热解纤维素菌的ack和ldh基因以使之失活。
除了对表达asRNA递送载体的菌株进行抗生素选择外,还可在包含几种毒性代谢产物类似物例如氟乙酸钠(SFA)、溴乙酸(BAA)、氯醋酸(CAA)、5-氟乳清酸(5-FOA)和氯乳酸的任一种的条件培养基上选择此类菌株。将化学诱变剂(包括但不限于使用乙烷甲基磺酸酯(ethane methylsulfonate)(EMS))与反义寡核苷酸(asRNA)的表达结合起来用于产生一个或多个基因被部分、基本上或完全缺失、沉默、失活或下调的菌株。
实施例
通过参考被包括只为了举例说明本发明的某些方面和实施方案而不意欲限定本发明的下列实施例,将更容易地理解刚才进行一般性描述的本发明。
实施例1
用于嗜温和嗜热纤维素分解、木聚糖分解生物的定制转座子(CustomTransposon)的产生
本发明提供了用于产生用于纤维素分解和/或木聚糖分解和/或嗜热生物的定制转座子的方法。为了实现该目的,将把来自宿主生物的天然启动子融合至已确定在该生物中起作用的选择标记。将该片段克隆入载体pMODTM-2<MCS>(Biotechnologies)上携带的EZ-Tn5TM转座子。例如,将热解纤维梭菌的gapDH启动子融合至mLs药物标记以及cat基因,然后将其亚克隆入载体pMODTM-2<MCS>。
商业转座子缺少热稳定性药物标记和纤维素分解和/或木聚糖分解和/或嗜热生物的天然启动子。mLs和cat标记已在嗜热细菌中起作用,gapDH启动子调控关键的糖酵解酶并且应当持续表达。上述药物标记和gapDH启动子的组合将极大地提高产生功能性转座子的概率。该方法可用于其他纤维素分解和/或木聚糖分解和/或嗜热生物。
实验设计
图26是获自Biotechnologies用户手册(其通过引用合并入本文)的图,代表pMODTM-2<MCS>的bp 250-550。在上面部分中,黑色箭头标记的ME表示界定转座子的19bp嵌合末端。EcoRI和HindIII位点界定多克隆位点,其由黑框标记的MCS表示。在下面部分中,显示了与MCS相联系的DNA序列和限制性内切酶。
下列引物将被用于扩增来自pMQ87-gapDH-cat和pMQ87-gapDH-mls的启动子融合片段:GGCGgaattc CTT GGT CTG ACAATC GAT GC(SEQ ID NO:19);GGCGgaattcTATCAGTTATTACCCACTTTTCG(SEQ ID NO:20)。小写字母表示经工程改造的EcoRI限制性位点。产生的扩增子的大小将为大约1.9kb。标准分子方法将使扩增子能用EcoRI来降解,并且被克隆入pMODTM-2<MCS>的唯一的EcoRI位点。如厂商所描述的,将产生转座子和随后转座体(transpososome),并且将其导入宿主生物。
实施例2
用于对纤维素分解和木聚糖分解菌株进行工程改造的构建体
本发明提供了用于通过突变编码代谢途径(所述代谢途径使碳流转离乙醇)的关键酶的基因将目的生物基因工程改造为CBP的组合物和方法。设计单交换型敲除构建体以将大片段的外源DNA***目的基因,从而将所述基因部分、基本上或完全缺失、沉默、失活或下调。设计双交换型敲除构建体以从染色体上部分、基本上或完全缺失、沉默、失活或下调目的基因,或用基因的突变拷贝例如被抗生素抗性盒破坏的基因的形式替代染色体上的目的基因。
单交换敲除载体的设计需要将目的基因的内部片段克隆入基于质粒的***。理想地,该载体将携带在宿主菌株中表达但不会在宿主菌株中复制的选择标记。因此,当导入宿主菌株后,质粒将不会复制。如果将细胞置于选择质粒上携带的标记的条件培养基中,只有已找到保持质粒的方式的细胞才将能生长。因为质粒不能作为自主DNA元件复制,因此,质粒将得到保持的最可能的方式是通过重组至宿主染色体上。重组最可能发生的位置在质粒和宿主染色体之间的同源区上。
备选地,复制型质粒可用于产生单交换破坏。可在条件培养基上选择已接受敲除载体的细胞,然后在不存在选择的情况下进行传代。在不存在由条件培养基提供的正向选择的情况下,许多生物将丢失质粒。在质粒被***宿主染色体的情况下,质粒在不存在选择的情况下将不会丢失。然后可将细胞返回条件培养基,只有已通过染色体整合保持标记的细胞将能够生长。将设计基于PCR的方法来筛选包含位于染色体上的标记的生物。
双交换型敲除载体的设计需要至少将侧连(flanking)目的基因的DNA(大约1kb)克隆入质粒并且在一些情况下可包括克隆目的基因。可将选择标记置于侧翼DNA之间,或如果克隆目的基因,则对于所述基因,将标记置于内部。理想地,所使用的质粒不能在宿主菌株中复制。在将质粒导入宿主并且在针对标记条件化的培养基上选择后,只有已将同源DNA重组入染色体的细胞才将能生长。需要两个重组事件来用选择标记替代目的基因。
备选地,复制型质粒可用于产生双交换型基因替代。可在条件培养基上选择已接受敲除载体的细胞,然后在不存在选择的情况下进行传代。在不存在由条件培养基提供的正向选择的情况下,许多生物将丢失质粒。在药物标记被***宿主染色体的情况下,其在不存在选择的情况下将不会丢失。然后可将细胞返回条件培养基,并且只有已通过染色体整合而保留标记的细胞将能够生长。将设计基于PCR的方法来筛选包含位于染色体上的标记的生物。
除了抗生素选择方案外,还可使用几个毒性代谢产物类似物例如氟乙酸钠(SFA)、溴乙酸(BAA)、氯醋酸(CAA)、5-氟乳清酸(5-FOA)和氯乳酸来选择通过同源重组或基于转座子的策略产生的突变体。将使用化学诱变剂(包括但不限于乙烷甲基磺酸酯(EMS))与使用同源重组或基于转座子的策略的定向诱变方案结合使用。
解纤维梭菌敲除构建体
乙酸激酶(来自解纤维梭菌的公开的基因组的基因131):
单交换
解纤维梭菌的乙酸激酶基因的长度为1,110bp。通过PCR扩增横跨核苷酸91-752的662bp内部片段(SEQ ID NO:21),然后将其克隆入具有不同选择标记的***载体和复制型载体。选择标记可包括提供红霉素和氯霉素抗性的标记。将此类质粒用于破坏ack基因。ack基因的图谱和通过PCR扩增的用于基因破坏的区域示于图19中。下面所示的SEQ IDNO:21的下划线标示的部分相应于为侧连敲除片段的EcoRI位点的位点。
gaattctgcgacagaatagggattgacaattcctttataaagcaatcaaggggttcagaagaggctgttatttt
gaataaagagctaaagaatcacaaagatgcaatagaggctgttatttctgcactgactgacgataatatgggc
gttataaaaaacatgtccgaaatatcagcagtgggacacagaatagtacacggcggtgaaaaattcaacagt
tctgtagttatagatgaaaacgttatgaatgcagtaagagagtgtatagacgttgcaccgcttcataatccgcc
gaatattataggtatagaggcttgccagcagattatgcccaatatacctatggtagctgtatttgataccactttc
cacagctccatgcctgattatgcatacctttacgcattgccatatgaactttatgaaaagtacggtataagaaaa
tatggtttccacggaacatcacacaaatatgttgcagaaagagcttctgcaatgcttgataagtctttgaacga
attaaagataattacatgccatcttgggaacggttcaagtatttgtgctgttaacaagggtaaatcaattgatact
tccatgggctttacacctttgcagggacttgcaatgggtacaagaagcggtacaatagaccctgaagttgtta
cgaattc
在“ack KO引物”的设计过程中对此类位点进行工程改造,从而将使所述片段随后能够克隆入许多载体。
双交换
为了构建用于解纤维梭菌的ack基因的双交换载体,将克隆大约1kb的侧连ack基因的各侧的DNA。在侧翼DNA之间***选择标记。选择标记可包括提供红霉素和氯霉素抗性的选择标记。ack基因的3’侧翼区域在可获得的基因组草图中是不可获得的。为了获得该DNA,将使用试剂盒例如来自Clontech的GenomeWalker。
乳酸脱氢酶(解纤维梭菌的公开的基因组的基因2262和2744):
单交换
解纤维梭菌的ldh基因的长度是951bp(对于基因2262)(SEQ IDNO:22)和932bp(对于基因2744)(SEQ ID NO:23)。通过PCR扩增各基因的5’末端附近的大约500bp内部片段,并且将其克隆入具有不同选择标记的***载体和复制型载体。选择标记可包括提供药物抗性例如对红霉素和氯霉素的抗性的选择标记。此类质粒将被用于破坏ldh 2262和ldh2744基因。例如,ldh 2262基因的图谱和通过PCR扩增的用于基因破坏的区域示于图20中。
双交换
为了构建用于解纤维梭菌的ldh基因的双交换敲除载体,将克隆大约1kb的侧连ldh基因的各侧的DNA。将选择标记***侧翼DNA之间。选择标记可包括提供药物抗性例如对红霉素和氟霉素抗性的选择标记。
图21提供了解纤维梭菌ldh(2262)双交换敲除片段的实例。
在下面所示的序列(SEQ ID NO:24)中,mLs基因(选择标记)以下划线标示,侧翼DNA是剩下的序列。在引物设计过程中,将对上述片段的5’和3’末端的限制性位点进行工程改造,以使其可被克隆入许多复制型和非复制型载体。相同的策略将用于产生缺失ldh 2744的载体。
gacgcatacaggttgtaacacccatttcccttagcttttcgggagatgaataaaacaaactttccgggtccttta
ccacaccgcccacataaagagctatgccgcatgaaagaaacgatatgttatcatttttttcgtaaactgttattt
ccgaacccggataaagctttaccatattattaactgctgccgtccctgcatgtgtacaccctataaccactattt
tcatatacatcctcctttgtttgcttgtaaatatatcccatatataccacctaaatatattttataaacaaattcggta
tatcattcttttggtaaataaaaagtacatccgatattagaatgtacctaaaaaaaattattattttattgtatatgct
ttatctgttttcattatatggtttgctatccattctacggtaaaatcaagtaattccattaagtactgatcctgatcct
tgtctatcctgctataatccgtattactgattttctcaataaaatcatggtgttcaactttgtgggagagaagcttg
cgatatcctatgctatgcatgtattcttcttcataggtaaaatgaaagacagtgtaatcttttagttccgtaattag
ccgtacaatttcatcatatttgtctgtaataagctgatttttcgtggcctcataaatttccgaagcaatctggaata
gtttcttatgctgttcgtcgattttctcaattccaagaataaattcgtctctccattctatcatatggaccctcctaa
attgtaatgtataccaagattatacatacttcctagaatataaacaatacaaggataaaattttaatatcgtatac
ctacataaatgactaacttaaagctctctaaaacttcttttttattatttctatactactaaaatcaaaaatattctct
aaagtatttctacaaatgttgtttttgcaacaaagtagtatacttttgcacccagaatgttttgttataacttacaaa
ttaggggtatatttatagtaaatactaaatggaagagtaggatattgattatgaacgagaaaaatataaaacac
agtcaaaactttattacttcaaaacataatatagataaaataatgacaaatataagattaaatgaacatgataat
atctttgaaatcggctcaggaaaagggcattttacccttgaattagtacagaggtgtaatttcgtaactgccatt
gaaatagaccataaattatgcaaaactacagaaaataaacttgttgatcacgataatttccaagttttaaacaa
ggatatattgcagtttaaatttcctaaaaaccaatcctataaaatatttggtaatataccttataacataagtacgg
atataatacgcaaaattgtttttgatagtatagctgatgagatttatttaatcgtggaatacgggtttgctaaaaga
ttattaaatacaaaacgctcattggcattatttttaatggcagaagttgatatttctatattaagtatggttccaaga
gaatattttcatcctaaacctaaagtgaatagctcacttatcagattaaatagaaaaaaatcaagaatatcacac
aaagataaacagaagtataattatttcgttatgaaatgggttaacaaagaatacaagaaaatatttacaaaaaa
tcaatttaacaattccttaaaacatgcaggaattgacgatttaaacaatattagctttgaacaattcttatctctttt
caatagctataaattatttaataagatcccctttacttcggatgcatgccgcaggcaggcatccgaagtagtttc
tccattatacaagtattctcttgagtacgtcgtcgcttctcagcagctgctttgctttttccctgttttccggcacat
ggagataagtgtatctgttaggcttaatagtgtgtgccatgtcaattgccttttcgaagtcatctgccttcattttta
aggtttccacaaaattgataaaacccgtatcagtcagaaattttactacccgctgatatctgtgttcttgaaccct
gctcataagataggttgcaatcccaacctgaattccatgaagctgaggtgtctccagcagcttatctaaagcat
gagatattagatgctcactaccgctggctggagcactgctgtctgctatctgcatggcaattccgctcattgtc
agagagtctaccatttcctttaaaaagaagttttctgtaacctgtgtgtagggcatccttacaatactgtttactga
ctttttagcaatcattgcagcaaaatcgtcaacctttgccgcattgttcctttcttcaaaataccagtcatacaca
gccgtaattttggatattatgtctccgagacctgaataaataaatttcataggtgcattttttaatacatctaaatc
cactaatattccaaatggcatcgaggcatgtacggaagtacgcctgccatttataatcaaagagcagcctga
gctggaaaaaccatcgtttgaggttgatgtaggtatactgataaaaggaagcttgtttaaaaaagctatatattt
ggctgcatcaagcacctttcctcctcctactccgaccactgcatcggttttggagggaatagtaaaagccttga
gcataagattttcaagctttatgtcatcatagtcgtaagtttcaagtactgcaagagattttcttgactttatggaat
ccagaatcttttcaccaaataagtcacgtattccctctccaaaaagtactacaacattactaattcctgccctttc
aatatgtgc
C.phytofermentans敲除构建体
对于乙酸激酶(来自C.phytofermentans的公开的基因组的基因327):
单交换
C.phytofermentans的乙酸激酶基因的长度是1,244bp。通过PCR扩增横跨核苷酸55-626的572bp内部片段,并且将其克隆入具有不同选择标记的***载体和复制型载体。使用的选择标记可包括为C.phytofermentans提供药物抗性的选择标记。此类质粒将被用于破坏ack基因。ack基因的图谱和通过PCR扩增的用于基因破坏的区域示于图22中。可在“ack KO引物”设计过程中对限制性位点进行工程改造,所述限制性位点将使所述片段随后能够克隆入许多载体。上述敲除片段的序列示为SEQ ID NO:25。
双交换
为了构建用于C.phytofermentans的ack基因的双交换型敲除载体,将克隆大约1kb的侧连ack基因的各侧的DNA。将选择标记***侧翼DNA之间。所使用的选择标记将包括为该菌株提供药物抗性的选择标记。以mLs基因作为假定的选择标记的假定的双交换型敲除构建体的实例示于图23中。
相应于图23中描述的片段(SEQ ID NO:26)的序列示于下面。mLs基因(假定的选择标记)以下划线标示,序列的剩下部分相应于侧翼DNA。在引物设计过程中,对上述片段的5’和3’末端的限制性位点进行工程改造以使其可被克隆入许多复制型和非复制型载体。
ctgagtgcaatgtaaaaaaggatgcctcaagtattcttgaaacatccttatattatactacaaaatcataaagta
aattactcagctgtagcaatgatctcttttttgttgtaagatccacaagctttacaaactctatgaggcatcataa
gtgcaccacacttgctgcatttcactaagtttggagcagtcatcttccagtttgcacgacgactatctcttctag
ctttggaatgtttattctttggacaaatagctcccattgattacacctccttaaacttgttaaaaatatctcggata
gcagacattcttgggtctagttctgtacggtcacacccgcactctccttcatttaggttagcaccgcagaccttg
cagattcctttacagtcttctttgcacagaaccttcattgggaaaccaatcaagacttcttcatagataagtttat
ctacgtctaaatcatatccggaaacaaaatttgtttcatctaaatcctcggtacgctgttcctctgttttcgataca
tcaatctctgtagccacgtcgatgtcttgttggatggtttcttccttcaaacaacgatcgcaaggaacggctaac
gctaatttcgtttttgcttccaccagaatttttcggccacctagattagttaatctaagtttaaccggttctttatag
gtaatagaataaccgacaccatttaattcgaatatatcaaattcaatcggtgcagtgtattctttgagaccattag
gaacattcatgacttcagacatttgtatcagcataagtaactcctgtctaaaaaaacgcataatgtaagcgccc
aaaaattcacactgttagtattataaacgcttaaaataggtttgtcaactcctaactgttaaaaatgtcagaattg
tgtaaccatattttctcttcattatcgttcttcccttattaaataatttatagctattgaaaagagataagaattgttc
aaagctaatattgtttaaatcgtcaattcctgcatgttttaaggaattgttaaattgattttttgtaaatattttcttgta
ttctttgttaacccatttcataacgaaataattatacttctgtttatctttgtgtgatattcttgatttttttctatttaatc
tgataagtgagctattcactttaggtttaggatgaaaatattctcttggaaccatacttaatatagaaatatcaact
tctgccattaaaaataatgccaatgagcgttttgtatttaataatcttttagcaaaccc gtattccacgattaaata
aatctcatcagctatactatcaaaaacaattttgcgtattatatccgtacttatgttataaggtatattaccaaatat
tttataggattggtttttaggaaatttaaactgcaatatatccttgtttaaaacttggaaattatc gtgatcaacaag
tttattttctgtagttttgcataatttatggtctatttcaatggcagttacgaaattacacctctgtactaattcaagg
gtaaaatgcccttttcctgagccgatttcaaagatattatcatgttcatttaatcttatatttgtcattattttatctata
ttatgttttgaagtaataaagttttgactgtgttttatatttttctc gttcattgtatttctccttataatgttcttaaattc
atttatcacggggcaacttaatatatccgaaatatagttcttctatatcgttcccccagtataatgattattatacta
tttaatcttcaacttaacaattggagtttccagttaagaaataataatttaatgccaaagcggatattcgcaatcc
gcttacgctacttgctcataacctcaacaggcaatgaagctaagttaattatttactctgtgcctgaacagcagt
gattgcaacaacaccaacgatatcatcagaagaacaacctcttgataaatcatttactggagctgcaataccc
tgagttaatggtccataagcttctgcctttgcaagacgctgtgttaacttatatccaatgttaccagcatcaaggt
ctgggaagattaatacgttagcttttccagcaatatcactaccaggagcttttgaagcacctacactaggaacg
attgctgcatctaactggaactcgccgtcgatcttatattctgggtataattcatttgcaatcttagttgcttctaca
accttatcaacatctgcatgctttgcgcttccctttgttgaatgagaaagcatagctacgataggttcagagcca
actaattgttcaaaactcttcgctgtggaaccagcgattgctgctaactcttcagcatttggattctgatttaaac
cagcatcagagaaaaggaaagttccatttgcgcccatatcacaattaggtactaccattacgaagaaagcag
aaactaacttagtatttggagcagtttttaaaatctgaagacatggtcttaaggtatctgctgtagagtgacaag
caccagatactaaaccatctgcatcgcccatcttaaccatcattacaccgtatgtaatgtagtctgttgttaaaa
gctcttttgctttttcaggggtcatgccttttgcctgtctaagttctacaagcttgttaatgtaagc对于乳酸脱氢酶(来自C.phytofermentans的公开的基因组的基因1389和2971)
单交换
C.phytofermentans的ldh基因的长度是978bp(对于基因1389)(SEQID NO:27)和960bp(对于基因2971)(SEQ ID NO:28)。通过PCR扩增各基因的5’末端附近的大约500bp内部片段,并且将其克隆入具有不同选择标记的***载体和复制型载体。所使用的选择标记可包括提供药物抗性的选择标记。此类质粒将被用于破坏ldh 1389和ldh 2971基因。例如,ldh 1389基因的图谱和通过PCR扩增的用于基因破坏的区域示于图24中。
双交换
为了构建用于C.phytofermentans的ldh基因的双交换型敲除载体,将克隆大约1kb的侧连ldh基因的各侧的DNA。将选择标记***侧翼DNA之间。所使用的选择标记将包括为该菌株提供药物抗性的选择标记。以mLs基因作为假定的选择标记的假定的双交换型敲除构建体的实例示于图25中。
相应于图25中描述的片段的序列在下面示于SEQ ID NO:29。mLs基因(选择标记)以下划线标示,序列的剩下部分相应于侧翼DNA。在引物设计过程中,对上述片段的5’和3’末端的限制性位点进行工程改造以使其可被克隆入许多复制型和非复制型载体。相同的策略可用于产生缺失ldh 2971的载体。
tggaatctcactatgcaccaatgtggtactaaattatatctttatctatggaaaattaggttttccgcgaatggag
atagagggagctgccattgctactttaatttgtagaattcttgagagtattttagttgttatttatatgtataagggt
gagaaggtacttaagatgagactttcttatatttttaagagatctaaacagtattttcgctctttggctcgttatagt
gcgccagtgcttatgagtgaggttaactgggggcttgggattgctgttcagtctgcaatcattgggcgtatggg
tgttagttttcttacagccgccagcttcattaatgtagtacaacagttagccggaatcattctgattggtattggtg
tgggttcgagcattataatagggaatttgattggtgagggaaaagagcatgaggcgagaatgctagccaata
agttaatacgtatcagtatgatactcggaggaattgttgcttttgcagtaatcttactacgtccaatcgctcctaa
ctttattgaggcgtctaaggaaacagcggatttaattcgtcagatgctatttgtttcggcttacctcttattcttcca
agccttatctgtattaactatggccggaatattacgtggtgcaggggataccctttactgtgcaacctttgatgtt
ttgaccttatgggtactaaaacttggaggaggtttgcttgcaaccatagtacttcatcttccacctgtatgggttt
actttatcttaagtagcgatgagtgtgttaaagcgctatttacggtaccgcgggtcttaaagggacgttggattc
atgatacaacactgcattaagatttcatatgtccagatatttttgcacagtagcataattactagagcttattccta
taatattcataggttttgatggtccattttacgttacgatagcatatattacatcaaaaccaattctatataagatga
ggttatagtatgaacgagaaaaatataaaacacagtcaaaactttattacttcaaaacataatatagataaaat
aatgacaaatataagattaaatgaacatgataatatctttgaaatcggctcaggaaaagggcattttacccttg
aattagtacagaggtgtaatttcgtaactgccattgaaatagaccataaattatgcaaaactacagaaaataaa
cttgttgatcacgataatttccaagttttaaacaaggatatattgcagtttaaatttcctaaaaaccaatcctataa
aatatttggtaatataccttataacataagtacggatataatacgcaaaattgtttttgatagtatagctgatgaga
tttatttaatcgtggaatacgggtttgctaaaagattattaaatacaaaacgctcattggcattatttttaatggca
gaagttgatatttctatattaagtatggttccaagagaatattttcatcctaaacctaaagtgaatagctcacttat
cagattaaatagaaaaaaatcaagaatatcacacaaagataaacagaagtataattatttcgttatgaaatggg
ttaacaaagaatacaagaaaatatttacaaaaaatcaatttaacaattccttaaaacatgcaggaattgacgatt
taaacaatattagctttgaacaattcttatctcttttcaatagctataaattatttaataagaagtaataggaaataa
tactcgaattattctgcaatctgttctaaaaaataaaattaagaaattactatagcaagccaggttaaaattacta
gcttgctatttttgtgcatttagtacagttttgattattaaagaataaatttaataactattttgcaataagttattgac
tatttcacaagttagtgttactatacaagtatgaaataaagatacataaaaaaataaataatatgaaacataaatt
catgacatgcggaatagaatgaaagaatattatgtcggttcctaatactaaatggatataacaatctattgaaa
cacttatggggtgtaagtgtggagagaatttctaaagcgccaaaagactctacatatgaaattctaaagcttca
cacgggaataatctaatttatgtatcttattatcataattcaggaaggtagtgtgaaaatataaaaattagttttcc
tgtttcattcaggcagtagcatttcttaaacaaatttgctatgcattgggtgttatctgaaaaacaaaaagcaattt
tctcacaacttatttctgaacaacaatggtattaaaaatttggaggaggattttactatgaaaaaaacggtaaca
ttactgttggttctgaccatggtggtaagcttatttgcagcatgtggtaagaaaaatggatcaagcgaaaccgg
cacaaaagatcctgtggcaacaagcggtgcaaaagaacctgacaaacaagatccaggcaataaagagcct
gaaaaacaagaccctgttaaaatcaagatttattactctgataatgcaaccttaccatttaaagaagattggtta
gttataaaggaagctgagaagagatttaatgttgatttcgatttcgaagtaattccaattgcagattatcaaaca
aaagtttctttaacattaaatacaggaaataacgctccagatgtcatcctttatcagtcaacgcagggagagaa
tgcatct。
Cald.Kristjanssonii和粪堆梭菌leptospartum亚种
就我们所知,上述微生物的基因组测序列还未进行,并且如果已经进行,其还未对公众开放。基于我们的实验结果,此类生物是纤维素分解和木聚糖分解生物。需要来自此类生物的编码关键代谢酶的基因的DNA序列以对其进行基因工程改造和将碳流转向乙醇。此类酶包括这样的酶如乙酸激酶和乳酸脱氢酶。为了获得此类基因的序列,将对此类生物的基因组进行测序。
由于可以使用基因组序列,上述酶的保守性质可用于发现编码基因和侧翼DNA。此类序列将可用于设计用于使用单交换和双交换策略的定向诱变(targeted mutagenesis)的构建体。此类策略可与上述策略相同。我们还将确定在此类生物中可用作选择标记的抗生素和效果最好的转化方案。
实施例3
解纤维梭菌的转化
将细胞在厌氧条件下于50mL含有4g/l纤维二糖的GS培养基中培养(在34℃下温育)至0.8的OD。在收获后,用含有500mM蔗糖和5mM MOPS的清洗缓冲液(pH被调节至7)以等体积清洗其3次。在最后的清洗后,将细胞沉淀重悬浮于等体积的清洗缓冲液中,将10ul细胞悬浮液等分置于具有1mm电极间距的标准电穿孔杯中。加入1ul质粒DNA。调节质粒的浓度以确保质粒对细胞的摩尔比在1∶1和10∶1之间。使用5ms脉冲,穿过样品的场强为7kV/cm(测量的)。使用常规脉冲发生器。立即用用于初始培养的相同培养基以1000∶1稀释样品,然后让其恢复直至生长重新开始,通过OD(24-48h)的提高来确定生长。将恢复的样品以50∶1稀释,然后置于含有15ug/mL红霉素或15ug/mL氯霉素的选择培养基中并且让其生长5至6天。检测展示在选择培养中生长的样品以确认它们事实上是解纤维梭菌并且确认它们是具有质粒的。
实施例4
用于对分解纤维素的菌株进行工程改造的构建体
纤维素是生物质的主要成分中的一种,其可被潜在用作通过使用热解纤维梭菌进行的发酵产生燃料乙醇的底物。然而,在该过程中,许多能量和碳源被用于形成副产品乙酸盐和乳酸盐。对热解纤维梭菌中纤维素利用的代谢途径进行工程改造是使乳酸盐和乙酸盐产量减少至最少和使能量及碳流有利于乙醇形成所必需的。
乙酸激酶是在热解纤维梭菌中形成乙酸的纤维素利用的代谢途径中的重要的酶,其由ack基因编码。ack基因的失活可破坏乙酸激酶,从而导致乙酸盐的减少或消除。
乳酸脱氢酶是在热解纤维梭菌中形成乳酸盐的纤维素利用的代谢途径中的重要的酶,其由ldh基因编码。ldh基因的失活可破坏乳酸脱氢酶,从而导致乳酸盐产生的减少或消除。
基于质粒pIKM1的热解纤维梭菌中ack基因的失活
为了敲除ack基因,在质粒pIKM1的多克隆位点(MCS)上构建载体,其中将编码氯霉素乙酰转移酶的cat基因***包括ack和pta基因(编码磷酸转乙酰酶)的3055bp的DNA片段,从而导致476bp的ack基因和399bp的pta基因的敲除,并且分别在mLs基因的两侧形成1025bp和1048bp的侧翼区域(图7)。pNW33N包含从凝结芽孢杆菌和金黄色葡萄球菌(Staphylococcus aureus)分离的并且预期在***菌株(包括热解纤维梭菌)中稳定复制的pBC1复制子。在质粒pIKM1上构建的ack敲除载体的序列示于SEQ ID NO:1。
基于复制型质粒pNW33N的热解纤维梭菌中ack基因的失活
为了敲除ack基因,在复制型质粒pNW33N的多克隆位点(CMS)上构建载体,其中将大内环酯、林肯酰胺和链阳性菌素B(MLSB)抗性基因mLs***3345bp的DNA片段,该片段包括ack基因、pta基因(编码磷酸转乙酰酶)和未知的上游基因,从而导致855bp的ack基因的敲除和在mLs基因的两侧上形成1195bp和1301bp的侧翼区域(图8)。pNW33N包含从凝结芽孢杆菌和金黄色葡萄球菌分离的并且预期在***菌株(包括热解纤维梭菌)中稳定复制的pBC1复制子。在质粒pNW33N上构建的ack敲除载体的序列示于SEQ ID NO:2。基于质粒pIKM1的热解纤维梭菌中ldh基因的失活
为了敲除ldh基因,在复制型质粒pIKM1的多克隆位点(CMS)上构建载体,其中将编码氯霉素乙酰转移酶的cat基因***3188bp的DNA片段,该片段包括ldh和mdh基因(编码苹果酸脱氢酶),从而导致包括ldh和mdh基因的部分的1171bp的DNA片段的敲除和分别在mLs基因的两侧上形成894bp和1123bp的侧翼区域(图9)。在质粒pIKM1上构建的ldh敲除载体的序列示于SEQ ID NO:3。
基于质粒pNW33N的热解纤维梭菌中的ldh基因的失活
为了敲除ldh基因,在复制型质粒pNW33N的多克隆位点(CMS)上构建载体,其中将大内环酯、林肯酰胺(lincosamide)和链阳性菌素B(MLSB)抗性基因mLs***2523bp的DNA片段,该片段包括ldh基因和mdh基因(编码苹果酸脱氢酶),从而导致489bp的ldh基因的敲除和在mLs基因的两侧上形成1034bp和1000bp的侧翼区域(图10)。pNW33N包含从从凝结芽孢杆菌和金黄色葡萄球菌分离的并且预期在其他***菌株(包括热解纤维梭菌)中稳定复制的pBC1复制子。在质粒pNW33N上构建的ldh敲除载体的序列示于SEQ ID NO:4。
基于质粒pUC19的热解纤维梭菌中的ldh基因的失活
为了敲除ldh基因,在复pUC 19质粒的多克隆位点(CMS)上构建载体,其中将编码氯霉素乙酰转移酶的基因(cat基因)***717bp的ldh基因片段,从而导致在cat基因的两侧上形成245bp和255bp的侧翼区(图11)。pUC19大肠杆菌质粒载体,其包含不能在***菌株(包括热解纤维梭菌)中扩增的pMB1起始区。可构建相似的载体,其中mLs基因两侧连接ldh基因片段。在质粒pUC19上构建的ldh敲除载体的序列示于SEQ ID NO:5。
热解纤维梭菌和C.straminisolvens木糖异构酶和木酮糖激酶的表达(预测实施例(prophetic example))
为了在热解纤维梭菌中表达木糖异构酶和木酮糖激酶,从解糖梭菌中克隆木糖异构酶和木酮糖激酶基因并且将其置于热解纤维梭菌gapDH启动子的控制之下。将该盒包含在基于pNW33N主链的热解纤维梭菌复制型质粒中,从而产生pMU340(图35)SEQ ID NO:74。在转移入热解纤维梭菌后,可就在木糖上生长的能力测定所得的转化。可使用C.kristajanssonii木糖异构酶和木酮糖激酶基因建立类似的构建体。也就在C.straminsolvens中的功能性检测此类构建体。
丙酮酸脱羧酶和乙醇脱氢酶在热解纤维梭菌和C.straminisolvens中的表达(预测实施例)
为了在热解纤维梭菌中表达丙酮酸脱羧酶和乙醇脱氢酶,从来源运动发酵单孢菌和棕榈发酵细菌在克隆丙酮酸脱羧酶基因,以及从来源运动发酵单孢菌克隆乙醇脱氢酶基因。将此类基因(pdc和adh)以操纵子的形式从热解纤维梭菌pta-ack启动子表达。将该盒包含在基于pNW33N主链的热解纤维梭菌复制型质粒(图36和37),SEQ ID NOS:75和76中。在转移入热解纤维梭菌中后,可就提高的乙醇产量和/或乙醛产量筛选所得的转化,以测量表达的酶的功能性。也就在C.straminsolvens中的功能性检测此类构建体。
实施例5
C.straminisolvens用于在装有CTFUD培养基的血清瓶中发酵1%产物的浓度特征谱和比例示于图27。在第3天产生大约2g/L的总产物,乙醇占总产物的大约50%。图27显示对于1%使用C.straminisolvens.产生的产物浓度特征谱。乙醇对乙酸盐的比例描述为E/A,乙醇对总产物的比例描述为E/T。
实施例6
用于嗜温和嗜热纤维素分解、木聚糖分解生物的经工程改造的第II族内含子
在许多细菌基因组中发现的可移动的第II族内含子是催化RNA和逆转录转座元件。它们使用称为逆转录转座的移动机制,其中被切离的内含子RNA直接逆剪接入DNA靶位点,然后由内含子编码的蛋白质进行逆转录。可移动的乳酸乳球菌(Lactococcus lactis)Ll.LtrB第II族内含子已被开发成称为TargetronTM载体的基因工具,其可从Sigma Aldritch(Catalog # TA0100)商购获得。该产品和其用途是美国专利5,698,421、5,804,418、5,869,634、6,027,895、6,001,608以及6,306,596和/或其他未决的美国和外国专利申请(由InGex,LLC.控制的)中的一个或多个的客体。
可将包含逆转录转座所必需的所有序列的Targetrons盒(图28和29)亚克隆入能够在嗜温或嗜热性纤维素分解生物中复制的载体。可通过用任何宿主或种特异性组成型或诱导型启动子替代lac启动子来修饰Targetron盒。可通过天然识别序列的定点诱变来进一步修饰所述盒以使第II族内含子被再靶向***目的基因,从而产生基因敲除。例如,可重新设计第II族内含子来在任何嗜温或嗜热纤维素分解生物中敲除乳酸脱氢酶或乙酸激酶。表4描述了将内含子再靶向解纤维梭菌乙酸激酶(SEQ ID NO:21)的***位置和引物的实例。表5描述了将内含子再靶向解纤维梭菌乳酸脱氢酶(SEQ ID NO:21)的***位置和引物的实例。
用于将Ll.Ltrb内含子再靶向解纤维梭菌Ack基因中的***物(SEQID NO:21)的载体的实例描述于图28中。pMU367的载体序列(解纤维梭菌乙酸激酶KO载体)的载体序列是SEQ ID NO:30。
将Ll.Ltrb内含子再靶向解纤维梭菌LDH2744基因中的***物(SEQID NO:23)的载体的实例描述于图29中。pMU367(解纤维梭菌乳酸脱氢酶KO载体)的载体序列示于SEQ ID NO:31。
表4
预测的***位置(SEQ ID NO:62) | ATTTACCTGGCTGGGAATACTGAGACATAT-内含子-GTCATTGAGGCCGTA |
IBS1诱变引物(SEQ ID NO:63) | AAAAAAGCTTATAATTATCCTTAATTTCCTACTACGTGCGCCCAGATAGGGTG |
EBS1d诱变引物(SEQ ID NO:64) | CAGATTGTACAAATGTGGTGATAACAGATAAGTCTACTACTGTAACTTACCTTTCTTTGT |
EBS2诱变引物(SEQ ID NO:65) | TGAACGCAAGTTTCTAATTTCGGTTGAAATCCGATAGAGGAAAGTGTCT |
表5
预测的***位置(SEQ ID NO:66) | TTAAATGTTGATAAGGAAGCTCTTTTCAAT-内含子-GAAGTTAAGGTAGCA |
IBS1诱变引物(SEQ ID NO:67) | AAAAAAGCTTATAATTATCCTTAGCTCTCTTCAATGTGCGCCCAGATAGGGTG |
EBS1d诱变引物(SEQ ID NO 68) | CAGATTGTACAAATGTGGTGATAACAGATAAGTCTTCAATGATAACTTACCTTTCTTTGT |
EBS2诱变引物(SEQ ID NO:69) | TGAACGCAAGTTTCTAATTTCGATTAGAGCTCGATAGAGGAAAGTGTCT |
实施例7
嗜热厌氧杆菌和高温厌氧芽孢杆菌菌株的转化(预测实施例)
将用下列方案转化Thermoanaerobacter pseudoethanolicus 39E,解糖好热厌氧杆菌JW/SL-YS485,解糖好热厌氧杆菌B6A-RI和Thermoanaerobacter sp.菌株59。在55℃下于40mL具有下列改变:5g/L纤维二糖取代纤维素,1.8g/L K2HPO4,无谷胱甘肽和0.5g/L L-半胱氨酸-HCl的DSMZ M122培养基(http://www.dsmz.de/microorganisms/media_list.php)中培养细胞直至达到0.6至0.8的光密度。然后收获细胞,在室温下用40mL 0.2M纤维二糖清洗2次。将细胞在100uL的等分中重悬浮于0.2M纤维二糖中,向1mm隙宽的电击杯(electroportation cuvette)中的样品中加入0.1至1ug的质粒DNA。将1.8kV,25μF,200Ω,大约3-6ms的指数式脉冲(Bio-Rad Instruments)用于电击杯,然后将细胞在新鲜的M122中稀释100至200倍,并且在55℃下温育12至16小时。然后将恢复的细胞在装有含有培养基(含有选择剂例如200μg/mL卡那霉素)的新鲜琼脂的陪替氏平皿(petri-plate)中稀释25至100倍。当培养基固化后,将板在55℃下温育24至72小时以形成集落。可通过PCR就位点特异性重组的显示检测集落。
实施例8
经工程改造的嗜热厌氧杆菌和高温厌氧芽孢杆菌菌株的发酵性能
表6描述了经工程改造的嗜热厌氧杆菌和高温厌氧芽孢杆菌菌株的发酵性能。在不振荡的条件下将培养物在55℃下于M122中培养24小时。下列缩写用于表6中:纤维二糖(CB)、葡萄糖(G)、乳酸(LA)、乙酸(AA)和乙醇(Etoh)。值以克/升表示。YS485-解糖好热厌氧杆菌JW/SL-YS485,B6A-RI-解糖好热厌氧杆菌B6A-RI,39E-Thermoanaerobacter pseudoethanolicus 39E.
表6
实施例9
用于工程改造分解纤维素和分解木聚糖的菌株的构建体-
反义RNA技术实施例
通过电穿孔和甲砜霉素选择将携带靶向编码乳酸脱的热解纤维梭菌基因(Cthe_1053)的反义RNA盒的复制型质粒(图38)转移入热解纤维梭菌1313。观察到的该质粒的转化效率等于亲本载体pMU102的转化效率。质粒的序列示于SEQ ID NO:61。asRNA盒描述于图38中并且进行如下组织:(i)以图38中显示的方向将整个1827bp盒克隆入pMU102的多克隆位点,(ii)将天然启动子区域包含在所述盒的前600bp内,(iii)将ldh开放阅读框架的前877bp以反义方向融合至天然启动子,(iv)将大约300个另外的bp包含在asRNA ldh区域的下游。
通过在6ug/mL甲砜霉素存在的情况下(以保持质粒)在M122C培养基上培养标准培养物(standing cultures)来就改变的终产物形成筛选所得的甲砜霉素抗性集落,如图39中所示的。9个随机选择的甲砜霉素抗性转化体的初步筛选显示4个培养物与野生型相比,展示出较低水平的乳酸盐产量。此外,将构建携带被导向两个ldh基因的反义RNA的构建体以同时部分地、基本上或完全缺失、沉默、失活或下调两个基因。
实施例10
SEQ ID NOS:44、45和46是来自解糖好热厌氧杆菌YS485、热解纤维梭菌ATCC 27405和Clostridium phytofermentans的丙酮酸甲酸裂解酶激(aka甲酸乙酰转移酶,EC.2.3.1.54,pfl)基因。Pfl催化丙酮酸至乙酰-CoA和甲酸的转化(图34)。pfl的缺失将导致甲酸产生的消除,以及可在一些嗜热菌株中导致乙酸收率的减少,结果导致乙醇得率的提高。
图40至45中描述的SEQ ID NOS:47-52显示了pfl敲除质粒,每2个用于上面列出的3种生物。各生物具有经设计用于部分、基本上或完全缺失、沉默、失活或下调pfl酶的单交换和双交换型质粒。使用与pfl基因的内部区段同源的单DNA序列(400bp至1000bp)设计单交换型质粒,使用与pfl基因的上游区域(5’)和下游区域(3’)同源的2个DNA序列(400至1000bp)设计双交换质粒。所有质粒经设计使用可最易获得的用于在给定的生物中进行选择的抗生素标记。可将质粒保持在大肠杆菌中和通过DNA合成合同公司(contract company)例如Codon Devices或DNA 2.0来构建质粒。
通过引用合并
所有本文中引用的美国专利和美国公开专利申请通过引用合并入本文。
等同实施方案
通过只使用常规实验,本领域技术人员将承认或能够确定本文中描述的特定实施方案的许多等同实施方案。此类等同实施方案意欲包括在下列权利要求中。
序列表
序列1:在质粒pIKM1上构建的ack敲除载体的序列
agctttggctaacacacacgccattccaaccaatagttttctcggcataaagccatgctctgac
gcttaaatgcactaatgccttaaaaaaacattaaagtctaacacactagacttatttacttcgt
aattaagtcgttaaaccgtgtgctctacgaccaaaagtataaaacctttaagaactttcttttt
tcttgtaaaaaaagaaactagataaatctctcatatcttttattcaataatcgcatcagattgc
agtataaatttaacgatcactcatcatgttcatatttatcagagctcgtgctataattatacta
attttataaggaggaaaaaataaagagggttataatgaacgagaaaaatataaaacacagtcaa
aactttattacttcaaaacataatatagataaaataatgacaaatataagattaaatgaacatg
ataatatctttgaaatcggctcaggaaaagggcattttacccttgaattagtacagaggtgtaa
tttcgtaactgccattgaaatagaccataaattatgcaaaactacagaaaataaacttgttgat
cacgataatttccaagttttaaacaaggatatattgcagtttaaatttcctaaaaaccaatcct
ataaaatatttggtaatataccttataacataagtacggatataatacgcaaaattgtttttga
tagtatagctgatgagatttatttaatcgtggaatacgggtttgctaaaagattattaaataca
aaacgctcattggcattatttttaatggcagaagttgatatttctatattaagtatggttccaa
gagaatattttcatcctaaacctaaagtgaatagctcacttatcagattaaatagaaaaaaatc
aagaatatcacacaaagataaacagaagtataattatttcgttatgaaatgggttaacaaagaa
tacaagaaaatatttacaaaaaatcaatttaacaattccttaaaacatgcaggaattgacgatt
taaacaatattagctttgaacaattcttatctcttttcaatagctataaattatttaataagta
agttaagggatgcataaactgcatcccttaacttgtttttcgtgtacctattttttgtgaatcg
attatgtcttttgcgcattcacttcttttctatataaatatgagcgaagcgaataagcgtcgga
aaagcagcaaaaagtttcctttttgctgttggagcatgggggttcagggggtgcagtatctgac
gtcaatgccgagcgaaagcgagccgaagggtagcatttacgttagataaccccctgatatgctc
cgacgctttatatagaaaagaagattcaactaggtaaaatcttaatataggttgagatgataag
gtttataacgaatttgtttgttctaatttttcactcattttgttctaatttcttttaacaaatg
ttcttttttttttagaacagttatgatatagttagaatagtttaaaataaggagtgagaaaaag
atgaaagaaagatatggaacagtctataaaggctctcagaggctcatagacgaagaaagtggag
aagtcatagaggtagacaagttataccgtaaacaaacgtctggtaacttcgtaaaggcatatat
agtgcaattaataagtatgttagatatgattggcggaaaaaaacttaaaatcgttaactatatc
ctagataatgtccacttaagtaacaatacaatgatagctacaacaagagaaatagcaaaagcta
caggaacaagtctacaaacagtaataacaacacttaaaatcttagaagaaggaaatattataaa
aagaaaaactggagtattaatgttaaaccctgaactactaatgagaggcgacgaccaaaaacaa
aaatacctcttactcgaatttgggaactttgagcaagaggcaaatgaaatagattgacctccca
ataacaccacgtagttattgggaggtcaatctatgaaatgcgattaagcttggctgcaggtcga
taaacccagcgaaccatttgaggtgataggtaagattataccgaggtatgaaaacgagaattgg
acctttacagaattactctatgaagcgccatatttaaaaagctaccaagacgaagaggatgaag
aggatgaggaggcagattgccttgaatatattgacaatactgataagataatatatcttttata
tagaagatatcgccgtatgtaaggatttcagggggcaaggcataggcagcgcgcttatcaatat
atctatagaatgggcaaagcataaaaacttgcatggactaatgcttgaaacccaggacaataac
cttatagcttgtaaattctatcataattgtggtttcaaaatcggctccgtcgatactatgttat
acgccaactttcaaaacaactttgaaaaagctgttttctggtatttaaggttttagaatgcaag
gaacagtgaattggagttcgtcttgttataattagcttcttggggtatctttaaatactgtaga
aaagaggaaggaaataataaatggctaaaatgagaatatcaccggaattgaaaaaactgatcga
aaaataccgctgcgtaaaagatacggaaggaatgtctcctgctaaggtatataagctggtggga
gaaaatgaaaacctatatttaaaaatgacggacagccggtataaagggaccacctatgatgtgg
aacgggaaaaggacatgatgctatggctggaaggaaagctgcctgttccaaaggtcctgcactt
tgaacggcatgatggctggagcaatctgctcatgagtgaggccgatggcgtcctttgctcggaa
gagtatgaagatgaacaaagccctgaaaagattatcgagctgtatgcggagtgcatcaggctct
ttcactccatcgacatatcggattgtccctatacgaatagcttagacagccgcttagccgaatt
ggattacttactgaataacgatctggccgatgtggattgcgaaaactgggaagaagacactcca
tttaaagatccgcgcgagctgtatgattttttaaagacggaaaagcccgaagaggaacttgtct
tttcccacggcgacctgggagacagcaacatctttgtgaaagatggcaaagtaagtggctttat
tgatcttgggagaagcggcagggcggacaagtggtatgacattgccttctgcgtccggtcgatc
agggaggatatcggggaagaacagtatgtcgagctattttttgacttactggggatcaagcctg
attgggagaaaataaaatattatattttactggatgaattgttttagtacctagatttagatgt
ctaaaaagctttttagacatctaatcttttctgaagtacatccgcaactgtccatactctgatg
ttttatatcttttctaaaagttcgctagataggggtcccgagcgcctacgaggaatttgtatcg
actctagaggatccctcagcgaagctccactatgtttcaaaatgtcagatatatcaattttcat
caaagtcacctcttaaaaccgacaaggactattatactaactaataaccctcatgtcaagaatt
atatgacagattggcttaaataacaaaaataattttgtttagttaaattcggaatttcttctta
atattattaacatattccacatattaatacaagaaaaaacccggcaaaaaaataaaaaaatttt
ataagcccgtttcctaaaaaaacaggcttgtaaaattataacgcatcttttataagttttttac
aagtcttaaagtctcccttgcaatctcaagctcctcatttgtcgggataaccaaagtctttact
ttcgcatcgggagcactgatatccgcttctttgcctttcacttcatttttatccaaatctattt
taattccgaaaaagtccatatccttcaaaacttctcttcttatataagcattgttttcgccgat
acctgcagtgaataccaccgcatcaacgccgttcagcactgcaatatattttccaatatatttc
ctaacaccatagcagaaaatatccaatgccagctgcgccctgtcatctcccttttctgcggcat
cctgaacatctctgaaatcactgcttacacctgaaattccaagcacacctgatttcttgttaag
gaaattgtttatatcgttaatattcattttttccttttccatcaaataagttataaccgcaggg
tcaacattgccgcttctggtacccatgcacaacccctgcagaggagtaaatcccattgaggtgt
caacggattttccgccttttaccgcacaaatacttgctccgtttccaagatggcaggttatcag
cttcaggctctcaataggtttgcccagcatctgagccgccctgtgggccacatatttgtgggaa
gttccgtggaatccgtattttctcaatttatacttctcatatatctcataagggagggcataaa
tatatgcatgctagttcaacaaacgggattgacttttaaaaaaggattgattctaatgaagaaa
gcagacaagtaagcctcctaaattcactttagataaaaatttaggaggcatatcaaatgaactt
taataaaattgatttagacaattggaagagaaaagagatatttaatcattatttgaaccaacaa
acgacttttagtataaccacagaaattgatattagtgttttataccgaaacataaaacaagaag
gatataaattttaccctgcatttattttcttagtgacaagggtgataaactcaaatacagcttt
tagaactggttacaatagcgacggagagttaggttattgggataagttagagccactttataca
atttttgatggtgtatctaaaacattctctggtatttggactcctgtaaagaatgacttcaaag
agttttatgatttatacctttctgatgtagagaaatataatggttcggggaaattgtttcccaa
aacacctatacctgaaaatgctttttctctttctattattccatggacttcatttactgggttt
aacttaaatatcaataataatagtaattaccttctacccattattacagcaggaaaattcatta
ataaaggtaattcaatatatttaccgctatctttacaggtacatcattctgtttgtgatggtta
tcatgcaggattgtttatgaactctattcaggaattgtcagataggcctaatgactggctttta
taacctgaggttttgctccaaccagcatctcaaaagatttggatgcagatattgcaatttcaga
aagctggtctgcatccggattttccaccaagccgcaatcggcatatacaaaggttccgttatga
ccatattcacagttgggtacaaccataacaaaaaaggatgatacgagttttgtccccggggccg
tctttaatatctgcaaagccggtctcaaagtatttgcagtggaattgacagcacccgccaccat
accatccgcttcacctttttttaccatcataactccataataaagagggtctttgatcgtttcc
cttgcggcttctatagtcatacccttcgattttctaagctcatacagtgtatttgcataatcct
ccaatttttcggaatttaaggaatcctctatcatcactccttcaagatcaatatcccccgccag
actcttaatctccttttcattgcctatcagtacaacctttgcaattccctttttcattatcatg
gatgcggctttaataaccctcagatccgtactttccggcaaaactatggtttttacgtctgatt
tcgccctttcaattatttgttccaaaaaactcataaattcttctcctttcataatcccaaaact
gttatcataaaaactgtatttgtaatacttataactatatattatcaccaggtaataataccta
ctcactataaacagctattttactgggttccaagcaactctaattatatacaaaatgttttttg
tatacaacaccctccttatctttttttcggctttagccataaataacggcaagtaactccaaaa
tacaggatatttcatgcttttagaaactttttattagtcttcttaattattcagattttgtggc
aattaaactttgcagctcctccaaatagttgtccagctcctcttctttaagattgctgagatat
gacaatctgtaatttttagcctttttggccatctctagcgcactctccgtcattcccaaatctt
tcaaaacacagctatagttatagtatgcgaattcactggccgtcgttttacaacgtcgtgactg
ggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgt
aatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggc
gcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactct
cagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgac
gcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccggga
gctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgat
acgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcactttt
cggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgc
tcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattca
acatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcaccca
gaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaac
tggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgag
cacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactc
ggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatc
ttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgc
ggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatg
ggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacg
agcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaact
acttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggacca
cttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtg
ggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatcta
cacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctca
ctgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaac
ttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatccc
ttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttga
gatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtgg
tttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgca
gataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagca
ccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgt
gtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggg
gggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgt
gagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggca
gggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcc
tgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagc
ctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctc
acatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagc
tgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagag
cgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgaca
ggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcatta
ggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataa
caatttcacacaggaaacagctatgaccatgattacgcca
序列2:在质粒pNW33N上构建的ack敲除载体的序列
aattctgttctggctttagaccatttacgctttgggtttgccatgcatcacacctcctcgtcac
catacaacaaaccttgtgtataaataaaatcttgtacacatggaaggtactcgattttgttttt
acttaaaacaaagctacactaattgtctgtaaaaaagtttttgaaaacttccagccgcggatct
atatcctcatttacgcatttgcactcatcgttgttaagatccgctccacacttcgggcaatatc
ccttgcaggcctcgtcacaaacctgctttgccggaagattcagtatgatattgtctatcattac
cttttccagctcgaggaacttaccatggtacgtataatattcctcgtcggttttgttactgccc
tcttctacaaagttttcctttacatcaatatgcatctttgattcaatatccttgaggcaccttg
agcattttgccctgtaatccgcccagagttcaccgtcaagttttataatccctccggcatttac
caaagtgcccttaaaagttaccggtttcgcaaagtcaaaatcctcagctataaaatcattaatt
ttaattgactcactaaagtccagtctcagcgaagctccactatgtttcaaaatgtcagatatat
caattttcatcaaagtcacctcttaaaaccgacaaggactattatactaactaataaccctcat
gtcaagaattatatgacagattggcttaaataacaaaaataattttgtttagttaaattcggaa
tttcttcttaatattattaacatattccacatattaatacaagaaaaaacccggcaaaaaaata
aaaaaattttataagcccgtttcctaaaaaaacaggcttgtaaaattataacgcatcttttata
agttttttacaagtcttaaagtctcccttgcaatctcaagctcctcatttgtcgggataaccaa
agtctttactttcgcatcgggagcactgatatccgcttctttgcctttcacttcatttttatcc
aaatctattttaattccgaaaaagtccatatccttcaaaacttctcttcttatataagcattgt
tttcgccgatacctgcagtgaataccaccgcatcaacgccgttcagcactgcaatatattttcc
aatatatttcctaacaccatagcagaaaatatccaatgccagctgctgcagtaatcgcatcaga
ttgcagtataaatttaacgatcactcatcatgttcatatttatcagagctcgtgctataattat
actaattttataaggaggaaaaaataaagagggttataatgaacgagaaaaatataaaacacag
tcaaaactttattacttcaaaacataatatagataaaataatgacaaatataagattaaatgaa
catgataatatctttgaaatcggctcaggaaaagggcattttacccttgaattagtacagaggt
gtaatttcgtaactgccattgaaatagaccataaattatgcaaaactacagaaaataaacttgt
tgatcacgataatttccaagttttaaacaaggatatattgcagtttaaatttcctaaaaaccaa
tcctataaaatatttggtaatataccttataacataagtacggatataatacgcaaaattgttt
ttgatagtatagctgatgagatttatttaatcgtggaatacgggtttgctaaaagattattaaa
tacaaaacgctcattggcattatttttaatggcagaagttgatatttctatattaagtatggtt
ccaagagaatattttcatcctaaacctaaagtgaatagctcacttatcagattaaatagaaaaa
aatcaagaatatcacacaaagataaacagaagtataattatttcgttatgaaatgggttaacaa
agaatacaagaaaatatttacaaaaaatcaatttaacaattccttaaaacatgcaggaattgac
gatttaaacaatattagctttgaacaattcttatctcttttcaatagctataaattatttaata
agtaagttaagggatgcataaactgcatcccttacagctgatactttagtgatgagcttccggt
attaataaccaaaatattcatttcaaaaactcactcccgtcttgttttttttaattttcctatt
cctaaacttcgataaacagatgtttttattaaacgctgcgcaacaccttcttcaatgtccggtt
ttaacagaatttatgccttgacatattgagcctgaaccgcagtaattgccgcaaccccgactat
atcctcggcactgcagcctcgtgacagatcatttaccggtcttgccaaaccttgtgttatcggg
ccgtaagcttcagcttttgccaatctctgtgtaagcttgtatgcaatatttccggcatcaagat
ccgggaaaataagaacattggcctttcctgcaacactgcttccctttgccttcgattttgccac
ttccggaacaatggcggcatccacctgaagttctccgtcaattgcaaggtggggagctttttcc
tttgcaagctgtgttgccttgattaccttttcggtcagctcacttttggcactgccgtaagaag
aataagaaagcattgccacctgaggttttgctccaaccagcatctcaaaagatttggatgcaga
tattgcaatttcagaaagctggtctgcatccggattttccaccaagccgcaatcggcatataca
aaggttccgttatgaccatattcacagttgggtacaaccataacaaaaaaggatgatacgagtt
ttgtccccggggccgtctttaatatctgcaaagccggtctcaaagtatttgcagtggaattgac
agcacccgccaccataccatccgcttcacctttttttaccatcataactccataataaagaggg
tctttgatcgtttcccttgcggcttctatagtcatacccttcgattttctaagctcatacagtg
tatttgcataatcctccaatttttcggaatttaaggaatcctctatcatcactccttcaagatc
aatatcccccgccagactcttaatctccttttcattgcctatcagtacaacctttgcaattccc
tttttcattatcatggatgcggctttaataaccctcagatccgtactttccggcaaaactatgg
tttttacgtctgatttcgccctttcaattatttgttccaaaaaactcataaattcttctccttt
cataatcccaaaactgttatcataaaaactgtatttgtaatacttataactatatattatcacc
aggtaataatacctactcactataaacagctattttactgggttccaagcaactctaggatcct
ctagagtcgacctgcaggcatgcaagcttggcgtaatcatggtcatagctgtttcctgtgtgaa
attgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctgggg
tgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcggga
aacctgtcgtgccagcccttcaaacttcccaaaggcgagccctagtgacattagaaaaccgact
gtaaaaagtacagtcggcattatctcatattataaaagccagtcattaggcctatctgacaatt
cctgaatagagttcataaacaatcctgcatgataaccatcacaaacagaatgatgtacctgtaa
agatagcggtaaatatattgaattacctttattaatgaattttcctgctgtaataatgggtaga
aggtaattactattattattgatatttaagttaaacccagtaaatgaagtccatggaataatag
aaagagaaaaagcattttcaggtataggtgttttgggaaacaatttccccgaaccattatattt
ctctacatcagaaaggtataaatcataaaactctttgaagtcattctttacaggagtccaaata
ccagagaatgttttagatacaccatcaaaaattgtataaagtggctctaacttatcccaataac
ctaactctccgtcgctattgtaaccagttctaaaagctgtatttgagtttatcacccttgtcac
taagaaaataaatgcagggtaaaatttatatccttcttgttttatgtttcggtataaaacacta
atatcaatttctgtggttatactaaaagtcgtttgttggttcaaataatgattaaatatctctt
ttctcttccaattgtctaaatcaattttattaaagttcatttgatatgcctcctaaatttttat
ctaaagtgaatttaggaggcttacttgtctgctttcttcattagaatcaatccttttttaaaag
tcaatcccgtttgttgaactactctttaataaaataatttttccgttcccaattccacattgca
ataatagaaaatccatcttcatcggctttttcgtcatcatctgtatgaatcaaatcgccttctt
ctgtgtcatcaaggtttaattttttatgtatttcttttaacaaaccaccataggagattaacct
tttacggtgtaaaccttcctccaaatcagacaaacgtttcaaattcttttcttcatcatcggtc
ataaaatccgtatcctttacaggatattttgcagtttcgtcaattgccgattgtatatccgatt
tatatttatttttcggtcgaatcatttgaacttttacatttggatcatagtctaatttcattgc
ctttttccaaaattgaatccattgtttttgattcacgtagttttctgtattcttaaaataagtt
ggttccacacataccaatacatgcatgtgctgattataagaattatctttattatttattgtca
cttccgttgcacgcataaaaccaacaagatttttattaatttttttatattgcatcattcggcg
aaatccttgagccatatctgacaaactcttatttaattcttcgccatcataaacatttttaact
gttaatgtgagaaacaaccaacgaactgttggcttttgtttaataacttcagcaacaacctttt
gtgactgaatgccatgtttcattgctctcctccagttgcacattggacaaagcctggatttaca
aaaccacactcgatacaactttctttcgcctgtttcacgattttgtttatactctaatatttca
gcacaatcttttactctttcagcctttttaaattcaagaatatgcagaagttcaaagtaatcaa
cattagcgattttcttttctctccatggtctcacttttccactttttgtcttgtccactaaaac
ccttgatttttcatctgaataaatgctactattaggacacataatattaaaagaaacccccatc
tatttagttatttgtttggtcacttataactttaacagatggggtttttctgtgcaaccaattt
taagggttttcaatactttaaaacacatacataccaacacttcaacgcacctttcagcaactaa
aataaaaatgacgttatttctatatgtatcaagaatagaaagaactcgtttttcgctacgctca
aaacgcaaaaaaagcactcattcgagtgctttttcttatcgctccaaatcatgcgattttttcc
tctttgcttttctttgctcacgaagttctcgatcacgctgcaaaacatcttgaagcgaaaaagt
attcttcttttcttccgatcgctcatgctgacgcacgaaaagccctctaggcgcataggaacaa
ctcctaaatgcatgtgaggggttttctcgtccatgtgaacagtcgcatacgcaatattttgttt
cccatactgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctctt
ccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctca
ctcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagca
aaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctcc
gcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggact
ataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccg
cttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgct
gtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgt
tcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgac
ttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgcta
cagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgc
tctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccacc
gctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaag
aagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggat
tttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagtttt
aaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgagg
cacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagat
aactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgc
tcaccggctccagatttatcagcaataaaccagccagcccgatatgggaaacaaaatattgcgt
atgcgactgttcacatggacgagaaaacccctcacatgcatttaggagttgttcctatgcgcct
agagggcttttcgtgcgtcagcatgagcgatcggaagaaaagaagaatactttttcgcttcaag
atgttttgcagcgtgatcgagaacttcgtgagcaaagaaaagcaaagaggaaaaaatcgcatga
tttggagcgataagaaaaagcactcgaatgagtgctttttttgcgttttgagcgtagcgaaaaa
cgagttctttctattcttgatacatatagaaataacgtcatttttattttagttgctgaaaggt
gcgttgaagtgttggtatgtatgtgattcaataatttcttttactcgctcgttatagtcgatcg
gttcatcattcaccaaatcataattttcatgtgaccgttctttatcaatatcgggattcgtttt
actttcccgttctctctgattgtgaaattg
序列3:在质粒pIKM1上构建的ldh敲除载体的序列
agctttggctaacacacacgccattccaaccaatagttttctcggcataaagccatgctctgac
gcttaaatgcactaatgccttaaaaaaacattaaagtctaacacactagacttatttacttcgt
aattaagtcgttaaaccgtgtgctctacgaccaaaagtataaaacctttaagaactttcttttt
tcttgtaaaaaaagaaactagataaatctctcatatcttttattcaataatcgcatcagattgc
agtataaatttaacgatcactcatcatgttcatatttatcagagctcgtgctataattatacta
attttataaggaggaaaaaataaagagggttataatgaacgagaaaaatataaaacacagtcaa
aactttattacttcaaaacataatatagataaaataatgacaaatataagattaaatgaacatg
ataatatctttgaaatcggctcaggaaaagggcattttacccttgaattagtacagaggtgtaa
tttcgtaactgccattgaaatagaccataaattatgcaaaactacagaaaataaacttgttgat
cacgataatttccaagttttaaacaaggatatattgcagtttaaatttcctaaaaaccaatcct
ataaaatatttggtaatataccttataacataagtacggatataatacgcaaaattgtttttga
tagtatagctgatgagatttatttaatcgtggaatacgggtttgctaaaagattattaaataca
aaacgctcattggcattatttttaatggcagaagttgatatttctatattaagtatggttccaa
gagaatattttcatcctaaacctaaagtgaatagctcacttatcagattaaatagaaaaaaatc
aagaatatcacacaaagataaacagaagtataattatttcgttatgaaatgggttaacaaagaa
tacaagaaaatatttacaaaaaatcaatttaacaattccttaaaacatgcaggaattgacgatt
taaacaatattagctttgaacaattcttatctcttttcaatagctataaattatttaataagta
agttaagggatgcataaactgcatcccttaacttgtttttcgtgtacctattttttgtgaatcg
attatgtcttttgcgcattcacttcttttctatataaatatgagcgaagcgaataagcgtcgga
aaagcagcaaaaagtttcctttttgctgttggagcatgggggttcagggggtgcagtatctgac
gtcaatgccgagcgaaagcgagccgaagggtagcatttacgttagataaccccctgatatgctc
cgacgctttatatagaaaagaagattcaactaggtaaaatcttaatataggttgagatgataag
gtttataaggaatttgtttgttctaatttttcactcattttgttctaatttcttttaacaaatg
ttcttttttttttagaacagttatgatatagttagaatagtttaaaataaggagtgagaaaaag
atgaaagaaagatatggaacagtctataaaggctctcagaggctcatagacgaagaaagtggag
aagtcatagaggtagacaagttataccgtaaacaaacgtctggtaacttcgtaaaggcatatat
agtgcaattaataagtatgttagatatgattggcggaaaaaaacttaaaatcgttaactatatc
ctagataatgtccacttaagtaacaatacaatgatagctacaacaagagaaatagcaaaagcta
caggaacaagtctacaaacagtaataacaacacttaaaatcttagaagaaggaaatattataaa
aagaaaaactggagtattaatgttaaaccctgaactactaatgagaggcgacgaccaaaaacaa
aaatacctcttactcgaatttgggaactttgagcaagaggcaaatgaaatagattgacctccca
ataacaccacgtagttattgggaggtcaatctatgaaatgcgattaagcttggctgcaggtcga
taaacccagcgaaccatttgaggtgataggtaagattataccgaggtatgaaaacgagaattgg
acctttacagaattactctatgaagcgccatatttaaaaagctaccaagacgaagaggatgaag
aggatgaggaggcagattgccttgaatatattgacaatactgataagataatatatcttttata
tagaagatatcgccgtatgtaaggatttcagggggcaaggcataggcagcgcgcttatcaatat
atctatagaatgggcaaagcataaaaacttgcatggactaatgcttgaaacccaggacaataac
cttatagcttgtaaattctatcataattgtggtttcaaaatcggctccgtcgatactatgttat
acgccaactttcaaaacaactttgaaaaagctgttttctggtatttaaggttttagaatgcaag
gaacagtgaattggagttcgtcttgttataattagcttcttggggtatctttaaatactgtaga
aaagaggaaggaaataataaatggctaaaatgagaatatcaccggaattgaaaaaactgatcga
aaaataccgctgcgtaaaagatacggaaggaatgtctcctgctaaggtatataagctggtggga
gaaaatgaaaacctatatttaaaaatgacggacagccggtataaagggaccacctatgatgtgg
aacgggaaaaggacatgatgctatggctggaaggaaagctgcctgttccaaaggtcctgcactt
tgaacggcatgatggctggagcaatctgctcatgagtgaggccgatggcgtcctttgctcggaa
gagtatgaagatgaacaaagccctgaaaagattatcgagctgtatgcggagtgcatcaggctct
ttcactccatcgacatatcggattgtccctatacgaatagcttagacagccgcttagccgaatt
ggattacttactgaataacgatctggccgatgtggattgcgaaaactgggaagaagacactcca
tttaaagatccgcgcgagctgtatgattttttaaagacggaaaagcccgaagaggaacttgtct
tttcccacggcgacctgggagacagcaacatctttgtgaaagatggcaaagtaagtggctttat
tgatcttgggagaagcggcagggcggacaagtggtatgacattgccttctgcgtccggtcgatc
agggaggatatcggggaagaacagtatgtcgagctattttttgacttactggggatcaagcctg
attgggagaaaataaaatattatattttactggatgaattgttttagtacctagatttagatgt
ctaaaaagctttttagacatctaatcttttctgaagtacatccgcaactgtccatactctgatg
ttttatatcttttctaaaagttcgctagataggggtcccgagagccccatactcatgagcagtc
ttgttacagctatgccggcagcacctgaaccgtttacaacaacttctatatcctcgattttctt
gttgacaagctttaatgcattgatcattgctgcaacagtaacaacggctgtaccgtgctggtca
tcatggaatattggaatgtcacattcctctttgagtcttctttctatttcaaagcatctcggag
cggatatatcttcgaggtttataccgccaaagcttccggagatgagcttgattgtctttacaat
ttcatctacgtcttttgatttgatacagagcggaaatgcgtccacatcaccaaacttcttgaag
agtacgcatttaccttccataacaggcattccggcttcaggtcctatgtctccgagccctaaaa
ccgccgtaccgtcggtaataaccgctaccaggttccaacgtcttgtatattcataagaaagatt
aacatctttctgaattgcaagacatggttctgcaacacccggtgtataagcaagcgacaactct
tccttggttgaaacaggtaccttgtgtataacctcaattttacccttccactcaccgtgaagcc
ttagtgattcttttctgtaatccatttgattctacctccaaattatattattaaatatctgcga
tattaatgcacaattataaattcttaacttcgttcaatacttttttaacctgctccgctgagaa
tcttaaagcttcttcttcttcaggagtcagattaaattggagaacttcctgaacaccttcggaa
tttacgatggatggaaggcttattgcaacatcttctattccatacatgccgtttataacggttc
ctacggttcttattgtattctgattcttaaggagtgtttcaactattgtgttgattgaaactgc
aataccatagtatgttgcacctttgttcttgataatggttgcacccgcagttttaacatcttca
gcgatttttttcttgtcttcttctgtgaaattgcatttcggatcatcgatatattcgttgatat
ttttaccggcgatatgtgtgcagctccacaacggaagctgtgaatcaccgtgttcgcctattat
gtagccgtgtacatagttcaacaaacgggattgacttttaaaaaaggattgattctaatgaaga
aagcagacaagtaagcctcctaaattcactttagataaaaatttaggaggcatatcaaatgaac
tttaataaaattgatttagacaattggaagagaaaagagatatttaatcattatttgaaccaac
aaacgacttttagtataaccacagaaattgatattagtgttttataccgaaacataaaacaaga
aggatataaattttaccctgcatttattttcttagtgacaagggtgataaactcaaatacagct
tttagaactggttacaatagcgacggagagttaggttattgggataagttagagccactttata
caatttttgatggtgtatctaaaacattctctggtatttggactcctgtaaagaatgacttcaa
agagttttatgatttatacctttctgatgtagagaaatataatggttcggggaaattgtttccc
aaaacacctatacctgaaaatgctttttctctttctattattccatggacttcatttactgggt
ttaacttaaatatcaataataatagtaattaccttctacccattattacagcaggaaaattcat
taataaaggtaattcaatatatttaccgctatctttacaggtacatcattctgtttgtgatggt
tatcatgcaggattgtttatgaactctattcaggaattgtcagataggcctaatgactggcttt
tataatgtacatttattggtaacattgtcttttgggtttttctttcttatatccgttcttgccg
ccgcggtttcggaaaaatttgaaatattgcttgtttcccttcttttgttggtacttttgatacc
ttatattgcccattattacaaactggagaacggagttcagaggctttatgagctttataacaaa
attgatgaaaaatgtgtaaggaaaaacaagaccgcctgagttctcacccagacggtcggtattg
gcagtttcactttcgttagtcgatgtttttcatgccggcaaagaaattattttcttgcaagaac
ctttttcagttttgcaaatcttggaagaccatcttcgataggaggtcttgattctccctgaatt
aacggaagagcatacttaataaagtcttctgtaaggcctgctccgtcaggtttaatccattcca
acggaactttcttctcagtatttgcaacttcactgaggttcagaagcttgatattgcacttgta
ttcaggaccttccgctctttcaaaagcaaccatgtagtctgttttcccttcaacggcatattgt
acggctgcctgtcctgcaagataagcttcatttacgtcggtaagagaagctacgtgagctgcgc
atctttggagaaggctgaattcaatgccgcgaacctttgcgccggtcttctctttaacaatgtt
agccagtgttgaagcaagaccgcccaactgtgcatgtccaaaggagtcttttgttttcgcaagg
tctgaaccgtattcggaaatatattttccgtttttgtctttgataccttcagatacggctacaa
taacctttccgttttctttgtagattcttgtcacatcttcaacaaatttgtctatgtcaaagga
aagctcgggtaccgagctcgaattcactggccgtcgttttacaacgtcgtgactgggaaaaccc
tggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaa
gaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcctgatgc
ggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaat
ctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctga
cgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgt
gtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatt
tttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaat
gtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagac
aataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccg
tgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctg
gtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctca
acagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaa
agttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgc
atacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatg
gcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaactt
acttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcat
gtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgaca
ccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactct
agcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgc
tcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcg
gtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggg
gagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaag
cattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcattttt
aatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtga
gttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttt
tttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgc
cggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaa
tactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctaca
tacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccg
ggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtg
cacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatga
gaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaa
caggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtt
tcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaa
aacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttct
ttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgc
tcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaata
cgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccg
actggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcacccca
ggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcac
acaggaaacagctatgaccatgattacgcca
序列4:在质粒pNW33N上构建的ldh敲除载体的序列
aattcagccccatactcatgagcagtcttgttacagctatgccggcagcacctgaaccgtttac
aacaacttctatatcctcgattttcttgttgacaagctttaatgcattgatcattgctgcaaca
gtaacaacggctgtaccgtgctggtcatcatggaatattggaatgtcacattcctctttgagtc
ttctttctatttcaaagcatctcggagcggatatatcttcgaggtttataccgccaaagcttcc
ggagatgagcttgattgtctttacaatttcatctacgtcttttgatttgatacagagcggaaat
gcgtccacatcaccaaacttcttgaagagtacgcatttaccttccataacaggcattccggctt
caggtcctatgtctccgagccctaaaaccgccgtaccgtcggtaataaccgctaccaggttcca
acgtcttgtatattcataagaaagattaacatctttctgaattgcaagacatggttctgcaaca
cccggtgtataagcaagcgacaactcttccttggttgaaacaggtaccttgtgtataacctcaa
ttttacccttccactcaccgtgaagccttagtgattcttttctgtaatccatttgattctacct
ccaaattatattattaaatatctgcgatattaatgcacaattataaattcttaacttcgttcaa
tacttttttaacctgctccgctgagaatcttaaagcttcttcttcttcaggagtcagattaaat
tggagaacttcctgaacaccttcggaatttacgatggatggaaggcttattgcaacatcttcta
ttccatacatgccgtttataacggttcctacggttcttattgtattctgattcttaaggagtgt
ttcaactattgtgttgattgaaactgcaataccatagtatgttgcacctttgttcttgataatg
gttgcacccgcagttttaacatcttcagcgatttttttcttgtcttcttctgtgaaattgcatt
tcggatcatcgattaatcgcatcagattgcagtataaatttaacgatcactcatcatgttcata
tttatcagagctcgtgctataattatactaattttataaggaggaaaaaataaagagggttata
atgaacgagaaaaatataaaacacagtcaaaactttattacttcaaaacataatatagataaaa
taatgacaaatataagattaaatgaacatgataatatctttgaaatcggctcaggaaaagggca
ttttacccttgaattagtacagaggtgtaatttcgtaactgccattgaaatagaccataaatta
tgcaaaactacagaaaataaacttgttgatcacgataatttccaagttttaaacaaggatatat
tgcagtttaaatttcctaaaaaccaatcctataaaatatttggtaatataccttataacataag
tacggatataatacgcaaaattgtttttgatagtatagctgatgagatttatttaatcgtggaa
tacgggtttgctaaaagattattaaatacaaaacgctcattggcattatttttaatggcagaag
ttgatatttctatattaagtatggttccaagagaatattttcatcctaaacctaaagtgaatag
ctcacttatcagattaaatagaaaaaaatcaagaatatcacacaaagataaacagaagtataat
tatttcgttatgaaatgggttaacaaagaatacaagaaaatatttacaaaaaatcaatttaaca
attccttaaaacatgcaggaattgacgatttaaacaatattagctttgaacaattcttatctct
tttcaatagctataaattatttaataagtaagttaagggatgcataaactgcatcccttaatcg
atgagaacaagttcatttgcggtttgccgcaaagccattgtgaaggctgcagacgcacctacaa
aaccagcaccaatgattgcaacttttgacctactttttaccatttccataccattcctttcaat
tacccagtatatttaacggttagttcgtttataaatttgagattaattctttaaattttaactg
tgaacccggttcacaggtattatcattaatttcagtatatgtgtttaataaaaattagtgaaaa
tttgcaactgcaagcatttaaaattgtaaacgataaataaatccaggcaacaaatttcccccat
tttaaatagcccagttaaacacattgataacattttaacattattttatatctgcgtccataac
tgaaaaagggaaatccattactttatgaaatcaaattttgaagttatcaagaaattatgacgat
tttctccgtggcatgcaagatttcgcgatatttcattcgtttatattaattttttatgaaaact
gcggtttgggctgacaattgcgatggaagtttcaattagactttttgtcaaatattatgtataa
taatattatctataataatgtatgaaaaaattgtcctaagatggaagacgggggtggtttcata
tatggttaaatatctgaagaggcaggaagagttggtagaagaagctctctcaaaggataactgt
tctgactgggaaagtttgagaaattatcataagtcccaaattgaatttttgcagcatgagagac
ttgtacatttattggtaacattgtcttttgggtttttctttcttatatccgttcttgccgccgc
ggtttcggaaaaatttgaaatattgcttgtttcccttcttttgttggtacttttgataccttat
attgcccattattacaaactggagaacggagttcagaggctttatgagctttataacaaaattg
atgaaaaatgtgtaaggaaaaacaagaccgcctgagtggatcctctagagtcgacctgcaggca
tgcaagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaatt
ccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaac
tcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagccctt
caaacttcccaaaggcgagccctagtgacattagaaaaccgactgtaaaaagtacagtcggcat
tatctcatattataaaagccagtcattaggcctatctgacaattcctgaatagagttcataaac
aatcctgcatgataaccatcacaaacagaatgatgtacctgtaaagatagcggtaaatatattg
aattacctttattaatgaattttcctgctgtaataatgggtagaaggtaattactattattatt
gatatttaagttaaacccagtaaatgaagtccatggaataatagaaagagaaaaagcattttca
ggtataggtgttttgggaaacaatttccccgaaccattatatttctctacatcagaaaggtata
aatcataaaactctttgaagtcattctttacaggagtccaaataccagagaatgttttagatac
accatcaaaaattgtataaagtcgctctaacttatcccaataacctaactctccgtcgctattg
taaccagttctaaaagctgtatttgagtttatcacccttgtcactaagaaaataaatgcagggt
aaaatttatatccttcttgttttatgtttcggtataaaacactaatatcaatttctgtggttat
actaaaagtcgtttgttggttcaaataatgattaaatatctcttttctcttccaattgtctaaa
tcaattttattaaagttcatttgatatgcctcctaaatttttatctaaagtgaatttaggaggc
ttacttgtctgctttcttcattagaatcaatccttttttaaaagtcaatcccgtttgttgaact
actctttaataaaataatttttccgttcccaattccacattgcaataatagaaaatccatcttc
atcggctttttcgtcatcatctgtatgaatcaaatcgccttcttctgtgtcatcaaggtttaat
tttttatgtatttcttttaacaaaccaccataggagattaaccttttacggtgtaaaccttcct
ccaaatcagacaaacgtttcaaattcttttcttcatcatcggtcataaaatccgtatcctttac
aggatattttgcagtttcgtcaattgccgattgtatatccgatttatatttatttttcggtcga
atcatttgaacttttacatttggatcatagtctaatttcattgcctttttccaaaattgaatcc
attgtttttgattcacgtagttttctgtattcttaaaataagttggttccacacataccaatac
atgcatgtgctgattataagaattatctttattatttattgtcacttccgttgcacgcataaaa
ccaacaagatttttattaatttttttatattgcatcattcggcgaaatccttgagccatatctg
acaaactcttatttaattcttcgccatcataaacatttttaactgttaatgtgagaaacaacca
acgaactgttggcttttgtttaataacttcagcaacaaccttttgtgactgaatgccatgtttc
attgctctcctccagttgcacattggacaaagcctggatttacaaaaccacactcgatacaact
ttctttcgcctgtttcacgattttgtttatactctaatatttcagcacaatcttttactctttc
agcctttttaaattcaagaatatgcagaagttcaaagtaatcaacattagcgattttcttttct
ctccatggtctcacttttccactttttgtcttgtccactaaaacccttgatttttcatctgaat
aaatgctactattaggacacataatattaaaagaaacccccatctatttagttatttgtttggt
cacttataactttaacagatggggtttttctgtgcaaccaattttaagggttttcaatacttta
aaacacatacataccaacacttcaacgcacctttcagcaactaaaataaaaatgacgttatttc
tatatgtatcaagaatagaaagaactcgtttttcgctacgctcaaaacgcaaaaaaagcactca
ttcgagtgctttttcttatcgctccaaatcatgcgattttttcctctttgcttttctttgctca
cgaagttctcgatcacgctgcaaaacatcttgaagcgaaaaagtattcttcttttcttccgatc
gctcatgctgacgcacgaaaagccctctaggcgcataggaacaactcctaaatgcatgtgaggg
gttttctcgtccatgtgaacagtcgcatacgcaatattttgtttcccatactgcattaatgaat
cggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgac
tcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggt
tatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccag
gaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcac
aaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttc
cccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgc
ctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtg
taggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgcct
tatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagc
cactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtgg
cctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttacct
tcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttt
tgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttct
acggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaa
aaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatata
tgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgt
ctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggct
taccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatc
agcaataaaccagccagcccgatatgggaaacaaaatattgcgtatgcgactgttcacatggac
gagaaaacccctcacatgcatttaggagttgttcctatgcgcctagagggcttttcgtgcgtca
gcatgagcgatcggaagaaaagaagaatactttttcgcttcaagatgttttgcagcgtgatcga
gaacttcgtgagcaaagaaaagcaaagaggaaaaaatcgcatgatttggagcgataagaaaaag
cactcgaatgagtgctttttttgcgttttgagcgtagcgaaaaacgagttctttctattcttga
tacatatagaaataacgtcatttttattttagttgctgaaaggtgcgttgaagtgttggtatgt
atgtgattcaataatttcttttactcgctcgttatagtcgatcggttcatcattcaccaaatca
taattttcatgtgaccgttctttatcaatatcgggattcgttttactttcccgttctctctgat
tgtgaaattg
序列5:在质粒pUC19上构建的ldh敲除载体的序列
gacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttctta
gacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaata
cattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaa
ggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgcct
tcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgca
cgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaag
aacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattga
cgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactca
ccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataa
ccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaac
cgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaat
gaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgca
aactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggc
ggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaa
tctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccct
cccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagat
cgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatata
ctttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgata
atctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaa
gatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaa
ccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaa
ctggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccacca
cttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgct
gccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgc
agcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccga
actgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggac
aggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacg
cctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatg
ctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggcc
ttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgta
ttaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagt
gagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcat
taatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatg
tgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtg
tggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagctt
gtagttggtgcaggctttgtaggttccaccacagcttatacattgatgctcagcggacttatat
ctgaaattgtactgatagacataaatgcaaaaaaagccgacggagaagtcatggacttaaatca
cggcatgccttttgtaaggcccgttgaaatttatcgtggtgactacaaagactgtgccggatcc
gacatagtaatcattaccgccggtgccaaccaaaaagaaggcgaaacgagaatagatcttagtt
caacaaacgggattgacttttaaaaaaggattgattctaatgaagaaagcagacaagtaagcct
cctaaattcactttagataaaaatttaggaggcatatcaaatgaactttaataaaattgattta
gacaattggaagagaaaagagatatttaatcattatttgaaccaacaaacgacttttagtataa
ccacagaaattgatattagtgttttataccgaaacataaaacaagaaggatataaattttaccc
tgcatttattttcttagtgacaagggtgataaactcaaatacagcttttagaactggttacaat
agcgacggagagttaggttattgggataagttagagccactttatacaatttttgatggtgtat
ctaaaacattctctggtatttggactcctgtaaagaatgacttcaaagagttttatgatttata
cctttctgatgtagagaaatataatggttcggggaaattgtttcccaaaacacctatacctgaa
aatgctttttctctttctattattccatggacttcatttactgggtttaacttaaatatcaata
ataatagtaattaccttctacccattattacagcaggaaaattcattaataaaggtaattcaat
atatttaccgctatctttacaggtacatcattctgtttgtgatggttatcatgcaggattgttt
atgaactctattcaggaattgtcagataggcctaatgactggcttttataatgtacatgcttat
attattggcgaacacggtgacaccgaagttgcggcctggagtcttgcaaatattgcgggaattc
ccatggatcgctactgtgacgaatgccatcagtgcgaggagcagatttcccggaataaaatata
tgaaagtgttaaaaatgcagcttatgaaatcatcaggaacaaaggtgcaacctattatgccgta
gcccttgccgtaagaagaatcgttgaagccattgtactgcaggtcgactctagaggatccccgg
gtaccgagctcgaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgtta
cccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccg
caccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcctgatgcggtatttt
ctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctg
atgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttg
tctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagagg
ttttcaccgtcatcaccgaaacgcgcga
序列6:解糖好热厌氧杆菌-木糖异构酶
ATGAATAAATATTTTGAGAACGTATCTAAAATAAAATATGAAGGACCAAAATCAAATAAT
CCTTATTCCTTTAAATTTTACAATCCAGAGGAAGTAATCGATGGCAAGACGATGGAGGAG
CATCTCCGCTTTTCTATAGCTTATTGGCACACTTTTACTGCTGATGGAACAGATCAATTT
GGCAAGGCTACTATGCAAAGACCATGGAACCACTACACAGATCCTATGGATATAGCGAAA
CGAAGGGTAGAAGCAGCATTTGAGTTTTTTGATAAGATAAATGCACCTTTCTTCTGCTTC
CATGATAGGGATATTGCCCCTGAAGGAGATACTCTTAGAGAGACAAACAAAAACTTAGAT
ACAATAGTTGCTATGATAAAGGATTACTTAAAGACCAGCAAGACAAAAGTTTTGTGGGGT
ACCGCAAATCTTTTCTCCAATCCGAGATTTGTACATGGTGCATCAACATCCTGCAATGCT
GACGTTTTTGCATATTCTGCAGCGCAAGTCAAAAAAGCCCTTGAGATTACTAAGGAGCTT
GGCCGCGAAAACTACGTATTTTGGGGTGGAAGAGAAGGGTACGAGACGCTTCTCAATACA
GATATGGAGTTAGAGCTTGATAACTTTGCAAGATTTTTGCACATGGCTGTTGACTATGCA
AAGGAAATCGGCTTTGAAGGTCAGTTCTTGATTGAGCCGAAGCCAAAGGAGCCTACAAAA
CATCAATACGACTTTGACGTGGCAAATGTATTGGCATTCTTGAGAAAATACGACCTTGAC
AAATATTTCAAAGTAAATATCGAAGCAAACCATGCGACATTGGCATTCCACGACTTCCAA
CATGAGCTAAGATACGCCAGAATAAACGGTGTATTAGGATCAATTGACGCAAATACAGGC
GACATGCTTTTGGGATGGGATACGGACCAGTTCCCTACAGATATACGCATGACAACGCTT
GCTATGTATGAAGTCATAAAGATGGGTGGATTTGACAAAGGTGGCCTTAACTTTGATGCA
AAAGTAAGACGTGCTTCATTTGAGCCAGAAGATCTTTTCTTAGGTCACATAGCAGGAATG
GATGCTTTTGCAAAAGGCTTTAAAGTTGCTTACAAGCTTGTGAAAGATGGCGTATTTGAC
AAGTTCATCGAAGAAAGATACGCAAGCTACAAAGAAGGCATTGGCGCTGATATTGTAAGC
GGTAAAGCTGACTTCAAGAGCCTTGAAAAGTATGCATTAGAGCACAGCCAGATTGTAAAC
AAATCAGGCAGACAAGAGCTATTAGAATCAATCCTAAATCAGTATTTGTTTGCAGAATAA
序列7:解糖好热厌氧杆菌-木酮糖激酶
ATGAGGGCGGCTTCATGCTTCATTAAAGCTGCCCTCAACAAAAATCATGGAGGTAAATGT
ATGTATTTTTTAGGGATAGATTTAGGGACATCATCAGTTAAGATAATACTGATGAATGAA
AGCGGCAATGTGGTATCAAGCGTTTCAAAAGAATATCCTGTGTACTATCCAGAGCCAGGC
TGGGCTGAGCAAAATCCAGAAGATTGGTGGAATGGCACAAGGGATGGAATAAGAGAGATT
ATTGCGAAAAGCGGCGTAAATGGCGATGAAATAAAGGGTGTTGGCTTAAGCGGGCAGATG
CATGGACTGGTGCTTTTAGACAAAGACAATAACGTTTTAACGCCAGCCATACTTTGGTGT
GACCAGAGGACACAGGAAGAATGCGACTACATCACAGAGAAAATAGGAAAAGAAGGCCTT
TTGAAGTACACAGGGAATAAAGCATTGACAGGTTTTACTGCACCAAAGATATTATGGGTA
AAGAAGCACCTTAAAGACGTATATGAAAGAATCGCTCATATCCTTTTGCCAAAAGATTAT
ATAAGGTTTAAATTGACAGGTGAGTACGCTACAGAAGTTTCAGATGCATCAGGTACACTT
CTTTTCGATGTGGAAAATAGAAGATGGTCAAAGGAAATGATAGACATATTTGAAATACCG
GAAAAAGCCCTTCCTAAGTGCTACGAATCAACAGATGTCACAGGGTATGTCACCAAAGAG
GCAGCAGATTTGACAGGGCTTCATGAAGGGACTATTGTCGTAGGCGGTGGTGGTGACCAA
GCCAGCGGCGCTGTAGGCACTGGCACGGTGAAAAGCGGCATAGTGTCCATCGCATTAGGA
ACTTCAGGCGTCGTATTTGCATCACAGGACAAGTACGCAGCAGATGATGAGCTTAGGCTT
CACTCATTCTGCCATGCAAACGGCAAATGGCATGTGATGGGTGTCATGCTTTCGGCTGCA
TCATGTCTTAAATGGTGGGTAGATGATGTAAATAATTACAAGACCGATGTTATGACATTT
GATGGACTCTTAGAAGAAGCAGAGAAGGTGAAGCCAGGCAGTGATGGATTGATATTCTTG
CCATACCTGATGGGTGAAAGGACCCCTTACAGCGATCCTTATGCGAGAGGCAGCTTTGTA
GGTTTAACAATTACACACAATAGAAGCCACATGACAAGATCTATATTAGAAGGCGTCGCA
TTTGGACTTAGGGATTCGCTGGAGCTTATAAAGGCTTTAAATATACCTGTAAATGAAGCC
AGGGTAAGTGGTGGTGGTGCTAAAAGCAGGCTTTGGAGGCAAATACTTGCCGATGTATTC
AATGTAAGGATAGACATGATAAATGCTACAGAAGGACCTTCATTTGGTGCAGCAATAATG
GCGTCTGTGGGATATGGCCTTTACAAAAATGTAGATGATGCATGCAATAGTTTAATAAAA
GTTACAGACAGCGTATATCCAATCAAAGAAAACGTCGAAAAGTACAACAAACTGTATCCA
ATCTACGTGAGCTTGTATTCAAGGCTTAAAGGCGCCTTTGAAGAAATTGGGAAGTTGGAT
TTGTAA
序列8:解糖好热厌氧杆菌-L-***糖异构酶
ATGATTATTGTGTACAAAGATGAAAAGCCCAGGATAGGTTTTTTGGGTATTATGCAGGAG
TTATACGATGATATGTTGCCTGGTATTACTGAAAGGCAAGAGATGTATGCACAACAGGTT
ATAGGTAGATTAGGTGATGTTGCTGATTTTTATTTCCCAGGTGCTGCAAAAAACAGAAAT
GATATAGAAAGGATAGTTAAGGAATTCAACGATAAAGATCTTGACGGAATAATGATCGTG
ATGCTGACATACGGACCAGCCACAAATCTTGTGAATGCTTTAAGAAACAATAGGCTTCCG
ATTATGCTGGCGAATATACAGCCAGAAAGCACTGTGACAGACGATTGGGATATGGGGGAC
TTGACCTACAACCAAGGTGTTCATGGTGCACAGGATACTTCAAATATTATTCTGAGAATG
GGCATAACTTGTCCTGTTATAACAGAAGATTGGCATTCTGATGAATTTAAAGATTTTGTG
AATGATTGGGCAAAAACTGTAAAGACAGTAAAAGCTTTGAGGAATATGAAGATAGCACAA
TTTGGAAGAATGCATGGTATGTATGACATAATGGGTGATGATGCAGCTTTTACAAGAAAA
TTGGGGCCGCAAATAAACCAGGAGTACATTGGCCAAGTTTTTAGATATATGGAAGAAGCT
ACAAATGAAGAAATTGACAAAGTGATAGAGGAAAACAAGAAGAACTTTTATATAGATCCT
AAATTAAGTGATGAGAGCCACAGATATGCTGCAAGGCTTCAAATAGGATTTAAGAAATTG
CTTGAGGAGAAAGGGTACTCTGGCTTTAGTGCTCACTTTGATGTGTTTAAAGGCGATGGA
AGATTTAAGCAGATACACATGATGGCAGCATCAAACTTGATGGCAGAAGGATATGGCTAT
GCGGCAGAGGGCGATGTAGTTACGGCAAGCCTGGTGGCAGCAGGTCATGTTTTGATAGGC
AATGCACACTTTACCGAGATGTATGCGATGGATTTTAAGAGAGATTCAATTTTGATGAGC
CACATGGGAGAGGGCAATTGGAAGATAGCCAGAAAAGATAGACCTATAAAATTAGTCGAT
CGAGAGCTTGGCATAGGAAAGCTTGATAATCCTCCAACAGTGGTGTTTATGGCTCAACCA
GGCATTGCGACATTGGCATCATTAGTGTCTTTAGAAGGCGAAAAATATAGACTTGTTGTT
TCAAAGGGAGAAATTTTAGATACAGAAGAAGCGAAAAATATAGAGATGCCGTATTTCCAT
TTTAGACCTGAAAACGGAGTTAGGGCTTGTCTAAATGGCTGGCTTAAAAATGGTGGTACA
CATCATCAGTGCTTGACATTAGGTGATGCTACTAAAAGATGGAAGCTTTTATGCGAATTA
TTAGATATCGAGTATGTTGAAGTGTAA
序列9:解糖好热厌氧杆菌-L-核酮糖-5-磷酸4-表异构酶
ATGTTAGAGAACCTAAAACAACGTGTATATAAAATGAACATGATGCTTCCTAAAAACAAT
TTAGTCACAATGACAAGCGGCAATGTCAGCGGAAGAGATCCTGAGACAAATCTTGTAGTC
ATAAAGCCCAGCGGAGTTTTGTACGATGAAATGACGCCAGATGATATGGTAGTCGTGGAT
TTGGATGGCAATGTGGTTGAGGGTAAGCTAAAACCATCTGTCGATACTGCTACACATCTT
TACGTCTACAGGCATAGAAATGATGTAAACGGCATTGTCCATACACACTCACCGTATGCT
ACAAGTTTTGCCGCACTTGGCCGGTCAATTCCGGTCTATCTTACAGCTATTGCAGACGAG
TTTGGATGCGCAATTCCTGTAGGGCCTTATGCCAAAATTGGCGGGGAAGAGATAGGAAAA
GCCATCGTAGATTATATAGGTGAGAGTCCTGCAATACTTATGAAAAATCACGGCGTTTTT
ACCATTGGCAATTCACCTGAAGCAGCCTTAAAAGCTGCTGTTATGGTAGAAGATACAGCT
AAGACGGTGCACTTATCACTGCTTTTAGGCACACCTGATGTAATACCAGATGAAGAAGTA
AAAAGAGCCCATGAAAGATATCTTACAAAATACGGTCAATGA
序列10:解纤维梭菌-木糖异构酶
ATGTCAGAAGTATTTAGCGGTATTTCAAACATTAAATTTGAAGGAAGCGGGTCAGATAAT
CCATTAGCTTTTAAGTACTATGACCCTAAGGCAGTTATCGGCGGAAAGACAATGGAAGAA
CATCTGAGATTCGCAGTTGCCTACTGGCATACTTTTGCAGCACCAGGTGCTGACATGTTC
GGTGCAGGATCATATGTAAGACCTTGGAATACAATGTCCGATCCTCTGGAAATTGCAAAA
TACAAAGTTGAAGCAAACTTTGAATTCATTGAAAAGCTGGGAGCACCTTTCTTCGCTTTC
CATGACAGGGATATTGCTCCTGAAGGCGACACACTCGCTGAAACAAATAAAAACCTTGAT
ACAATAGTTTCAGTAATTAAAGATAGAATGAAATCCAGTCCGGTAAAGTTATTATGGGGA
ACTACAAATGCTTTCGGAAACCCAAGATTTATGCATGGTGCATCAACTTCGCCAAACGCT
GACATATTTGCGTATGCAGCAGCTCAGGTTAAAAAGGCAATGGAAATCACAAAGGAATTA
GGCGGAGAAAACTATGTATTCTGGGGTGGTAGAGAAGGTTATGAAACTCTCTTGAATACA
GACATGAAGCTGGAACTTGATAATTTAGCAAGATTCTTGAAGATGGCTGTTGACTATGCT
AAGGAAATCGGTTTTGACGGACAATTCCTAATCGAACCAAAGCCAAAAGAACCAACTAAG
CACCAATATGATTTTGATACAGCTACAGTTATCGGCTTCCTGAAGACATATGGATTAGAC
CCATACTTCAAGATGAATATCGAAGCTAACCATGCTACATTAGCAGGACACACATTCCAA
CATGAGCTTGCTATGTGCAGAATCAACGACATGCTTGGAAGTATTGATGCTAACCAAGGT
GATGTAATGCTCGGATGGGATACAGACCAATTCCCAACGAACCTATATGATGCAACACTA
GCAATGGTGGAAGTATTAAAGGCCGGCGGATTGAAAAAGGGAGGTTTGAACTTCGACTCA
AAAGTTAGAAGAGGATCATTCGAACCATCAGACTTGTTCTATGGACATATTGCAGGTATG
GATACTTTTGCAAAGGGTCTTATCATAGCAAATAAGATCGTTGAGGACGGTAAGTTTGAT
GCATTTGTTGCTGACAGATACTCAAGCTACACAAATGGTATCGGAAAAGATATTGTTGAA
GGAAAAGTTGGCTTTAAGGAATTGGAGCAATATGCACTTACTGCAAAGATTCAGAACAAG
TCTGGACGTCAGGAAATGCTGGAAGCTTTGTTAAACCAGTATATCCTCGAAACAAAATAA
序列11:解纤维梭菌-木酮糖激酶
ATGAAGCATGAACTAAATGACGGGAGAAATGCTATTCTAAATGGAAAGACAGCAATTGGGATTGAACTCGGATCAACTAG
AATAAAAACGGTATTGATAGGTGCAGACAATGCACCTATCGCATCCGGTAGTCATGACTGGGAAAACAGCTATATCAATA
ATATTTGGACTTACAGCTTGGAAGATATCTGGAAAGGCGTACAGAGCAGCTATCAGGAAATGGTTAAAGATGTTAGGGAC
AAATTCGGAGTAAGTCTAAAGACAACCGGAGCAATAGGTTTTAGCGGAATGATGCACGGTTATATGGTTTTTGATAAGGA
AGGTAATCTTCTGACTCATTTCAGAACATGGCGTAACACTATAACTGCACAGGCTTCCGAGGAACTAACCAAGTTGTTTA
ATTATCCTATTCCTCAAAGGTGGAGCATTGCCCATCTTTACCAAGCCATACTGAACAATGAAGAGCATGTATCCAATATC
GATTTTATGACTACATTGGCCGGATTTATACACTGGAAGTTGACAGGAGAAAAAGTTCTTGGTGTCGGAGAGGCATCAGG
TGTTTTCCCAATAGATTTAGATACTAAGGATTTTAATTCAAGTATGATTAATCAGTTTAATGAGGCTACCACCAATCGAA
ATTTTTCATGGAAGCTTCAAAATATTCTTCCAAAAGTTTTGGTTTCGGGTACTGAAGCAGGTAGGCTGACAGAAGAAGGT
GCAAAGCTTCTTGATGTTACCGGGGAGCTTCAGGCGGGTATTCCTTTTTGTCCCCCTGAGGGAGATGCGGGAACCGGTAT
GGTTGCAACTAACAGCGTTGCTGTCCGTACAGGCAATGTGTCTGCCGGGACTTCTGTTTTTGCTATGGTTGTTCTCGAAA
AGGAATTATCCAAAGTGTATTCGGAAATTGACCTGGTGACTACACCTGACGCAAATCTTGTGGCTATGGTTCATTCAAAT
AATTGTACATCGGACTATGACGCATGGATGGGTATATTTGCTGAGGCAGTTAAGACCTTGGGCTTTGACGTGAAAAAACC
ACAGCTATATGATACCCTGCTGGGAGCCGCACTTCAAGGTGACCCTGATTGCGGAGGGTTGCTTGCGTACGGTTATATTT
CAGGTGAGCATATTACCCATTTTGAAGAAGGTCGCCCGATGGTTGTTCGTTCATCAAACAGCAAATTCAACCTGGCCAAC
TTTATCAGGGTCAATTTGTTTACATCTCTTGGAGCCTTGAAGACCGGTTTGGATATTCTTTTTCAAAAGGAAGCTGTTAA
AGTGGACGGTATTACCGGACACGGCGGTTTCTTTAAGACGAAGGAAGTAGGACAGAAGATTATGGCGGCTGCCTTTAATG
TCCCTGTATCTGTTATGAAGACTGCGGGTGAAGGCGGTGCATGGGGTATTGCCCTACTTGCTTCGTATATGATTAATAGG
GAAAGCTCACAGTCCTTGGAGGATTTTCTTAAACAAAATGTGTTTGGGGAAAGCCAAGGTGAGACTGTACAGCCAGATTC
GAAGGATGTTGACGGTTTCAACGAGTTTATGAAAAGGTACACAAAGGGACTGGGTATTGAAAGGGCTGCGATAAACTTCT
TGAACTGA
序列12:解纤维梭菌-L-***糖异构酶
ATGATAACCAAACAAAAACCAAGAATCGGATTTTTGGGCCTAATGCAGGGATTGTATGACGAATCACAGCCGGAACTGCC
GAAAATGCAGGAGGCATTTGCCAGAGAAGTGGTTGAACAATTAAAAGATGTGGCAGATATTGATTTTCCCGGTCCAGCAA
AAGAAAGAGAAGATATAGAAAGATATGTAAAATATTTCAATGATAAAGAGTACGATGGAATAATGATAGTAAATCTGTTG
TACAGTCCGGGAAATCGTTTAATACAGGCTATGAAGAATAATAATCTGCCAATATTGCTGGCTAATATTCAACCACTTCC
CGATGTTACATCAAACTGGGATTGGATTTTGTGCACAACTAATCAGGGAATTCATGGAATACAGGATACAAGTAATGTTC
TCATGCGTTGTGGTATTAAACCGGCTATTATAACAGATGATTGGAAGGCTGAATCCTTTAAAGCCTACTTTGAAGATTGG
GCATTGGCTGCCAACACGCATAACAGACTAAAAAAGACAAAGGTTGCGATTTTCGGCCGTATGCACAATATGGGTGACAT
ACTTGGTGATGATGCGGCATTGTGCAGAAAATTTGGTGTAGAGGCAAACCATGTAACAATCGGTCCGGTTTATTACAACA
TGGAAGGATTGTCAGATAAAGAAGTAGATGCCCAGATTGAGGAAGATAAAAAGAATTTTAAAATTGATCCTAATCTTCCT
GAAGAAAGTCATCGGTATGCTGCACGTATGCAATTAGCCTTTGAAAAATTCCTTAATGATAACGGTTATGAAGGTTTTTC
ACACTTCTTCAACATATACAAGGAAGACGGCAGGTTCAAACAAATACCGATATTGGCAGGCTCCAGTCTCCTTGCAAAAG
GTTATGGTTATTCGGCGGAAGGTGATACAAATGTACTTCTCATGACTGTGATCGGTCACATGATGATAGGGGATCCTCAT
TTTACTGAGATGTACTCCCTGGACTTTGGTAAGGATTCAGCAATGCTAAGCCATATGGGAGAAGGCAACTGGAAGGTTGC
AAGGAAGGATCGCGGAGTGACACTGATTGACAGGCCTCTTGATATTGGTGGTCTTGGTAATCCTCCGACACCAAAGTTCA
ACGTAGAACCAGGAACAGCTACCCTTGTTTCCCTCGTTGCAGTAGAAGGAGAAAAATACCAACTAATTGTATCAAAGGGT
ACTATCCTTGATACTGAGGACTTGCCAGATGTTCCTATGAACCATGCTTTTTTCAGACCGGATTCCGGCATCAAAAAGGC
TATGGACGAATGGTTAGCTAATGGTGGTACACATCACGAAGTACTATTCCTGGGTGATTTTAGAAGACGTTTTGAATTAT
TATGTAAATTCTTGACATAA
序列13:解纤维梭菌-L-核酮糖-5-磷酸4-表异构酶
ATGTTGGAACAACTAAAACAAGCGGTGTTGGAAGCCAATCTAGAGCTGCCTGAAAAAGGACTTGTAACATATACATGGGG
AAATGTAAGCGGTATCGACAGAGAAAGCAGACTTATTGCAATTAAACCCAGTGGTGTTGAGTATAATGTTATGACAGCTG
ATGATATTGTATTAATCGACCTTACAGGTAAAGTGGTGGAAGGAAAATTGAAGCCGTCTTCTGATGCACCAACACATGTA
GCTCTGTATAATGCATTTCCTGATATAGGAGGTGTAACACACACCCATTCCAGGTGGGCAACTGCTTTTGCACAGGCTGG
TATGGGGATTCCTGCTTACGGGACTACTCATGCGGATTACTTTTATGGTGAAATCCCATGTACTCGGGAAATGACAAAGG
ATGAGATTGAGTCCGATTATGAAGCAAATACCGGAACGGTGATAATAGAGACTTTTAAAGATTTAAATCCTAACTATATC
CCTGCCGTACTTGTAAAAAATCATGCACCTTTTACATGGGGAAAAAGTGCAGCGGAATCGGTTCATAATTCTGTTGTTTT
AGAAGAAGTAGCTATGATGGCTATTCAGTGCAGACAACTGAACCCAAATGTAACTCCCATGCCGCAGGTGCTGCTAGACA
AGCATTTTATGAGGAAGCACGGCCCGAAAGCTTATTACGGACAAAAATAA
序列14:C.phytofermentans-木糖异构酶
ATGAAAAATTACTTTCCAAATGTTCCAGAAGTAAAATACGAAGGCCCAAATTCAACGAATCCATTTGCTTTTAAATATTA
TGACGCAAATAAAGTTGTAGCGGGTAAAACAATGAAAGAGCACTGTCGTTTTGCATTATCTTGGTGGCATACTCTTTGTG
CAGGTGGTGCTGATCCATTCGGTGTAACAACTATGGATAGAACCTACGGAAATATCACAGATCCAATGGAACTTGCTAAG
GCAAAAGTTGACGCTGGTTTCGAATTAATGACTAAATTAGGAATTGAATTCTTCTGTTTCCATGACGCAGATATTGCTCC
AGAAGGTGATACTTTTGAAGAGTCAAAGAAGAATCTTTTTGAAATCGTTGATTACATCAAAGAGAAGATGGATCAGACTG
GTATCAAGTTATTATGGGGTACTGCTAATAACTTTAGTCATCCAAGATTTATGCATGGTGCTTCCACATCTTGCAACGCA
GACGTATTTGCATATGCTGCTGCTAAGATTAAGAATGCATTAGATGCAACAATTAAATTAGGCGGTAAAGGTTATGTATT
CTGGGGTGGTCGTGAAGGTTATGAAACACTTCTTAATACAGATTTAGGACTTGAGCTTGATAATATGGCTAGACTTATGA
AGATGGCTGTAGAGTATGGCCGTGCAAATGGTTTTGATGGCGACTTCTATATTGAGCCAAAGCCAAAGGAACCAACCAAG
CATCAATATGATTTTGATACAGCAACCGTACTTGCTTTCCTTCGCAAATATGGCTTAGAAAAAGATTTCAAGATGAACAT
TGAAGCAAACCATGCTACTCTTGCAGGTCATACCTTTGAACATGAACTTGCAATGGCTAGAGTTAATGGTGCATTTGGTT
CTGTAGATGCAAACCAGGGTGATCCAAACCTTGGATGGGATACGGATCAATTCCCAACTGATGTTCATAGTGCAACTCTT
GCAATGCTTGAAGTACTTAAGGCTGGTGGATTCACTAACGGCGGACTTAACTTTGATGCAAAGGTAAGACGTGGTTCCTT
CGAATTTGATGATATTGCATACGGTTATATTGCAGGAATGGATACTTTTGCACTTGGTTTAATTAAGGCTGCTGAGATTA
TCGACGATGGTAGAATCGCAAAATTTGTAGATGATCGTTATGCAAGCTATAAAACAGGAATTGGTAAAGCAATTGTGGAT
GGAACTACATCTCTTGAAGAATTAGAGCAGTATGTTTTAACACATAGTGAACCAGTAATGCAGAGTGGTCGTCAGGAAGT
TCTTGAAACAATCGTAAATAATATTTTATTTAGATAA
序列15:C.phytofermentans-木酮糖激酶
ATGGGCATGGAGCATTTTAAAGATGCGATTCTTACGGGTAAAACAACACTTGGAATTGAGCTTGGTTCCACTAGAATAAA
AGCTGTTTTAGTAAATGAAGAAAACGAACCAATTGCGTCAGGAAGCCATGATTGGGAAAATCAATATATTGATAATGTAT
GGACTTACAATCTGGATGATATCTGGAGGGGCGTTCAGAATAGTTATGGACAAATGACAAGTGATGTTAAGAATAAGTAC
GGAGTAGAACTTACAACAATTGGAGCCATTCGTTTTAGTGGAATGATGCATGGCTATATGGCTTTTGACGAAAGTGGAGA
GTTACTTGTACCATTTCGTACCTGGAGAAATACAATAACAGGACCAGCATCCGAGCAGTTGACCAATGTATTTCAGTATC
AAATTCCACAACGTTGGAGTATTGCCCATCTATATCAAGCTATCTTAAATGGGGAATCCCACGTGAAAAATATTAGATTC
CTGACAACATTGGCAGGATATATTCACTGGAAGCTAACAGGAGAAAAAGTATTAGGAGTCGGAGAAGCATCTGGAATGTT
TCCAATCGATATAAATACGAAAGATTTTAATAAATCAATGTTAGCTCAGTTTAATGAACTGGTTGCTTCGAATGACTATT
CATGGAAAATAGAAGATATTCTACCGAAAGTACTAGTTGcAGGAGAGTCTGCTGGAGTATTAACCGAAGAAGGAGTAAAA
CTTCTTGATGTTTCAGGTAAATTAAAAGCAGGAATTCCTCTTTGTCCGCCGGAAGGAGATGCTGGAACTGGTATGGTAGC
AACCAACAGCGTAGcAAAGAGAAcTGGTAATGTATCTGCTGGTACTTCTGTATTTGCAATGGCTGTATTAGAAAAAGAGC
TTTCAAAAGTTTACGAAGAAATTGACCTTGTGACGACTCCAAGCGGAGATCTTGTGGCTATGGTGCACTGCAATAACTGT
ACTTCTGATTTGAATGCGTGGGTTTCTATCTTTAAAGAATTTGCTTCGGCAATGGGCATGGAAGCTGATATGTCAAAGAT
ATTCTCAACGCTATACAATAAGGCGTTAGAAGGCAATGCAGAGTGTGGAGGCTTACTCGCATACAATTATTTTTCCGGTG
AACATATAACACACTTTGAAGAAGGCCGCCCATTGTTTGTAAGAACTCCAGAGAGTAAGTTTAACCTTGCGAATTTCATG
AGAGTTCATCTATTCACAGCACTTGGTGCTTTAAAGATAGGTCTTGATATCCTATTAAAACAAGAATCAGTACAATTGGA
TGAGATTTTTGGTCATGGTGGATTATTTAAGACGAAAGATGTCGGACAAAAAATTATGGCTGGTGCAATCAATGTTCCTG
TTTCTGTGATGGAGACTGCCGGAGAAGGCGGAGCATGGGGAATCGCAATCTTGGCTTCTTATATGATTTCTAAGGAAGAA
GGTCAGTCCTTAGATGAGTATCTTTCTAAACATGTATTCCAAGGAAAGACAGGTAGCAAGATGCAGCCGGATCCAAGGGA
TGTAGAAGGTTTTGAACAGTTTATCAAACGATATATTGACGGACTTGAAATTGAGCGTAAAGCAGTGGAGATATTATAA
热解纤维梭菌启动子
序列16:三糖磷酸异构酶
actttgccaacggtacaagggaagttgcaagagcggttgccgagtccggagcaatttcaa
taatcggaggcggagattctgccgcagctatagaacagcttggttttgccgataagatta
cccacatttcaaccggaggcggcgcgtctttggagtttcttgaaggaaaagtattgccgg
gaattgatgtattaatggataaataaggagagaagaggtcatgagtagaaaagttattgc
(atg..TPI基因的起始)
序列17:GAPDH基因
aattactgtatctctctggcattgccaggttttaataaagattaaaattattgactagaa
ataaaaaaattgtccataatattaatggacaaaaaaacaaagaattacatcaaaggaaga
taaaaatactttgttaaaaaattaattattttttatctaaactattgaaaatgaaaataa
aataatataaaatgaatcatagtgcaagagatacttgccagaggatgaatattttactgc
attcatgctttatggcagctaatagaggcattaaattaaattttaatttacaataggagg
cgatattaatggcagtaaaaattggtatcaacggttttggacgtatcggtcgtcttgtgt
tcagggccagtctcaacaacccgaacgttgaggttgtaggtataaacgacccatttattg
(atg...基因的起始)
序列18:LDH
ctgccccattaaaagctcggttccaaccgctaatatctccgcattcatattgaaagaccc
cttaaatttaaactttttgtaacttattatatcaattagtgttataaaataaaagggaaa
aagaattaaaatcaaaggtttcaagagcagccgtatcacccgtaaaagtttcagccgatt
caacctttttacacataaaactttcaaaaattgatgacttacaattatcaagtaggatat
aatattactaatgctaaacagttattgataaaggaggaaggaatatgaacaataacaaag
(ag...:基因的起始)
序列19
GGCGgaattc CTT GGT CTG ACA ATC GAT GC
序列20
GGCGgaattc TATCAGTTATTACCCACTTTTCG
序列21
gaattctgcgacagaatagggattgacaattcctttataaagcaatcaaggggttcagaagagg
ctgttattttgaataaagagctaaagaatcacaaagatgcaatagaggctgttatttctgcact
gactgacgataatatgggcgttataaaaaacatgtccgaaatatcagcagtgggacacagaata
gtacacggcggtgaaaaattcaacagttctgtagttatagatgaaaacgttatgaatgcagtaa
gagagtgtatagacgttgcaccgcttcataatccgccgaatattataggtatagaggcttgcca
gcagattatgcccaatatacctatggtagctgtatttgataccactttccacagctccatgcct
gattatgcatacctttacgcattgccatatgaactttatgaaaagtacggtataagaaaatatg
gtttccacggaacatcacacaaatatgttgcagaaagagcttctgcaatgcttgataagtcttt
gaacgaattaaagataattacatgccatcttgggaacggttcaagtatttgtgctgttaacaag
ggtaaatcaattgatacttccatgggctttacacctttgcagggacttgcaatgggtacaagaa
gcggtacaatagaccctgaagttgttacgaattc
序列22:ldh基因-2262
ATGAAAAATAAATCTATAAATAAAATAGTAATTGTAGGTACGGGTTTTGTCGGTTCAACAACTG
CCTATACTTTAATGGTCAGCGGACTAGTTTCCGAGATTGTACTTATTGACCGTAACACAAGCAA
AGCCGAAGGAGAGGCAATGGATATGAATCACGGTATGCCCTTTGTAAGACCTGTCAGAATATAC
AAAGGTGATTATCCTGATTGCAAAGGTGCTGATATTGTTGTAATAACAGGTGGAGCAAACCAGA
AGCCCGGTGAAACCAGAATTGACCTTGTAAATAAAAATACTGAAGTTTTTAAAGACATTGTTGG
AAATATCATTAAATACAATACAGACTGTATTTTACTTGTTGTTACAAACCCGGTTGATATCTTA
ACCTATGTAACATACAAATTATCCGGATTTCCCAAAAACAGAGTTATAGGCTCCGGAACAGTTC
TTGATACTGCACGTTTCAAATATATGCTTGGTGAACACATGGGAGTTGACCCAAGAAACGTTCA
TGCTTATATAATCGGTGAACATGGAGATACAGAGGTACCTACATGGAGTCTGGCATCCATAGCC
GGGATACCGATGGATGCTTATTGCAAGGAATGTAAATCCTGTGATGCTGAAAACTTTAAGAGTG
AAACTTTTGACAAAGTAAAAAATGCAGCTTATGAAATTATTGATAGAAAAAATGCAACCTACTA
CGCCGTTGCTCTTGCAGTAAGAAGAATTGTAGAGGCTATCGTTCGTAATGAAAACTCCATATTG
ACGGTATCAAGCCTATTCGAAGGAGAATACGGCCTCAATGACATATGTCTCAGTATTCCCAGCC
AGGTAAATTC GGAGGGTGTTTCAAGGATTTTGAATATTCCTCTGAGCAGTGAGGAAACAGGTTT
ACTTAATAAATCTGCCCAGGCCTTGAAACAGGTTATCAGTGGGCTGAATTTATAA
序列23:ldh基因-2744
ATGGGTTTTAAAGTTGCGATCATAGGAGCAGGATTTGTTGGAGCATCAGCTGCGTATGCGATGT
CTATAAACAACTTGGTTTCTGAATTGGTATTAATTGATGTAAATAAAGAGAAGGCTTATGGTGA
AGCACTTGATATCAGCCATGGCTTATCATTCTCAGGAAATATGACAGTTTATTCCGGCGACTAT
TCTGATGTTAAGGATTGTGATGTTATAGTTGTAACTGCAGGGGCAGCAAGAAAACCGGGAGAAA
CTCGTTTGGACCTTGCTAAAAAGAATACTATGATCATGAAGAGCATAGTTACTGATATAATGAA
GTACTACAATAAGGGTGTTATTGTAAGTGTATCAAATCCTGTTGATGTATTGGCATATATGACA
CAAAAGTGGTCAGGATTGCCTGCAAATAAAGTTATAGGATCAGGAACAGTTCTTGACAGTGCAA
GACTGAGAACTCATATCAGTCAGGCATTGGATGTAGACATTGCTAACGTTCACGGTTATATTGT
TGGTGAACATGGTGATTCTCAGTTGCCATTATGGAGTGCAACACATATAGCAGGAGTACAATTT
GACGACTATGTAAAAGCTACTGGCTTAAATGTTGATAAGGAAGCTCTTTTCAATGAAGTTAAGG
TAGCAGGTGCAACTATTATTAAGAACAAGGGAGCAACTTACTACGGTATAGCTCTTTCAATTAA
CAGAATAGTTGAATCAATCCTGAAGGACTTCAATACTATTATGCCTGTTGGTACAGTTCTTGAC
GGACAGTACGGATTAAAGGATGTTTTATTAAACGTTCCTACGATAGTTGGCGGAAACGGAGCTG
AAAAAGTTCTTGAAGTGAACATTACAGATGCAGAATTACAACTTTTGAAGCATTCAGCTGAACA
GGTTAGGGCAGTTATTAACGAAGTTAAAGACATATAA
序列24
gacgcatacaggttgtaacacccatttcccttagcttttcgggagatgaataaaacaaactttc
cgggtcctttaccacaccgcccacataaagagctatgccgcatgaaagaaacgatatgttatca
tttttttcgtaaactgttatttccgaacccggataaagctttaccatattattaactgctgccg
tccctgcatgtgtacaccctataaccactattttcatatacatcctcctttgtttgcttgtaaa
tatatcccatatataccacctaaatatattttataaacaaattcggtatatcattcttttggta
aataaaaagtacatccgatattagaatgtacctaaaaaaaattattattttattgtatatgctt
tatctgttttcattatatggtttgctatccattctacggtaaaatcaagtaattccattaagta
ctgatcctgatccttgtctatcctgctataatccgtattactgattttctcaataaaatcatgg
tgttcaactttgtgggagagaagcttgcgatatcctatgctatgcatgtattcttcttcatagg
taaaatgaaagacagtgtaatcttttagttccgtaattagccgtacaatttcatcatatttgtc
tgtaataagctgatttttcgtggcctcataaatttccgaagcaatctggaatagtttcttatgc
tgttcgtcgattttctcaattccaagaataaattcgtctctccattctatcatatggaccctcc
taaattgtaatgtataccaagattatacatacttcctagaatataaacaatacaaggataaaat
tttaatatcgtatacctacataaatgactaacttaaagctctctaaaacttcttttttattatt
tctatactactaaaatcaaaaatattctctaaagtatttctacaaatgttgtttttgcaacaaa
gtagtatacttttgcacccagaatgttttgttataacttacaaattaggggtatatttatagta
aatactaaatggaagagtaggatattgattatgaacgagaaaaatataaaacacagtcaaaact
ttattacttcaaaacataatatagataaaataatgacaaatataagattaaatgaacatgataa
tatctttgaaatcggctcaggaaaagggcattttacccttgaattagtacagaggtgtaatttc
gtaactgccattgaaatagaccataaattatgcaaaactacagaaaataaacttgttgatcacg
ataatttccaagttttaaacaaggatatattgcagtttaaatttcctaaaaaccaatcctataa
aatatttggtaatataccttataacataagtacggatataatacgcaaaattgtttttgatagt
atagctgatgagatttatttaatcgtggaatacgggtttgctaaaagattattaaatacaaaac
gctcattggcattatttttaatggcagaagttgatatttctatattaagtatggttccaagaga
atattttcatcctaaacctaaagtgaatagctcacttatcagattaaatagaaaaaaatcaaga
atatcacacaaagataaacagaagtataattatttcgttatgaaatgggttaacaaagaataca
agaaaatatttacaaaaaatcaatttaacaattccttaaaacatgcaggaattgacgatttaaa
caatattagctttgaacaattcttatctcttttcaatagctataaattatttaataagatcccc
tttacttcggatgcatgccgcaggcaggcatccgaagtagtttctccattatacaagtattctc
ttgagtacgtcgtcgcttctcagcagctgctttgctttttccctgttttccggcacatggagat
aagtgtatctgttaggcttaatagtgtgtgccatgtcaattgccttttcgaagtcatctgcctt
catttttaaggtttccacaaaattgataaaacccgtatcagtcagaaattttactacccgctga
tatctgtgttcttgaaccctgctcataagataggttgcaatcccaacctgaattccatgaagct
gaggtgtctccagcagcttatctaaagcatgagatattagatgctcactaccgctggctggagc
actgctgtctgctatctgcatggcaattccgctcattgtcagagagtctaccatttcctttaaa
aagaagttttctgtaacctgtgtgtagggcatccttacaatactgtttactgactttttagcaa
tcattgcagcaaaatcgtcaacctttgccgcattgttcctttcttcaaaataccagtcatacac
agccgtaattttggatattatgtctccgagacctgaataaataaatttcataggtgcatttttt
aatacatctaaatccactaatattccaaatggcatcgaggcatgtacggaagtacgcctgccat
ttataatcaaagagcagcctgagctggaaaaaccatcgtttgaggttgatgtaggtatactgat
aaaaggaagcttgtttaaaaaagctatatatttggctgcatcaagcacctttcctcctcctact
ccgaccactgcatcggttttggagggaatagtaaaagccttgagcataagattttcaagcttta
tgtcatcatagtcgtaagtttcaagtactgcaagagattttcttgactttatggaatccagaat
cttttcaccaaataagtcacgtattccctctccaaaaagtactacaacattactaattcctgcc
ctttcaatatgtgc
序列25
ccaaggtgacaaacgataacttttgagttatttacatctaagccagcaagcgtggttgctcttt
tagaaacatagctgtgacttgttccgtggaaaccatatcttcttaccttatatttatcatagta
ctcaaatggaataccataaaggtaagcttcttttggcattgtctgatggaatgcagtatcaaaa
acagctaccattggtacatttggcataattgatttacaagcgttgataccaataaggtttgctg
ggttgtgtaaaggtgcaagatcattacactcttcaattgcatttaagacttcatcattgattac
tacggaatgagcaaatttctcaccaccatgtactactctatgtccaacagcgttgatttcatct
aaggacttaatcacaccataattttcattcataagagcagcgattacatttttaatagcaacct
catggtttggaagtgcatcctcaagaactaccttctcaccgtcagctgacttgtgagtaagacg
gccatcaataccgattctttcacaaagacctactgctaatgcttgctctgtcacagagtc
序列26
ctgagtgcaatgtaaaaaaggatgcctcaagtattcttgaaacatccttatattatactacaaa
atcataaagtaaattactcagctgtagcaatgatctcttttttgttgtaagatccacaagcttt
acaaactctatgaggcatcataagtgcaccacacttgctgcatttcactaagtttggagcagtc
atcttccagtttgcacgacgactatctcttctagctttggaatgtttattctttggacaaatag
ctcccattgattacacctccttaaacttgttaaaaatatctcggatagcagacattcttgggtc
tagttctgtacggtcacacccgcactctccttcatttaggttagcaccgcagaccttgcagatt
cctttacagtcttctttgcacagaaccttcattgggaaaccaatcaagacttcttcatagataa
gtttatctacgtctaaatcatatccggaaacaaaatttgtttcatctaaatcctcggtacgctg
ttcctctgttttcgatacatcaatctctgtagccacgtcgatgtcttgttggatggtttcttcc
ttcaaacaacgatcgcaaggaacggctaacgctaatttcgtttttgcttccaccagaatttttc
ggccacctagattagttaatctaagtttaaccggttctttataggtaatagaataaccgacacc
atttaattcgaatatatcaaattcaatcggtgcagtgtattctttgagaccattaggaacattc
atgacttcagacatttgtatcagcataagtaactcctgtctaaaaaaacgcataatgtaagcgc
ccaaaaattcacactgttagtattataaacgcttaaaataggtttgtcaactcctaactgttaa
aaatgtcagaattgtgtaaccatattttctcttcattatcgttcttcccttattaaataattta
tagctattgaaaagagataagaattgttcaaagctaatattgtttaaatcgtcaattcctgcat
gttttaaggaattgttaaattgattttttgtaaatattttcttgtattctttgttaacccattt
cataacgaaataattatacttctgtttatctttgtgtgatattcttgatttttttctatttaat
ctgataagtgagctattcactttaggtttaggatgaaaatattctcttggaaccatacttaata
tagaaatatcaacttctgccattaaaaataatgccaatgagcgttttgtatttaataatctttt
agcaaacccgtattccacgattaaataaatctcatcagctatactatcaaaaacaattttgcgt
attatatccgtacttatgttataaggtatattaccaaatattttataggattggtttttaggaa
atttaaactgcaatatatccttgtttaaaacttggaaattatcgtgatcaacaagtttattttc
tgtagttttgcataatttatggtctatttcaatggcagttacgaaattacacctctgtactaat
tcaagggtaaaatgcccttttcctgagccgatttcaaagatattatcatgttcatttaatctta
tatttgtcattattttatctatattatgttttgaagtaataaagttttgactgtgttttatatt
tttctcgttcattgtatttctccttataatgttcttaaattcatttatcacggggcaacttaat
atatccgaaatatagttcttctatatcgttcccccagtataatgattattatactatttaatct
tcaacttaacaattggagtttccagttaagaaataataatttaatgccaaagcggatattcgca
atccgcttacgctacttgctcataacctcaacaggcaatgaagctaagttaattatttactctg
tgcctgaacagcagtgattgcaacaacaccaacgatatcatcagaagaacaacctcttgataaa
tcatttactggagctgcaataccctgagttaatggtccataagcttctgcctttgcaagacgct
gtgttaacttatatccaatgttaccagcatcaaggtctgggaagattaatacgttagcttttcc
agcaatatcactaccaggagcttttgaagcacctacactaggaacgattgctgcatctaactgg
aactcgccgtcgatcttatattctgggtataattcatttgcaatcttagttgcttctacaacct
tatcaacatctgcatgctttgcgcttccctttgttgaatgagaaagcatagctacgataggttc
agagccaactaattgttcaaaactcttcgctgtggaaccagcgattgctgctaactcttcagca
tttggattctgatttaaaccagcatcagagaaaaggaaagttccatttgcgcccatatcacaat
taggtactaccattacgaagaaagcagaaactaacttagtatttggagcagtttttaaaatctg
aagacatggtcttaaggtatctgctgtagagtgacaagcaccagatactaaaccatctgcatcg
cccatcttaaccatcattacaccgtatgtaatgtagtctgttgttaaaagctcttttgcttttt
caggggtcatgccttttgcctgtctaagttctacaagcttgttaatgtaagc
序列27:ldh基因-1389
atggcgattacaataaaccgaagtaaagttattgttgtgggtgcaggtttagttggtacttcaa
cggcgtttagtctaattacgcaaagtgtttgtgatgaggttatgttgatagatatcaatcgtgc
taaggcgcatggggaagtaatggatttgtgtcatagtatcgagtatttaaatcgaaatgttttg
gtaacggaaggagattatacagactgtaaggacgctgatattgttgtaataactgcagggcctc
cgccaaaaccaggacagtcgcggcttgatactcttgggttatccgcagatattgtgagcacgat
tgtggaacctgtcatgaagagtgggttcaatggaatattcttagtcgtgacgaatccggtggat
tcgattgctcaatatgtttatcaattatcggggcttccaaagcaacaagttcttggaactggaa
cagcgattgactctgcaagattaaaacactttattggagatattttacatgtagatcctagaag
catacaggcttatacgatgggagagcatggagattctcaaatgtgtccttggtcgcttgttacg
gttggcggtaaaaatattatggacatcgtacgggataacaaagagtattccgatattgacttta
atgaaatcttatataaggttaccagggtaggttttgatattttatcagtgaagggtactacttg
ttatggaatagcgtcagcagctgtggggattataaaagcaattctttatgatgagaattccatc
cttccggtctctaccttattggagggggaatatggtgagtttgatgtatatgcaggggtaccat
gcattctaaatcgtttcggcgtgaaggatgtagtggaagtaaatatgacagaagtagagttaaa
tcaattccgagcctctgttcacgttgtgagggaagctattgaaaacttaaaagacagagataaa
aaggcattatttttataa
序列28:ldh基因-2971
TTATGATAGCGTCAATGCATACTGAAAATTTTCTTTCATTGTTCTACAAGAAGCATCGAATTTT
CCCTTTTCTTCAGGTGTCAAATTTAGCTCAATGATTTCTTCTACACCATGAATTCCAAGTACCG
TAGGAACAGATGCATAGACATCATGCTGGCCATACTCACCATTTAAGAGAGTAGATACTGGTAA
TACCTTCTTCTCATCTGAGAAAATGGCTCGTGTAACCTCAGCTAGTGATGCACCAATACCAAAT
TCCGTTGAGCCTTTTCCAGTTAGGATATGCCATCCCCCTGCTCTAGCTTCATCAGAAAGCTTAG
AAAGATCAATCTGCCCATATTTTTCAGGTTTTTCCTTGATTAGTTCCAAAATTGGTTTTCCAGC
TATAGATACCGTTGACCATGCAACCATCTGGCTTTCTCCGTGTTCTCCAAGAACAAATCCATAG
ATTGATTTTTGATCAATTTCAACAGCATCTGCAATTGCTCTTCTAAGTCTGGCAGAGTCTAGTA
CCGTACTTGTTGAAATAATTTTATTGGATGAGTACTGAAGTAAATGCTGTAAATAATGTGTTAT
TACATCTGCTGGATTTGAAATGCTAACAATCATACCATCAAAACCTGAATTTTTGATATGCCAA
GCTACCTCTTTAATAATTAGAGCAGTATTCGTAAGGGTACTCATTCTTGTTTCACCCTTATTTT
TATCTGGATTGGTTCCTACTGCAATCACCATGAGATCTGCATCAGCTGCATCACTATAATCACC
CGATTTTACCTTAACTCTGTGTGGTAGGTATACTGTAGCATCGTAGATATCCAGTGCTTGTGCT
TTCGCTTTTTCTCTATCAATATCAATAAAGATAATTTCTTCTGCAAGCCCCTGCTCTGCCAGTG
CATATCCAGCATGAGATCCTACGTGACCTGCTCCGATAATAATGACTTTTCTTGGTTTTGCCAT
序列29
tggaatctcactatgcaccaatgtggtactaaattatatctttatctatggaaaattaggtttt
ccgcgaatggagatagagggagctgccattgctactttaatttgtagaattcttgagagtattt
tagttgttatttatatgtataagggtgagaaggtacttaagatgagactttcttatatttttaa
gagatctaaacagtattttcgctctttggctcgttatagtgcgccagtgcttatgagtgaggtt
aactgggggcttgggattgctgttcagtctgcaatcattgggcgtatgggtgttagttttctta
cagccgccagcttcattaatgtagtacaacagttagccggaatcattctgattggtattggtgt
gggttcgagcattataatagggaatttgattggtgagggaaaagagcatgaggcgagaatgcta
gccaataagttaatacgtatcagtatgatactcggaggaattgttgcttttgcagtaatcttac
tacgtccaatcgctcctaactttattgaggcgtctaaggaaacagcggatttaattcgtcagat
gctatttgtttcggcttacctcttattcttccaagccttatctgtattaactatggccggaata
ttacgtggtgcaggggataccctttactgtgcaacctttgatgttttgaccttatgggtactaa
aacttggaggaggtttgcttgcaaccatagtacttcatcttccacctgtatgggtttactttat
cttaagtagcgatgagtgtgttaaagcgctatttacggtaccgcgggtcttaaagggacgttgg
attcatgatacaacactgcattaagatttcatatgtccagatatttttgcacagtagcataatt
actagagcttattcctataatattcataggttttgatggtccattttacgttacgatagcatat
attacatcaaaaccaattctatataagatgaggttatagtatgaacgagaaaaatataaaacac
agtcaaaactttattacttcaaaacataatatagataaaataatgacaaatataagattaaatg
aacatgataatatctttgaaatcggctcaggaaaagggcattttacccttgaattagtacagag
gtgtaatttcgtaactgccattgaaatagaccataaattatgcaaaactacagaaaataaactt
gttgatcacgataatttccaagttttaaacaaggatatattgcagtttaaatttcctaaaaacc
aatcctataaaatatttggtaatataccttataacataagtacggatataatacgcaaaattgt
ttttgatagtatagctgatgagatttatttaatcgtggaatacgggtttgctaaaagattatta
aatacaaaacgctcattggcattatttttaatggcagaagttgatatttctatattaagtatgg
ttccaagagaatattttcatcctaaacctaaagtgaatagctcacttatcagattaaatagaaa
aaaatcaagaatatcacacaaagataaacagaagtataattatttcgttatgaaatgggttaac
aaagaatacaagaaaatatttacaaaaaatcaatttaacaattccttaaaacatgcaggaattg
acgatttaaacaatattagctttgaacaattcttatctcttttcaatagctataaattatttaa
taagaagtaataggaaataatactcgaattattctgcaatctgttctaaaaaataaaattaaga
aattactatagcaagccaggttaaaattactagcttgctatttttgtgcatttagtacagtttt
gattattaaagaataaatttaataactattttgcaataagttattgactatttcacaagttagt
gttactatacaagtatgaaataaagatacataaaaaaataaataatatgaaacataaattcatg
acatgcggaatagaatgaaagaatattatgtcggttcctaatactaaatggatataacaatcta
ttgaaacacttatggggtgtaagtgtggagagaatttctaaagcgccaaaagactctacatatg
aaattctaaagcttcacacgggaataatctaatttatgtatcttattatcataattcaggaagg
tagtgtgaaaatataaaaattagttttcctgtttcattcaggcagtagcatttcttaaacaaat
ttgctatgcattgggtgttatctgaaaaacaaaaagcaattttctcacaacttatttctgaaca
acaatggtattaaaaatttggaggaggattttactatgaaaaaaacggtaacattactgttggt
tctgaccatggtggtaagcttatttgcagcatgtggtaagaaaaatggatcaagcgaaaccggc
acaaaagatcctgtggcaacaagcggtgcaaaagaacctgacaaacaagatccaggcaataaag
agcctgaaaaacaagaccctgttaaaatcaagatttattactctgataatgcaaccttaccatt
taaagaagattggttagttataaaggaagctgagaagagatttaatgttgatttcgatttcgaa
gtaattccaattgcagattatcaaacaaaagtttctttaacattaaatacaggaaataacgctc
cagatgtcat cctttatcagtcaacgcagggagagaatgcatct
序列30:pMU367的载体序列(C.cell乙酸激酶KO载体)
gaattcgagctcggtacccggggatcctctagagtcgacctgcaggcatgcaagcttggcactg
gccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcag
cacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgcgtagaggatcgagat
ctcgatcccgcgaaattaatacgactcactataggggaattgtgagcggataacaattcccctc
taggctcataacttcacgctcctgtatatatttttatttatttaaaaatgagtcaaaatttagg
aaatgattgcaatatgtataatatccaaatttccattcaaataaccaaagtaattttacctctt
tttatgagctatttcaatactttgttagtaaattaacatatatgagctgtatatggtttaatga
aaaaagttattttgaagggatattgtaaaaaacataatatattatatggataaattttacattt
gacttatcatatgttaatatatgtaatatgaatagctaatctaagcaggctactgcctagaaaa
aagcttataattatccttaatttcctactacgtgcgcccagatagggtgttaagtcaagtagtt
taaggtactactctgtaagataacacagaaaacagccaacctaaccgaaaagcgaaagctgata
cgggaacagagcacggttggaaagcgatgagttacctaaagacaatcgggtacgactgagtcgc
aatgttaatcagatataaggtataagttgtgtttactgaacgcaagtttctaatttcggttgaa
atccgatagaggaaagtgtctgaaacctctagtacaaagaaaggtaagttacagtagtagactt
atctgttatcaccacatttgtacaatctgtaggagaacctatgggaacgaaacgaaagcgatgc
cgagaatctgaatttaccaagacttaacactaactggggataccctaaacaagaatgcctaata
gaaaggaggaaaaaggctatagcactagagcttgaaaatcttgcaagggtacggagtactcgta
gtagtctgagaagggtaacgccctttacatggcaaaggggtacagttattgtgtactaaaatta
aaaattgattagggaggaaaacctcaaaatgaaaccaacaatggcaattttagaaagaatcagt
aaaaattcacaagaaaatatagacgaagtttttacaagactttatcgttatcttttacgtccag
atatttattacgtggcgacgcgttgggaaatggcaatgatagcgaaacaacgtaaaactcttgt
tgtatgctttcattgtcatcgtcacgtgattcataaacacaagtgaatgtcgacagtgaatttt
tacgaacgaacaataacagagccgtatactccgagaggggtacgtacggttcccgaagagggtg
gtgcaaaccagtcacagtaatgtgaacaaggcggtacctccctacttcancatatcattttctg
cagccccctagaaataattttgtttaactttaagaaggagatatacatatatggctagatcgtc
cattccgacagcatcgccagtcactatggcgtgctgctagcgctatatgcgttgatgcaatttc
tatgcactcgtagtagtctgagaagggtaacgccctttacatggcaaaggggtacagttattgt
gtactaaaattaaaaattgattagggaggaaaacctcaaaatgaaaccaacaatggcaatttta
gaaagaatcactaaaaattcacaagaaaatatagacgaagtttttacaagactttatcgttatc
ttttacgtccagatatttattacgtggcgtatcaaaatttatattccaataaaggagcttccac
aaaaggaatattagatgatacagcggatggctttagtgaagaaaaaataaaaaagattattcaa
tctttaaaagacggaacttactatcctcaacctgtacgaagaatgtatattgcaaaaaagaatt
ctaaaaagatgagacctttaggaattccaactttcacagataaattgatccaagaagctgtgag
aataattcttgaatctatctatgaaccggtattcgaagatgtgtctcacggttttagacctcaa
cgaagctgtcacacagctttgaaaacaatcaaaagagagtttggcggcgcaagatggtttgtgg
agggagatataaaaggctgcttcgataatatagaccacgttacactcattggactcatcaatct
taaaatcaaagatatgaaaatgagccaattgatttataaatttctaaaagcaggttatctggaa
aactggcagtatcacaaaacttacagcggaacacctcaaggtggaattctatctcctcttttgg
ccaacatctatcttcatgaattggataagtttgttttacaactcaaaatgaagtttgaccgaga
aagtccagaaagaataacacctgaatatcgggagctccacaatgagataaaaagaatttctcac
cgtctcaagaagttggagggtgaagaaaaagctaaagttcttttagaatatcaagaaaaacgta
aaagattacccacactcccctgtacctcacagacaaataaagtattgaaatacgtccggtatgc
ggacgacttcattatctctgttaaaggaagcaaagaggactgtcaatggataaaagaacaatta
aaactttttattcataacaagctaaaaatggaattgagtgaagaaaaaacactcatcacacata
gcagtcaacccgctcgttttctgggatatgatatacgagtaaggagatctggaacgataaaacg
atctggtaaagtcaaaaagagaacactcaatgggagtgtagaactccttattcctcttcaagac
aaaattcgtcaatttatttttgacaagaaaatagctatccaaaagaaagatagctcatggtttc
cagttcacaggaaatatcttattcgttcaacagacttagaaatcatcacaatttataattctga
actccgcgggatttgtaattactacggtctagcaagtaattttaaccagctcaattattttgct
tatcttatggaatacagctgtctaaaaacgatagcctccaaacataagggaacactttcaaaaa
ccatttccatgtttaaagatggaagtggttcgtgggggatcccgtatgagataaagcaaggtaa
gcagcgccgttattttgcaaattttagtgaatgtaaatccccttatcaatttacggatgagata
agtcaagctcctgtattgtatggctatgcccggaatactcttgaaaacaggttaaaagctaaat
gttgtgaattatgtgggacgtctgatgaaaatacttcctatgaaattcaccatgtcaataaggt
caaaaatcttaaaggcaaagaaaaatgggaaatggcaatgatagcgaaacaacgtaaaactctt
gttgtatgctttcattgtcatcgtcacgtgattcataaacacaagtgagatatctcgagcaccc
gttctcggagcactgtccgaccgctttggccgccgcccagtcctgctcgcttcgctacttggag
ccactatcgactacgcgatcatggcgaccacacccgtcctgtggatcgccaagctcgccgatgg
tagtgtggggtctccccatgcgagagtagggaactgccaggcatcaaataaaacgaaaggctca
gtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggaca
aatccgccgggagcggatttgaacgttgcgaagcaacggcccggagggtggcgggcaggacgcc
cgccataaactgccaggcatcaaattaagcagaaggccatcctgacggatggcctttttgcgtt
tctacaaactcttcctgtcgtcatatctacaagccatcccgcccttcccaacagttgcgcagcc
tgaatggcgaatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccg
catatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccg
ccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctg
tgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacg
aaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacg
tcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacatt
caaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaa
gagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcct
gtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgag
tgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacg
ttttccaatgatgagcacttttaaattaaaaatgaagttttaaaacttcatttttaatttaaat
taaaaatgaagttttatcaaaaaaatttccaataatcccactctaagccacaaacacgccctat
aaaatccccctttaatcccactttgagacacatgtaatattactttacgccctagtatagtgat
aattttttacattcaatgccacgcaaaaaaataaaggggcactataataaaagttccttcggaa
ctaactaaagtaaaaaattatctttacaacctccccaaaaaaaagaacaggtacaaagtaccct
ataatacaagcgtaaaaaaatgagggtaaaaataaaaaaataaaaaaataaaaaaataaaaaaa
taaaaaaaataaaaaaataaaaaaataaaaaaataaaaaaataaaaaaataaaaaaataaaaaa
ataaaaaaatataaaaataaaaaaatataaaaataaaaaaatataaaaataaaaaaatataaaa
ataaaaaaataaaaaaatataaaaataaaaaaataaaaaaatataaaaatattttttatttaaa
gtttgaaaaaaatttttttatattatataatctttgaagaaaagaatataaaaaatgagccttt
ataaaagcccattttttttcatatacgtaatatgacgttctaatgtttttattggtacttctaa
cattagagtaatttctttatttttaaagcctttttctttaagggcttttattttttttcttaat
acatttaattcctctttttttgttgcttttcctttagcttttaattgctcttgataattttttt
tacctctaatattttctcttctcttatattcctttttagaaattattattgtcatatatttttg
ttcttcttctgtaatttctaataactctataagagtttcattcttatacttatattgcttattt
ttatctaaataacatctttcagcacttctagttgctcttataacttctctttcacttaaatgtt
gtctaaacatactattaagttctaaaacatcatttaatgccttctcaatgtcttctgtaaagct
acaaagataatatctatataaaaataatataagctctctgtgtccttttaaatcatattctctt
agttcacaaagttttattatgtcttgtattcttccataatataaacttctttctctataaatat
aatttattttgcttggtctaccctttttcctttcatatggttttaattcaggtaaaaatccatt
ttgtatttctcttaagtcataaatatattcgtactcatctaatatattgactactgtttttgat
ttagagtttatacttcctggaactcttaatattctggttgcatctaaggcttgtctatctgctc
caaagtattttaattgattatataaatattcttgaaccgctttccataatggtaatgctttact
aggtactgcatttattatccatattaaatacattcctcttccactatctattacatagtttggt
ataggaatactttgattaaaataattcttttctaagtccattaatacctggtctttagttttgc
cagttttataataatccaagtctataaacagtgtatttaactcttttatattttctaatcgcct
acacggcttataaaaggtatttagagttatatagatattttcatcactcatatctaaatctttt
aattcagcgtatttatagtgccattggctatatccttttttatctataacgctcctggttatcc
accctttacttctactatgaatattatctatatagttctttttattcagctttaatgcgtttct
cacttattcacctccccttctgtaaaactaagaaaattatatcatattttcaataattattaac
tattcttaaactcttaataaaaaatagagtaagtccccaattgaaacttaatctattttttatg
ttttaatttattatttttattaaaatattttaaactaaattaaatgattctttttaatttttta
ctatttcattccataatatattactataattatttacaaataatatttcttcatttgtaatatt
tagatgatttactaattttagtttttatatattaaataattaatgtataatttatataaaaaat
caaaggagcttataaattatgattatttccaaagatactaaagatttaattttttcaattttaa
caatactttttgtaatattatgtttaaatttaattgtatttttttcatataataaagccgttga
agtaaaccaatccattttccttatgatgttattattaaatttaagttttataataatatcttta
ttatatttattgtttttaaaaaaactagtgaaatttccggctttattaaacttatttttaggaa
ttttattttcattttcatctttacaggatttgattatatctttaaatatgttttatcaaatatt
atctttttctaaatttatatatatttttattatatttattattatatatattttatttttaagt
ttctttctaacagctattaaaaagaaacttaaaaataaaaacacgtactctaaaccaataaata
aaactatttttattattgctgccttgattggaatagtttttagtaaaattaatttcaatattcc
acaatattatattataagctagctttgcattgtacttttcaatcgcttcacgaatgcggttatc
tccgaaagataaagtcttttcatcttccttgatgaagataagattttctccgtctccgccggca
gaattgaagcggggtactacggtatcgtctgcgtcatcttccgttgtctgatagatgatagtca
taggctcattttcttccgtttcggtaaaggggataggttcgccctttgagagcagggcggcgat
ggaaagcattaacttgcttttcccatcgcccggatctccctgcaatagcgtaactttgccaaac
ggaatatacggataccacagccactttacttctttcggctcgatttcacttgccttgatgattt
caagaggtacgctgaaattcatttcgttttcatttagtttcattttttcttgttctccttttct
ctgaaaatataaaaaccacagattgatactaaaaccttggttgtgttgcttttcggggcttaaa
tcaaggaaaaatccttgttttaagcctttcaaaaagaaacacaaggtctttgtactaacctgtg
gttatgtataaaattgtagattttagggtaacaaaaaacaccgtatttctacgatgtttttgct
taaatacttgtttttagttacagacaaacctgaagttgaattcatatttattaaattaagcgta
tatactattgaaaatgtttttgaaatattataaaattaactttggtttaggaaaagtaaccagt
tcttttgtcgataagcattaatttgcttgactaattaataaaaaacttaggaggtaacactaat
ggtattcgagaaaattgacaagaacagttggaacagaaaagaatactttgatcactattttgct
agtgtaccttgcacatacagtatgactgtaaaggttgatataacacagattaaagagaagggaa
tgaaattgtaccctgcaatgctttattacatagcaatgatagtaaacagacatagtgaatttag
gaccgctatcaatcaggatggtgaacttggaatttatgatgaaatgattccatcatatactata
ttccataatgacaccgagacattctcaagtctttggactgaatgcaagtcagattttaagtcat
ttcttgcagattatgaatctgatactcaaagatacggtaataatcaccgtatggaaggaaaacc
taatgcacctgagaatattttcaatgtttccatgataccttggtcaacatttgacggatttaat
ctgaatctgcaaaaaggctacgattacttaatccctatctttacaatgggcaagtattataagg
aagacaataaaatcatccttccccttgcaatccaggtacatcatgcagtatgtgatggatttca
tatttgtcgttttgtaaatgaactgcaagaattaataaattcctaactcgagggcagtagcgcg
gtggtcccacctgaccccatgccgaactcagaagtgaaacgataaaacgaaaggctcagtcgaa
agactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccg
ccgggagcggatttgaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccat
aaactgccaggcatcaaattaagcagaaggccatcctgacggatggccttttttattgtaaatt
ccggtaacccttgtagcttagtgggaatttgtaccccttatcgatacaaattccccgtaggcgc
tagggacactttttcactcgttaaaaagttttgagaatattttatatttttgttcatgtaatca
ctccttcttaattacaaatttttagcatctaatttaacttcaattcctattatacaaaatttta
agatactgcactatcaacacactcttaagtttgcttctaagtcttatttccataacttctttta
cgtttccgggtacaattcgtaatcatgtcatagctgtttcctgtgtgaaattcttatccgctca
caattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgag
ctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccag
aaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaa
atcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatctt
cttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagc
ggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcaga
gcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctg
tagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataa
gtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctga
acggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctac
agcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaag
cggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttat
agtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggc
ggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttt
tgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgag
tgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcgg
aagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggca
cgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcact
cattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcg
gataacaatttcacacaggaaacagctatgaccatgattac
序列31:pMU367的载体序列C.cell LDH2272
gaattcgagctcggtacccggggatcctctagagtcgacctgcaggcatgcaagcttggcactg
gccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcag
cacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgcgtagaggatcgagat
ctcgatcccgcgaaattaatacgactcactataggggaattgtgagcggataacaattcccctc
taggctcataacttcacgctcctgtatatatttttatttatttaaaaatgagtcaaaatttagg
aaatgattgcaatatgtataatatccaaatttccattcaaataaccaaagtaattttacctctt
tttatgagctatttcaatactttgttagtaaattaacatatatgagctgtatatggtttaatga
aaaaagttattttgaagggatattgtaaaaaacataatatattatatggataaattttacattt
gacttatcatatgttaatatatgtaatatgaatagctaatctaagcaggctactgcctagaaaa
aagcttataattatccttagctctcttcaatgtgcgcccagatagggtgttaagtcaagtagtt
taaggtactactctgtaagataacacagaaaacagccaacctaaccgaaaagcgaaagctgata
cgggaacagagcacggttggaaagcgatgagttacctaaagacaatcgggtacgactgagtcgc
aatgttaatcagatataaggtataagttgtgtttactgaacgcaagtttctaatttcgattaga
gctcgatagaggaaagtgtctgaaacctctagtacaaagaaaggtaagttatcattgaagactt
atctgttatcaccacatttgtacaatctgtaggagaacctatgggaacgaaacgaaagcgatgc
cgagaatctgaatttaccaagacttaacactaactggggataccctaaacaagaatgcctaata
gaaaggaggaaaaaggctatagcactagagcttgaaaatcttgcaagggtacggagtactcgta
gtagtctgagaagggtaacgccctttacatggcaaaggggtacagttattgtgtactaaaatta
aaaattgattagggaggaaaacctcaaaatgaaaccaacaatggcaattttagaaagaatcagt
aaaaattcacaagaaaatatagacgaagtttttacaagactttatcgttatcttttacgtccag
atatttattacgtggcgacgcgttgggaaatggcaatgatagcgaaacaacgtaaaactcttgt
tgtatgctttcattgtcatcgtcacgtgattcataaacacaagtgaatgtcgacagtgaatttt
tacgaacgaacaataacagagccgtatactccgagaggggtacgtacggttcccgaagagggtg
gtgcaaaccagtcacagtaatgtgaacaaggcggtacctccctacttcancatatcattttctg
cagccccctagaaataattttgtttaactttaagaaggagatatacatatatggctagatcgtc
cattccgacagcatcgccagtcactatggcgtgctgctagcgctatatgcgttgatgcaatttc
tatgcactcgtagtagtctgagaagggtaacgccctttacatggcaaaggggtacagttattgt
gtactaaaattaaaaattgattagggaggaaaacctcaaaatgaaaccaacaatggcaatttta
gaaagaatcagtaaaaattcacaagaaaatatagacgaagtttttacaagactttatcgttatc
ttttacgtccagatatttattacgtggcgtatcaaaatttatattccaataaaggagcttccac
aaaaggaatattagatgatacagcggatggctttagtgaagaaaaaataaaaaagattattcaa
tctttaaaagacggaacttactatcctcaacctgtacgaagaatgtatattgcaaaaaagaatt
ctaaaaagatgagacctttaggaattccaactttcacagataaattgatccaagaagctgtgag
aataattcttgaatctatctatgaaccggtattcgaagatgtgtctcacggttttagacctcaa
cgaagctgtcacacagctttgaaaacaatcaaaagagagtttggcggcgcaagatggtttgtgg
agggagatataaaaggctgcttcgataatatagaccacgttacactcattggactcatcaatct
taaaatcaaagatatgaaaatgagccaattgatttataaatttctaaaagcaggttatctggaa
aactggcagtatcacaaaacttacagcggaacacctcaaggtggaattctatctcctcttttgg
ccaacatctatcttcatgaattggataagtttgttttacaactcaaaatgaagtttgaccgaga
aagtccagaaagaataacacctgaatatcgggagctccacaatgagataaaaagaatttctcac
cgtctcaagaagttggagggtgaagaaaaagctaaagttcttttagaatatcaagaaaaacgta
aaagattacccacactcccctgtacctcacagacaaataaagtattgaaatacgtccggtatgc
ggacgacttcattatctctgttaaaggaagcaaagaggactgtcaatggataaaagaacaatta
aaactttttattcataacaagctaaaaatggaattgagtgaagaaaaaacactcatcacacata
gcagtcaacccgctcgttttctgggatatgatatacgagtaaggagatctggaacgataaaacg
atctggtaaagtcaaaaagagaacactcaatgggagtgtagaactccttattcctcttcaagac
aaaattcgtcaatttatttttgacaagaaaatagctatccaaaagaaagatagctcatggtttc
cagttcacaggaaatatcttattcgttcaacagacttagaaatcatcacaatttataattctga
actccgcgggatttgtaattactacggtctagcaagtaattttaaccagctcaattattttgct
tatcttatggaatacagctgtctaaaaacgatagcctccaaacataagggaacactttcaaaaa
ccatttccatgtttaaagatggaagtggttcgtgggggatcccgtatgagataaagcaaggtaa
gcagcgccgttattttgcaaattttagtgaatgtaaatccccttatcaatttacggatgagata
agtcaagctcctgtattgtatggctatgcccggaatactcttgaaaacaggttaaaagctaaat
gttgtgaattatgtgggacgtctgatgaaaatacttcctatgaaattcaccatgtcaataaggt
caaaaatcttaaaggcaaagaaaaatgggaaatggcaatgatagcgaaacaacgtaaaactctt
gttgtatgctttcattgtcatcgtcacgtgattcataaacacaagtgagatatctcgagcaccc
gttctcggagcactgtccgaccgctttggccgccgcccagtcctgctcgcttcgctacttggag
ccactatcgactacgcgatcatggcgaccacacccgtcctgtggatcgccaagctcgccgatgg
tagtgtggggtctccccatgcgagagtagggaactgccaggcatcaaataaaacgaaaggctca
gtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggaca
aatccgccgggagcggatttgaacgttgcgaagcaacggcccggagggtggcgggcaggacgcc
cgccataaactgccaggcatcaaattaagcagaaggccatcctgacggatggcctttttgcgtt
tctacaaactcttcctgtcgtcatatctacaagccatcccgcccttcccaacagttgcgcagcc
tgaatggcgaatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccg
catatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccg
ccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctg
tgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacg
aaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacg
tcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacatt
caaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaa
gagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcct
gtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgag
tgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacg
ttttccaatgatgagcacttttaaattaaaaatgaagttttaaaacttcatttttaatttaaat
taaaaatgaagttttatcaaaaaaatttccaataatcccactctaagccacaaacacgccctat
aaaatcccgctttaatcccactttgagacacatgtaatattactttacgccctagtatagtgat
aattttttacattcaatgccacgcaaaaaaataaaggggcactataataaaagttccttcggaa
ctaactaaagtaaaaaattatctttacaacctccccaaaaaaaagaacaggtacaaagtaccct
ataatacaagcgtaaaaaaatgagggtaaaaataaaaaaataaaaaaataaaaaaataaaaaaa
taaaaaaaataaaaaaataaaaaaataaaaaaataaaaaaataaaaaaataaaaaaataaaaaa
ataaaaaaatataaaaataaaaaaatataaaaataaaaaaatataaaaataaaaaaatataaaa
ataaaaaaataaaaaaatataaaaataaaaaaataaaaaaatataaaaatattttttatttaaa
gtttcaaaaaaatttttttatattatataatctttgaagaaaagaatataaaaaatgagccttt
ataaaagcccattttttttcatatacgtaatatgacgttctaatgtttttattggtacttctaa
cattagagtaatttctttatttttaaagcctttttctttaagggcttttattttttttcttaat
acatttaattcctctttttttgttgcttttcctttagcttttaattgctcttgataattttttt
tacctctaatattttctcttctcttatattcctttttagaaattattattgtcatatatttttg
ttcttcttctgtaatttctaataactctataagagtttcattcttatacttatattgcttattt
ttatctaaataacatctttcagcacttctagttgctcttataacttctctttcacttaaatgtt
gtctaaacatactattaagttctaaaacatcatttaatgccttctcaatgtcttctgtaaagct
acaaagataatatctatataaaaataatataagctctctgtgtccttttaaatcatattctctt
agttcacaaagttttattatgtcttgtattcttccataatataaacttctttctctataaatat
aatttattttgcttggtctaccctttttcctttcatatggttttaattcaggtaaaaatccatt
ttgtatttctcttaagtcataaatatattcgtactcatctaatatattgactactgtttttgat
ttagagtttatacttcctggaactcttaatattctggttgcatctaaggcttgtctatctgctc
caaagtattttaattgattatataaatattcttgaaccgctttccataatggtaatgctttact
aggtactgcatttattatccatattaaatacattcctcttccactatctattacatagtttggt
ataggaatactttgattaaaataattcttttctaagtccattaatacctggtctttagttttgc
cagttttataataatccaagtctataaacagtgtatttaactcttttatattttctaatcgcct
acacggcttataaaaggtatttagagttatatagatattttcatcactcatatctaaatctttt
aattcagcgtatttatagtgccattggctatatccttttttatctataacgctcctggttatcc
accctttacttctactatgaatattatctatatagttctttttattcagctttaatgcgtttct
cacttattcacctccccttctgtaaaactaagaaaattatatcatattttcaataattattaac
tattcttaaactcttaataaaaaatagagtaagtccccaattgaaacttaatctattttttatg
ttttaatttattatttttattaaaatattttaaactaaattaaatgattctttttaatttttta
ctatttcattccataatatattactataattatttacaaataatatttcttcatttgtaatatt
tagatgatttactaattttagtttttatatattaaataattaatgtataatttatataaaaaat
caaaggagcttataaattatgattatttccaaagatactaaagatttaattttttcaattttaa
caatactttttgtaatattatgtttaaatttaattgtatttttttcatataataaagccgttga
agtaaaccaatccattttccttatgatgttattattaaatttaagttttataataatatcttta
ttatatttattgtttttaaaaaaactagtgaaatttccggctttattaaacttatttttaggaa
ttttattttcattttcatctttacaggatttgattatatctttaaatatgttttatcaaatatt
atctttttctaaatttatatatatttttattatatttattattatatatattttatttttaagt
ttctttctaacagctattaaaaagaaacttaaaaataaaaacacgtactctaaaccaataaata
aaactatttttattattgctgccttgattggaatagtttttagtaaaattaatttcaatattcc
acaatattatattataagctagctttgcattgtacttttcaatcgcttcacgaatgcggttatc
tccgaaagataaagtcttttcatcttccttgatgaagataagattttctccgtctccgccggca
gaattgaagcggggtactacggtatcgtctgcgtcatcttccgttgtctgatagatgatagtca
taggctcattttcttccgtttcggtaaaggggataggttcgccctttgagagcagggcggcgat
ggaaagcattaacttgcttttcccatcgcccggatctccctgcaatagcgtaactttgccaaac
ggaatatacggataccacagccactttacttctttcggctcgatttcacttgccttgatgattt
caagaggtacgctgaaattcatttcgttttcatttagtttcattttttcttgttctccttttct
ctgaaaatataaaaaccacagattgatactaaaaccttggttgtgttgcttttcggggcttaaa
tcaaggaaaaatccttgttttaagcctttcaaaaagaaacacaaggtctttgtactaacctgtg
gttatgtataaaattgtagattttagggtaacaaaaaacaccgtatttctacgatgtttttgct
taaatacttgtttttagttacagacaaacctgaagttgaattcatatttattaaattaagcgta
tatactattgaaaatgtttttgaaatattataaaattaactttggtttaggaaaagtaaccagt
tcttttgtcgataagcattaatttgcttgactaattaataaaaaacttaggaggtaacactaat
ggtattcgagaaaattgacaagaacagttggaacagaaaagaatactttgatcactattttgct
agtgtaccttgcacatacagtatgactgtaaaggttgatataacacagattaaagagaagggaa
tgaaattgtaccctgcaatgctttattacatagcaatgatagtaaacagacatagtgaatttag
gaccgctatcaatcaggatggtgaacttggaatttatgatgaaatgattccatcatatactata
ttccataatgacaccgagacattctcaagtctttggactgaatgcaagtcagattttaagtcat
ttcttgcagattatgaatctgatactcaaagatacggtaataatcaccgtatggaaggaaaacc
taatgcacctgagaatattttcaatgtttccatgataccttggtcaacatttgacggatttaat
ctgaatctgcaaaaaggctacgattacttaatccctatctttacaatgggcaagtattataagg
aagacaataaaatcatccttccccttgcaatccaggtacatcatgcagtatgtgatggatttca
tatttgtcgttttgtaaatgaactgcaagaattaataaattcctaactcgagggcagtagcgcg
gtggtcccacctgaccccatgccgaactcagaagtgaaacgataaaacgaaaggctcagtcgaa
agactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccg
ccgggagcggatttgaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccat
aaactgccaggcatcaaattaagcagaaggccatcctgacggatggccttttttattgtaaatt
ccggtaacccttgtagcttagtgggaatttgtaccccttatcgatacaaattccccgtaggcgc
tagggacactttttcactcgttaaaaagttttgagaatattttatatttttgttcatgtaatca
ctccttcttaattacaaatttttaccatctaatttaacttcaattcctattatacaaaatttta
agatactgcactatcaacacactcttaagtttgcttctaagtcttatttccataacttctttta
cgtttccgggtacaattcgtaatcatgtcatagctgtttcctgtgtgaaattcttatccgctca
caattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgag
ctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccag
aaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaa
atcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatctt
cttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagc
ggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcaga
gcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctg
tagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataa
gtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctga
acggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctac
agcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaag
cggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttat
agtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggc
ggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttt
tgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgag
tgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcgg
aagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggca
cgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcact
cattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcg
gataacaatttcacacaggaaacagctatgaccatgattac
序列32T.saccharolyticum_YS485_pta
gtgtatacaatatatttcttcttagtaagaggaatgtataaaaataaatattttaaaggaaggg
acgatcttatgagcattattcaaaacatcattgaaaaagctaaaagcgataaaaagaaaattgt
tctgccagaaggtgcagaacccaggacattaaaagctgctgaaatagttttaaaagaagggatt
gcagatttagtgcttcttggaaatgaagatgagataagaaatgctgcaaaagacttggacatat
ccaaagctgaaatcattgaccctgtaaagtctgaaatgtttgataggtatgctaatgatttcta
tgagttaaggaagaacaaaggaatcacgttggaaaaagccagagaaacaatcaaggataatatc
tattttggatgtatgatggttaaagaaggttatgctgatggattggtatctggcgctattcatg
ctactgcagatttattaagacctgcatttcagataattaaaacggctccaggagcaaagatagt
atcaagcttttttataatggaagtgcctaattgtgaatatggtgaaaatggtgtattcttgttt
gctgattgtgcggtcaacccatcgcctaatgcagaagaacttgcttctattgccgtacaatctg
ctaatactgcaaagaatttgttgggctttgaaccaaaagttgccatgctatcattttctacaaa
aggtagtgcatcacatgaattagtagataaagtaagaaaagcgacagagatagcaaaagaattg
atgccagatgttgctatcgacggtgaattgcaattggatgctgctcttgttaaagaagttgcag
agctaaaagcgccgggaagcaaagttgcgggatgtgcaaatgtgcttatattccctgatttaca
agctggtaatataggatataagcttgtacagaggttagctaaggcaaatgcaattggacctata
acacaaggaatgggtgcaccggttaatgatttatcaagaggatgcagctatagagatattgttg
acgtaatagcaacaacagctgtgcaggctcaa
序列33:T.saccharolyticum_YS485_ack
atgaaaattatgaaaatactggttattaattgcggaagttcttcgctaaaatatcaactgattg
aatcaactgatggaaatgtgttggcaaaaggccttgctgaaagaatcggcataaatgattccat
gttgacacataatgctaacggagaaaaaatcaagataaaaaaagacatgaaagatcacaaagac
gcaataaaattggttttagatgctttggtaaacagtgactacggcgttataaaagatatgtctg
agatagatgctgtaggacatagagttgttcacggaggagaatcttttacatcatcagttctcat
aaatgatgaagtgttaaaagcgataacagattgcatagaattagctccactgcacaatcctgct
aatatagaaggaattaaagcttgccagcaaatcatgccaaacgttccaatggtggcggtatttg
atacagcctttcatcagacaatgcctgattatgcatatctttatccaataccttatgaatacta
cacaaagtacaggattagaagatatggatttcatggcacatcgcataaatatgtttcaaatagg
gctgcagagattttgaataaacctattgaagatttgaaaatcataacttgtcatcttggaaatg
gctccagcattgctgctgtcaaatatggtaaatcaattgacacaagcatgggatttacaccatt
agaaggtttggctatgggtacacgatctggaagcatagacccatccatcatttcgtatcttatg
gaaaaagaaaatataagcgctgaagaagtagtaaatatattaaataaaaaatctggtgtttacg
gtatttcaggaataagcagcgattttagagacttagaagatgccgcctttaaaaatggagatga
aagagctcagttggctttaaatgtgtttgcatatcgagtaaagaagacgattggcgcttatgca
gcagctatgggaggcgtcgatgtcattgtatttacagcaggtgttggtgaaaatggtcctgaga
tacgagaatttatacttgatggattagagtttttagggttcagcttggataaagaaaaaaataa
agtcagaggaaaagaaactattatatctacgccgaattcaaaagttagcgtgatggttgtgcct
actaa tgaagaatacatgattgctaaagatactgaaaagattgtaaagagtataaaa
序列34:T.psuedoethanolicus_39E_ack
GCTAATGCTATCGGACCAATTTCTCAAGGTCTTGCAAAACCTATCAATGACTTGTCAAGAGGTT
GTAGTGTAGAAGATATTGTTAATGTTATAGCAATAACTTGTGTACAAGCTCAAGGGGTGCAAAA
ATAACTTTGAGGAGGCAGCGATTATGAAAATTTTAGTCATGAACTGTGGAAGCTCGTCATTAAA
AGTATCAATTGTTAGATATGGATAATGGGAAAGTGCTAGCGAAAGGATTGGCGGAAAGGATAGG
TATCAATGATTCTCTTTTAACTCATCAAGTAGAGGGCAAAGATAAAATAAAAATACAAAAAGAT
ATGAAAAATCATAAAGAAGCTATACAAATTGTTTTAGAGGCTTTAGTAGATAAAGAAATCGGAA
TATTAAAAGATATGAAAGAAATAGATGCAGTAGGACATAGAGTTGTGCACGGGGGAGAGTTTTT
TACTGATTCCGTATTGATTGACGATGAGGTAATCAAAAAATTAGAAGCATGTATTGACCTTGCA
CCTTTGCACAATCCTGCTAATATTGAGGGAATAAAAGCTTGTCGGCAGATAATGCCAGGGGTGC
CAATGGTAGCAGTTTTTGATACGGCTTTCCATCAAACAATGCCAGATTATGCGTATATTTATCC
CATTCCTTATGAATACTACGAAAAATATAGAATAAGAAGATATGGATTCCATGGGACTTCTCAT
AAATATGTATCTTTAAGAGCTGCTGAAATATTAAAGAGGCCTATTGAAGAGTTAAAAATTATTA
CTTGCCATTTAGGGAATGGGTCTAGTATTGCTGCGGTTAAAGGCGGTAAGTCGATAGATACAAG
TATGGGATTTACTCCATTAGAAGGGCTGGCTATGGGTACAAGGTCCGGAAATGTTGATCCTTCA
ATTATAACTTTCTTAATGGAAAAAGAAGGATTGACTGCAGAACAGGTTATAGATATACTTAATA
AGAAATCAGGTGTATACGGAATTTCAGGAATAAGTAATGACTTTAGAGATATAGAAAATGCAGC
TTTTAAAGAAGGGCATAAAAGGGCTATGTTGGCATTAAAAGTTTTCGCTTATAGGGTGAAAAAG
ACAATAGGTTCTTATACAGCTGCTATGGGTGGGGTTGATGTAATTGTGTTTACTGCTGGAGTTG
GAGAAAATGGACCAGAAATGAGAGAGTTTATTTTAGAGGATCTAGAGTTTTTAGGCTTTAAACT
GGACAAAGAGAAGAATAAGGTAAGAGGAAAAGAGGAAATTATATCTACAGAAGATTCAAAAGTT
AAAGTTATGGTTATTCCTACAAATGAAGAATATATGATTGCTAAAGATACTGAAAAATTGGTAA
AAGGTTTAAAGTAG
序列35:T.psuedoethanolicus_39E_pta
ATGGCAGTAATGGATAGTATCATACAAAAGGCTAAAGCTAATAAAAAAAGGATTGTGCTTCCTG
AGGGAAGTGAAGCTCGAACTTTAAAAGCTGCTGAAAAGGTTATTAAAGAAGGTATTGCTGATGT
AGTTTTATTAGGGAAGGAAGAAGAAATAAAAGAAAAAGCAAAGGGATTGGATATCTCGAAAGCA
GAAATTATAGACCCTGAAAAGTCGCCTCTTTTACAAAAATATGCTGAAGAATATTATAATTTGA
GAAAAACCAAAGGAGTTACAGAAGAACAGGCATATCAAATTATGAAAGACCCTATGTACTATGG
GTGCATGATGGTCAAATTAGACGATGTTGATGGTATGGTATCTGGGGCGATTCACGCTACTGCT
GATGTTTTCAGACCGGCTTTTCAAATTGTAAAAACTGCTGCAGGTGTCAAAGTAGTATCCAGCG
CCTTTATAATGGAAGTACCTAATTGTACTTATGGAAGCGATGGAGTATTTATTTTTGCTGATTG
TGCAATAAATCCTAATCCTAATGAAGAGGAATTAGCAGCAATTGCCATTGCTTCTGCCCATACT
GCAAAAGTCCTTGCTGGAATTGAGCCTAGAATTGCTATGCTGTCATTTTCTACTAAAGGAAGTG
CAAACCATGAATTAGTAGATAAGGTGAAAAATGCGACTAAAATCGCAAAAGAATTGGCGCCTGA
TTTGCTAATTGATGGTGAGCTTCAATTAGATGCTGCGATTGTCAAAGAAGTAGGAGAGTTAAAG
GCTCCAGGAAGTCCTGTAGCGGGGAATGCAAATGTGCTTATTTTCCCAGATTTGCAAGCGGGAA
ACATTGGATATAAGCTAGTGCAAAGACTTGCTAAAGCTAATGCTATCGGACCAATTTCTCAAGG
TCTTGCAAAACCTATCAATGACTTGTCAAGAGGTTGTAGTGTAGAAGATATTGTTAATGTTATA
GCAATAACTTGTGTACAAGCTCAAGGGGTGCAAAAATAACTTTGAGGAGGCAGCGATTATGAAA
ATTTTAGTCATGAACTGTGGAAGCTCGTCATTAAAAGTATCAATTGTTAGATATGGATAATGGG
AAAGTGCTAGCGAAAGGATTGGCGGAAAGGATAGGTATCAATGATTCTCTTTTAACTCATCAAG
TAGAGGGCAAAGATAAAATAAAAATACAAAAAGATATGAAAAAT
序列36:Thermoanaerobacter_sp._strain_59_pta
gtgtatacaatatatttcttctttttagtaagaggaatgtataaaaataaatattttaaaggaa
gggacgatcttatgagcattattcaaaacatcattgaaaaagctaaaagtgataaaaagaaaat
tgttctgccggaaggtgcagaacccagaacattaaaagctgctgaaatagttttaaaagaagga
attgcagatttggtgcttcttggaaatgaagatgagataagaaatgctgcaaaagacttggaca
tatctaaagctgaaatcattgatcctgtaaaatctgaaatgtttgataggtatgctaatgattt
ttatgagttaaggaagagcaaaggaatcacgttggaaaaagccagagaaacaatcaaggataat
atctattttggatgtatgatggttaaagaaggttatgctgatggattggtatctggcgctattc
atgctactgcagatttattaagacctgcatttcagataattaaaacggctccaggagcaaagat
agtatcaagcttttttataatggaagtgcctaattgtgaatatggtgaaaatggtgtattcttg
tttgctgattgcgcggtcaacccatcgcctaatgcagaagaacttgcttctattgctgtacaat
ctgctaatactgcaaagaatttgttgggctttgaaccaaaagttgctatgctatcattttccac
aaaaggtagtgcatcacatgaattagtagataaagtaagaaaagcgacagaaatagcaaaagaa
ttgatgccagatgttgctatcgacggtgaattgcaattggatgctgctcttgtcaaagaagttg
cagagctaaaagcgccaggaagcaaagttgcgggatgtgcaaatgtgcttatattccctgattt
acaagctggtaatataggatataagcttgtacagagattagctagcaaatgcaattggacctat
aacacaggaatgggtgcaccggttaatgatttatcaagaggatgcagctatagagatattgttg
acgtaatagcacacagctgtacaggctca
序列37:Thermoanaerobacter_sp._strain_59_ack
atgctaacggagaaaaatcaagataaaaaaagacatgaaagatcacaaagacgcaataaaattg
ttttagatgctttggtaagcagtgactacggcgttataaaggatatgtctgagatagatgctgt
aggacatagagttgttcacggaggagaatcttttacatcatcagttctcataaatgatgatgtg
ttaaaagcgataacagattgcatagaattagctccactgcacaatcctgccaatatagaaggaa
ttaaagcttgccagcaaatcatgccaaacgttccaatggtggcggtatttgatacagcctttca
tcagacaatgcctgattatgcatatctttatccaataccttatgaatactacacaaagtacagg
atcagaagatatggatttcatggcacatcgcataaatatgtttcaaatagggctgcagagattt
taaataaacctattgaagatttgaaaatcataacttgtcatcttggaaatggctccagcattgc
tgctgtcaaatatggtaaatcaattgacacaagcatgggatttacaccattagaaggtttggct
atgggtacacgatctggaagcatagacccatccattatttcgtatcttatggaaaaagaaaata
taagcgctgaagaagtagtaaatatattaaataaaaaatctggtgtttacggtatttcaggaat
aagcagcgattttagagacttagaagatgccgcctttaaaaatggagatgaaagagctcagttg
gctttaaatgtgtttgcatatcgagtaaagaagatgattggcgcttatgcagcagctatgggag
gcgtcgatgccattgtatttacagcaggtgttggtgaaaatggtcctgagatacgagaatttat
acttgatggattagagttcttagggttcagcttggataaagaaaaaaataaagtcagaggaaaa
gaaactattatatctacgccgaattcaaaagttagcgtgatggttgtgcccactaatgaagaat
acatgattgctaaagatactgaaaagattgtaaagagtataaaa
序列38:T.saccharolyticum_B6A-RI_pta
gtgtatacaatatatttcttctttttagtaagaggaatgtataaaaataaatattttaaaggaa
gggatgatcttatgagcattattcagaacatcattgaaaaagctaaaagcgataaaaagaaaat
tgttctgccagaaggtgcagaacccaggacattaaaagctgctgaaatagttttaaaagaagga
attgcagatttggtgcttcttggaaatgaagatgagataagaaatgcagcaaaagacttggaca
tatccaaagctgaaataattgaccctgtaaaatctgaaatgtttgataggtatgctaatgattt
ttacgaattaagaaagagcaagggaatcacattggaaaaagccagagaaacaatcaaggataat
atctattttggatgtatgatggttaaagaaggttatgctgatggattagtatctggcgctattc
atgctactgcagatttattaagacctgcatttcagataattaaaacagctccaggagcaaagat
agtatcaagcttttttataatggaagtgcctaattgtgaatatggtgaaaatggcgtattcttg
tttgctgattgtgcggtcaatccatcacctaatgcagaagaacttgcttctattgctgtacaat
ctgctaatactgcaaagaatttgttgggttttgaaccaaaagttgccatgctatcattttccac
aaaaggtagtgcatcacatgaattagtagacaaggtaagaaaagcgacagagatagcaaaggat
ttgatgccagatgttgctatcgatggtgaattgcaactggatgctgctattgttaaagaagttg
cagagctaaaagcaccgggaagcaaagttgcgggatgtgcaaatgtgcttatattccctgactt
acaagctggtaatataggatataagcttgtacagagattagctaaggcaaatgcaattggaccg
ataacgcaaggaatgggtgcaccagttaatgatttatcaagaggatgcagctataaagatattg
ttgacgtaatagcgacaacagctgtgcaggctcaa
序列39:T.saccharolyticum_B6A-RI_ack
atgaaaactatgaaaattctggttattaattgtggaagttcttcactaaaatatcaattgattg
aatcaattgatggaaatgtgctggcaaaaggccttgctgaaagaatcggcataaatgattccct
gttgacgcataatgctaacggagaaaaaatcaagataaaaaaagacatgaaagatcacaaagac
gcaataaaattggttttagatgctttggtaagtagcgactacggcgttataaaggatatgtctg
agatagatgctgtaggacatagagttgttcatggaggagagtcttttacatcatcagttcttat
aaatgatgaagtgttaaaggcaataacagattgtatagaattagctccactgcataatcctgct
aatatagaaggaattaaagcttgccagcaaatcatgccaaacgttccaatggtggcggtatttg
atacagcctttcatcaaacaatgcctgattatgcatatctttatccaataccttatgagtacta
cacaaagtacaggatcagaagatatggatttcatggcacgtcgcataaatatgtttcaagtagg
gctgcagagattttgaataaacctattgaagatttgaaaatcataacttgtcatcttggaaatg
gctccagtattgctgccgtcaaatatggtaaatcaattgacacaagcatgggatttacaccatt
agaaggtttggctatgggtacacgatctggaagtatagacccatccatcatttcttatcttatg
gaaaaagaaaatataagtgctgaagaggtagtaaatatattaaataaaaaatctggtgtttacg
gtatttcgggaataagcagcgattttagagatttagaagatgctgcctttaaaaatggagatga
aagagctcagttggccttaaatgtgtttgcatatcgagtaaagaagacgattggagcttatgca
gcagctatgggaggcgttgatgtcattgtatttacggcaggtgttggtgaaaatgggcctgaga
taagagaatttatacttgatggattggagttcttagggttcagcttggataaagaaaaaaataa
agtcagaggaaaggaaactattatatctacgccaaattcaaaaattagcgtgatggttgtgccg
actaatgaagaatatatgattgctaaagatactgaaaagattgtaaagagtataaaa
序列40:T.saccharolyticum_YS485
atgagcaaggtagcaataataggatctggttttgtaggtgcaacatcggcatttacgctggcat
taagtgggactgtgacagatatcgtgctggtggatttaaacaaggacaaggctataggcgatgc
actggacataagccatggcataccgctaatacagcctgtaaatgtgtatgcaggtgactacaaa
gatgtgaaaggcgcagatgtaatagttgtgacagcaggtgctgctcaaaagccgggagagacac
ggcttgaccttgtaaagaaaaatacagccatatttaagtccatgatacctgagcttttaaagta
caatgacaaggccatatatttgattgtgacaaatcccgtagatatactgacgtacgttacatac
aagatttctggacttccatggggcagagtttttggttctggcaccgttcttgacagctcaaggt
ttagataccttttaagcaagcactgcaatatagatccgagaaatgtccacggaaggataatcgg
cgagcatggtgacacagagtttgcagcatggagcataacaaacatatcgggtatatcatttaat
gagtactgcagcatatgcggacgcgtctgcaacacaaatttcagaaaggaagtagaagaagaag
tcgtaaatgctgcttacaagataatagacaaaaaaggtgctacatactatgctgtggcagttgc
agtaagaaggattgtggagtgcatcttaagagatgaaaattccatcctcacagtatcatctcca
ttaaatggacagtacggcgtgaaagatgtttcattaagcttgccatctatcgtaggcaggaatg
gcgttgccaggattttggacttgcctttatctgacgaagaagtggagaagtttaggcattcagc
aagtgtcatggcagatgtcataaaacaattagatata
序列41:Thermoanaerobacter_sp._菌株59
atgagtaaagtggccataataggttcaggatttgtaggtgctacatctgcatttacattggctc
taagtgggactgtgacagacattgttttagtagatttaaacaaggacaaggcgataggcgatgc
actggatattagccacggtataccgcttatacagcctgtaaatgtgtatgctggcgactacaag
gatatcgagggcgcagatgtagtagttgtaacagcaggtgcggctcaaaagccaggagagtcta
ggctggaccttgtaaaaaagaatacatctatattcaagtccatgatacctgaacttttaaaata
caatgataaagctatatacctgattgtaacaaatcctgttgatatattaacgtatgttacatac
aaaatagcgaaacttccgtgggggcgtgtattcggttcaggtactgtccttgacagttcccgat
ttaggtatcttttaagtaaacattgcaatattgatcctagaaatgtacatggaaggataattgg
agaacacggcgatacagaatttgcggcgtggagcataacaaatatttcaggaatatcatttaat
gagtactgcaatttgtgcggacgagtttgtaatacaaatttcagaaaggaagtggaagatgaag
ttgtcaatgcggcttacaaaattattgataaaaagggtgccacgtattacgctgtggctgtagc
agtaagaagaatagttgagtgtatcataagggatgaaaattcaattcttacagtttcatctcca
ttaaatggtcaatacggtgtaagagatgtatctttaagcttgccatcaattgtgggcaaaaatg
gtgttgcaagggttctggatttgcctttggctgatgacgaagttgagaagtttaaacattcggc
aagcgttatggctgatgttataaaacagttggacata
序列42:T.pseudoethanolicus_39E
ATGAACAAAATATCTATAATAGGTTCTGGATTTGTCGGTGCTACTACTGCATACACACTGGCTT
TGAGTGGGATTGCCAAAACTATTGTATTAATAGATATTAATAAAGACAAAGCAGAAGGCGATGC
TCTTGATATAAGCCACGGCGTACCGTTTATTAGTCCAGTTGAATTGTACGCGGGAGATTATAGT
GATGTTTCAGGTTCTGACATAATAATCATTACAGCGGGAGCAGCACAAAAACCGGGAGAAACCA
GACTTGACTTAGTGAAGAGAAATACGATGATTTTTAAAGACATAGTGGCAAAACTTATTAAAGT
AAATGACACAGCAATATACCTTATAGTTACAAATCCAGTAGATATTCTTACATACGTTACCTAT
AAAATATCTGGCTTGCCATACGGAAGAGTATTGGGGTCTGGCACAGTTCTCGACAGTGCGAGAT
TCAGATATCTTTTAAGCAAACATTGTAACATAGATCCGAGGAATATACACGGATATATAATTGG
GGAGCATGGCGATTCTGAGCTTGCAGCTTGGAGCATTACGAACATAGCAGGCATACCAATTGAT
AATTACTGCAATTTATGTGGAAAAGCATGTGAAAAAGATTTTAGAGAGGAGATTTTTAATAATG
TTGTAAGAGCTGCCTATACGATAATAGAAAAAAAGGGTGCGACATATTATGCGGTTGCTCTCGC
AGTAAGAAGAATCGTAGAAGCTATTTTCAGAGATGAAAATTCCATTTTGACTGTGTCATCTCCG
CTAACCGGCCAATATGGTGTTACAAATGTGGCTTTGAGCCTTCCCTCCGTTGTTGGACGAAATG
GAATCGTAAATATACTTGAATTACCACTTTCACAGGAAGAAATTGCTGCTTTTAGAAGATCAGC
CGAAGTTATCAAAAGTGTAATACAAGAGCTTGATATATAA
序列43:T.saccharolyticum_B6ARI
AGGCGATGCACTGGACATAAGCCATGGCATACCATTAATACAGCCTGTAAATGTGTATGCAGGT
GACTACAAAGATGTTGAAGGCGCGGACGTAATAGTTGTGACAGCAGGGGCTGCTCAAAAGCCAG
GTGAGACGAGGCTTGACCTTGTGAAGAAAAATACAGCTATATTTAAGTCCATGATACCTGAGCT
TTTAAAGTACAATGACAAGGCTATATATTTGATTGTCACAAATCCTGTAGACATACTGACGTAC
GTTACATACAAGATATCTGGACTTCCATGGGGCAGAGTTTTCGGTTCTGGCACTGTTCTTGACA
GTTCAAGGTTTAGGTACCTTTTAAGCAGGCACTGCAATATAGATTCCGAGAAATGTCCACGGAA
GGATAATCGGCGAGCATGGTGACACAGAGTTTGCAGCATGGAGCATAACAAACATATCTGGAAT
ATCATTTAATGAGTACTGCAGCATATGCGGGCGCATCTGCAACACAAATTTCAGAAAGGAAGTA
GAAGAAGAAGTCGTAAATGCTGCTTATAAGATAATAGACAAAAAAGGTGCTACATACTATGCTG
TCGCAGTTGCAGTAAGAAGGATTGTGGAGTGCATCTTAAGAGATGAAAATTCCAT
序列44:T.sacch._pfl
ATGATCAATGAATGGCGCGGGTTTCAGGAGGGCAAATGGCAAAAGACTATTGACGTTCAAGATT
TTATCCAGAAAAATTACACATTATACGAAGGCGATGATAGTTTTTTAGAAGGGCCTACAGAAAA
GACTATTAAGCTTTGGAACAAAGTTCTTGAGCTAATGAAGGAAGAACTGAAAAAAGGTGTGTTA
GATATTGATACAAAAACTGTATCGTCTATAACATCCCATGATGCGGGGTATATAGACAAAGATC
TTGAGGAAATAGTTGGATTGCAGACAGACAAACCTCTTAAAAGAGCTATAATGCCTTACGGTGG
CATAAGAATGGTCAAAAAAGCTTGCGAAGCTTATGGATATAAAGTGGACCCAAAAGTAGAAGAG
ATATTTACGAAGTACAGAAAGACCCACAATGATGGTGTATTTGATGCATATACTCCAGAAATAA
GAGCAGCAAGACATGCCGGCATAATAACAGGTCTTCCAGATGCATATGGCAGAGGAAGAATCAT
AGGTGATTACAGAAGAGTTGCTCTTTATGGAATTGATAGACTCATCGAAGAAAAGGAAAAAGAA
AAACTTGAGCTTGATTACGATGAATTTGATGAAGCAACTATTCGCTTGAGAGAAGAATTGACAG
AACAGATAAAAGCATTAAACGAAATGAAAGAGATGGCTTTAAAGTACGGTTATGACATATCAAA
GCCTGCAAAAAATGCAAAAGAAGCTGTGCAGTGGACTTACTTTGCCTTCCTTGCTGCTATAAAG
GAACAAAATGGTGCCGCTATGTCGCTGGGCAGAGTATCTACTTTTTTAGATATATACATTGAAA
GAGATCTTAAAGAAGGAACATTGACAGAGAAACAAGCACAAGAGTTAATGGATCACTTTGTCAT
GAAGCTTAGAATGGTGAGGTTCTTAAGGACTCCTGATTACAATGAACTATTTAGTGGCGATCCT
GTTTGGGTGACTGAATCAATTGGCGGTGTAGGCGTAGACGGAAGACCTCTTGTCACTAAAAATT
CATTCAGGATATTAAATACTTTATATAACTTAGGTCCTGCACCTGAGCCAAACTTGACGGTTTT
ATGGTCCAAAAACCTTCCTGAAAACTTTAAAAGATTCTGTGCCAAGGTATCAATAGATACAAGT
TCTATTCAATATGAAAATGACGACTTAATGAGGCCAATATACAATGACGACTATAGCATCGCCT
GCTGTGTGTCAGCTATGAAGACGGGAGAACAGATGCAATTTTTTGGAGCAAGGGCAAATCTCGC
GAAGGCGCTACTGTATGCTATAAACGGCGGTATCGATGAAAGGTATAAAACGCAAGTGGCACCA
AAATTTAATCCTATAACGTCTGAGTATTTAGACTACGATGAGGTAATGGCAGCATATGACAATA
TGTTAGAGTGGCTTGCAAAAGTGTATGTTAAAGCTATGAATATAATACACTACATGCACGATAA
ATACGCTTATGAAAGATCCCTTATGGCTTTGCATGATAGAGACATCGTAAGGACGATGGCTTTT
GGAATCGCAGGTCTTTCTGTTGCGGCAGATTCGTTAAGCGCCATAAAGTATGCTAAAGTAAAAG
CCATAAGAGATGAAAATGGCATAGCAATAGATTATGAAGTGGAAGGAGATTTCCCTAAGTTTGG
CAATGATGATGACAGGGTTGACTCAATAGCAGTTGACATTGTAGAAAGATTCATGAATAAGCTT
AAAAAGCACAAGACTTACAGAAACTCTATACCAACACTGTCTGTTTTGACAATAACGTCAAATG
TGGTGTACGGCAAAAAGACGGGTGCTACACCTGACGGAAGAAAAGCGGGAGAACCTTTTGCGCC
AGGCGCAAATCCGATGCACGGCAGAGATACAAAAGGTGCCATAGCATCAATGAATTCAGTATCA
AAAATACCTTATGACAGTTCATTGGATGGTATATCATACACATTTACGATTGTACCAAATGCGC
TTGGCAAGGATGACGAAGATAAAATTAATAATCTTGTAGGACTATTAGATGGATATGCATTTAA
TGCGGGGCACCACATAAACATCAATGTTTTAAACAGAGATATGTTGCTTGATGCTATGGAGCAT
CCTGAAAAATATCCGCAGCTTACTATAAGGGTTTCAGGGTATGCTGTCAATTTCAATAAATTAA
CGAGAGAGCAACAGTTGGAGGTTATATCCCGCACTTTTCACGAATCTATGTAG
序列45:c.therm._pfl
ATGGATGCATGGCGCGGATTTAATAAAGGCAACTGGTGCCAGGAAATTGACGTTCGTGATTTTA
TAATTAGAAATTATACTCCTTATGAAGGCGATGAAAGCTTTCTTGTAGGACCTACGGATAGAAC
GCGGAAACTTTGGGAGAAGGTTTCCGAACTGTTAAAGAAAGAACGGGAGAACGGCGGGGTATTG
GATGTTGATACCCATACAATTTCAACGATTACGTCTCATAAACCTGGATATATAGATAAAGAAC
TTGAAGTTATTGTCGGGCTTCAGACGGATGAGCCTTTAAAAAGAGCCATAATGCCGTTTGGCGG
TATACGTATGGTGATTAAGGGAGCCGAAGCTTATGGCCACAGTGTGGACCCTCAGGTTGTTGAA
ATATTCACAAAGTACAGAAAGACTCATAACCAGGGAGTTTATGATGTATATACTCCCGAAATGA
GAAAAGCCAAAAAAGCCGGGATTATTACAGGACTTCCCGACGCATACGGCAGAGGAAGAATAAT
TGGCGATTACAGAAGGGTTGCACTTTATGGCGTTGACAGGCTGATTGCTGAAAAAGAGAAAGAA
ATGGCAAGTCTTGAAAGAGATTACATTGACTATGAGACTGTTCGAGACAGAGAAGAAATAAGCG
AGCAGATTAAATCTTTAAAACAACTTAAAGAAATGGCTTTAAGTTACGGTTTTGACATATCTTG
TCCTGCAAAGGATGCCAGAGAAGCCTTTCAATGGTTGTATTTTGCATATCTTGCAGCAGTCAAG
GAACAGAACGGCGCGGCAATGAGTATTGGAAGAATTTCGACTTTCCTTGACATATACATTGAAA
GGGATCTCAAAGAAGGAAAACTCACGGAGGAGTTGGCTCAGGAACTGGTTGACCAGCTGGTTAT
AAAGCTGAGAATTGTGAGATTTTTGAGAACTCCTGAGTATGAAAAGCTCTTCAGCGGAGACCCC
ACTTGGGTAACCGAAAGTATCGGAGGTATGGCGCTGGATGGAAGAACGCTGGTTACAAAATCTT
CGTTCAGGTTTTTGCACACTCTTTTCAACCTGGGACATGCACCGGAGCCCAACCTTACAGTACT
TTGGTCCGTCAATCTTCCCGAAGGCTTTAAAAAGTACTGTGCAAAGGTATCAATTCATTCAAGC
TCCATCCAGTATGAAAGCGACGACATAATGAGGAAACACTGGGGAGACGATTATGGAATAGCAT
GCTGTGTTTCTGCTATGAGAATTGGAAAACAGATGCAGTTCTTCGGTGCAAGATGCAATCTTGC
AAAAGCTCTTCTTTACGCTATTAACGGCGGAAAGGATGAAATGACGGGAGAACAGATTGCTCCG
ATGTTTGCACCGGTGGAAACCGAATACCTTGATTACGAGGACGTAATGAAGAGGTTTGACATGG
TGCTTGACTGGGTGGCAAGGCTTTATATGAACACCCTCAATATAATTCACTACATGCATGACAA
ATATGCCTATGAGGCGCTGCAGATGGCATTGCATGACAAAGACGTGTTCAGGACGATGGCATGC
GGAATAGCCGGTTTGTCTGTGGTGGCAGACTCCCTTAGCGCGATAAAATATGCAAAGGTTAAAC
CGATACGCAATGAAAACAACCTCGTTGTTGACTACGAAGTTGAGGGTGATTATCCTAAATTCGG
AAATAACGACGAACGTGTTGATGAAATTGCAGTGCAAGTAGTAAAAATGTTCATGAACAAGCTT
AGAAAGCAAAGGGCTTACAGAAGTGCCACTCCGACCCTTTCCATACTTACCATAACTTCAAACG
TGGTATATGGAAAGAAAACCGGAAACACTCCTGACGGCAGAAAAGCTGGAGAACCTTTGGCGCC
GGGAGCAAATCCGATGCATGGAAGGGATATAAACGGAGCATTGGCTGTACTGAACAGTATTGCG
AAGCTTCCCTATGAATATGCCCAGGACGGCATTTCATATACTTTCTCCATAATTCCAAAAGCTC
TGGGAAGAGACGAGGAAACCAGAATAAACAATCTTAAATCAATGCTTGACGGATATTTCAAGCA
GGGCGGCCACCACATAAATGTAAATGTGTTTGAAAAAGAGACACTGTTAGATGCCATGGAACAT
CCGGAAAAATATCCACAACTTACCATAAGAGTGTCCGGGTATGCAGTGAACTTTATAAAGCTTA
CACGGGAGCAACAGCTGGATGTTATTAACAGAACGATTCACGGAAAGATTTAA
序列46:C.phyto._pfl
ATGATGACTTCAGTTATGAAACAGGAATGGGAAGGTTTTAAACAAGGTAGATGGATCACTTCAG
TAAATGTTCGAGACTTCATACAGAACAATTACACAATGTATGATGGTGATGAATCCTTTTTAGC
AGGTCCAACCGAAGCCACCAATAAACTATGGGCCCAGGTTATGGAGCTTTCAAAGCAGGAAAGT
GAGAAAGGTGGAGTCCTTGATATGGACACCAAGATAGTATCTACTATTGTTTCTCACGGTCCTG
GTTATTTAGATAAAGATATTGAAACAATTGTTGGTTTTCAGACCGATAAGCCATTTAAGAGATC
ACTACAGGTCTTTGGTGGTATTCGTATGGCACAGAGTGCTTGCCATGAATATGGATATGAGGTA
GACGAAGAGGTAGCACGTATTTTTACAGACTACCGCAAGACACATAATCAAGGTGTATTTGATG
CATACACTGACGAAATGAAGCTCGCTAGAAAATCAGCAATCATTACTGGTTTGCCTGATGCTTA
TGGTAGAGGTAGAATTATTGGCGATTACCGTCGAGTGGCACTTTACGGTACTGATTTACTTATT
GAAGACAAGAAAGAACAACTTACAACTTCCTTAAAGAGAATGACTAGTGATAATATTCGCTTAA
GAGAAGAATTAGCAGAACAAATTCGTGCATTAAAAGAATTAGCGAAGCTTGGTGAAATCTATGG
TTACGATATTACGAAGCCAGCAATAAATGCAAAGGAAGCAATTCAGTGGCTTTACTTTGGATAT
CTTGCAGCGGTAAAAGAGCAAAACGGTGCTGCAATGAGCTTAGGCCGTACTTCTACATTCCTTG
ATATTTATATCCAGAGAGATTTAGATAATGGTGTTATCACAGAAAAAGAAGCACAAGAGTATAT
CGATCATTTTATTATGAAACTTCGTCTAGTGAAGTTTGCAAGAACTCCAGAATACAATGCCTTA
TTCTCCGGTGACCCTACTTGGGTAACAGAAAGTATCGCTGGGATTGGTACAGATGGACGCCATA
TGGTAACAAAGACATCCTTCCGTTACCTTCATACGTTAGACAACCTTGGAACTGCTCCAGAACC
AAACATGACAGTTCTATGGTCAACTAGATTACCAAGATTATTTAAAGAGTACTGTGCTAAGATG
TCAATTAAGTCATCCTCTATTCAATACGAAAATGATGATATCATGCGTCCAACTCATGGTGATG
ATTATGCAATTGCTTGTTGTGTATCCTCTATGAAAATTGGTAAAGAGATGCAGTTCTTTGGAGC
ACGTGCAAATCTTGCTAAGTGTCTTCTTTACGCAATCAATGGTGGTGTAGATGAAGTTCTTAAA
ATTCAGGTTGGTCCAAAGTACCGTCCAGTTGAGGGTGAATACCTTAATTATGAGGACGTAATGT
CGAAATACAAAGATATGATGGAGTGGCTAGCAGAACTTTATGTGAATACTTTAAATGTAATCCA
CTACATGCATGATAAATATAGCTATGAAAGAATTCAAATGGCACTTCATGATCGTGAAGTAAAA
CGTTACTTTGCAACTGGTATTGCGGGTCTTTCTGTTGTAGCGGACTCTTTAAGTGCAATTAAGT
ATGCTAAGGTAAAAGTAATTCGTGATGAGAATGGCGTTGTAACCGATTACGAAATTGAAGGTGA
TTATCCAAAGTACGGCAACAATGATGATCGTGTAGACGATATCGCTGTACAGTTAGTGCATGAC
TTTATGAACATGATTCGCAAGCATCATACTTATCGTGATGGATACCCAACGATGTCAATCTTAA
CGATAACTTCTAATGTAGTTTATGGAAAGAAGACAGGTAATACTCCAGACGGACGTAAGAAGGG
TGAACCATTAGCACCAGGTGCTAACCCAATGCATCGTCGTGATACTCATGGTGCAGCAGCGTCC
CTAGCATCGGTAGCAAAGCTTCCATTCCGTGATGCGCAGGATGGTATTTCTAATACGTTCTCTA
TTGTACCAGGAGCATTAGGTAAGAATGATGTGTTATTTGCTGGAGACTTAGATTTAGACGATAT
GTCTGAGAACTAA
序列47:T.sacch.pfl KO单交换质粒
agagctataatgccttacggtggcataagaatggtcaaaaaagcttgcgaagcttatggatata
aagtggacccaaaagtagaagagatatttacgaagtacagaaagacccacaatgatggtgtatt
tgatgcatatactccagaaataagagcagcaagacatgccggcataataacaggtcttccagat
gcatatggcagaggaagaatcataggtgattacagaagagttgctctttatggaattgatagac
tcatcgaagaaaaggaaaaagaaaaacttgagcttgattacgatgaatttgatgaagcaactat
tcgcttgagagaagaattgacagaacagataaaagcattaaacgaaatgaaagagatggcttta
aagtacggttatgacatatcaaagcctgcaaaaaatgcaaaagaagctgtgcagtggacttact
ttgccttccttgctgctataaaggaacaaaatggtgccgctatgtcgctgggcagagtatctac
ttttttagatatatacattgaaagagatcttaaagaaggaacattgacagagaaacaagcacaa
gagttaatggatcactttgtcatgaagcttagaatggtgaggttcttaaggactcctgattaca
atgaactatttagtggcgatcctgtttgggtgactgaatcaattggcggtgtaggcgtagacgg
aagacctcttgtcactaaaaattcattcaggatattaaatactttatataacttaggtcctgca
cctgagccaaacttgacggttttatggtccaaaaaccttcctgaaggtcaatctatgaaatgcg
attaagcttggctgcaggtcgataaacccagcgaaccatttgaggtgataggtaagattatacc
gaggtatgaaaacgagaattggacctttacagaattactctatgaagcgccatatttaaaaagc
taccaagacgaagaggatgaagaggatgaggaggcagattgccttgaatatattgacaatactg
ataagataatatatcttttatatagaagatatcgccgtatgtaaggatttcagggggcaaggca
taggcagcgcgcttatcaatatatctatagaatgggcaaagcataaaaacttgcatggactaat
gcttgaaacccaggacaataaccttatagcttgtaaattctatcataattgtggtttcaaaatc
ggctccgtcgatactatgttatacgccaactttcaaaacaactttgaaaaagctgttttctggt
atttaaggttttagaatgcaaggaacagtgaattggagttcgtcttgttataattagcttcttg
gggtatctttaaatactgtagaaaagaggaaggaaataataaatggctaaaatgagaatatcac
cggaattgaaaaaactgatcgaaaaataccgctgcgtaaaagatacggaaggaatgtctcctgc
taaggtatataagctggtgggagaaaatgaaaacctatatttaaaaatgacggacagccggtat
aaagggaccacctatgatgtggaacgggaaaaggacatgatgctatggctggaaggaaagctgc
ctgttccaaaggtcctgcactttgaacggcatgatggctggagcaatctgctcatgagtgaggc
cgatggcgtcctttgctcggaagagtatgaagatgaacaaagccctgaaaagattatcgagctg
tatgcggagtgcatcaggctctttcactccatcgacatatcggattgtccctatacgaatagct
tagacagccgcttagccgaattggattacttactgaataacgatctggccgatgtggattgcga
aaactgggaagaagacactccatttaaagatccgcgcgagctgtatgattttttaaagacggaa
aagcccgaagaggaacttgtcttttcccacggcgacctgggagacagcaacatctttgtgaaag
atggcaaagtaagtggctttattgatcttgggagaagcggcagggcggacaagtggtatgacat
tgccttctgcgtccggtcgatcagggaggatatcggggaagaacagtatgtcgagctatttttt
gacttactggggatcaagcctgattgggagaaaataaaatattatattttactggatgaattgt
tttagtacctagatttagatgtctaaaaagctttttagacatctaatcttttctgaagtacatc
cgcaactgtccatactctgatgttttatatcttttctaaaagttcgctagataggggtcccgag
cgcctacgaggaatttgtatcgactctagaggatccccgggtaccgagctcgaattcactggcc
gcaagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattc
cacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaact
cacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcat
taatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgc
tcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggt
aatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaa
aaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacg
agcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagatacca
ggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatac
ctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctca
gttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccg
ctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactg
gcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttga
agtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagcc
agttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggt
ggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttga
tcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgag
attatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaa
agtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcag
cgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacg
ggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctcca
gatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttat
ccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatag
tttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggct
tcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaag
cggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcat
ggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgact
ggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccgg
cgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacg
ttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccact
cgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacag
gaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactctt
cctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaa
tgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacg
tctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcg
tctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcaca
gcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcg
ggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcg
gtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcagg
ctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaag
ggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaa
aacgacggcca
序列48:T.sacch.pfl KO双交换质粒
cccattgtgcaatcgatcaattgaaatagctattcaattgatttaagcaattttatttcttctt
catttaattctcgccattccccttcttttaaattttcatctaattttaattggcctattgaaag
cctttttaagtagacgacttttgagcctatcgcttcaaacattctttttatttgatgatattta
ccttctctgattgagacatatactttcgatgtactgcctgaagatattatttccagttttgccg
gcatagtcttgtaaccatcgtctaatagtatgccatctgaaaacaaagatacgtcatcttcatc
gataaaacccaaaacttctgcatagtattttttgaaaacgtgtttttttggcgataagagctta
tgagatagttcaccgtcatttgtaattaaaagtaatccctctgtgtctttatcaagccttcctg
ctgggaaaacctttcttgcctttatatggtgtggcaaaagatctacaacggttttttctgatgg
atcatatgttgcacagattacacctttcggtttattcatcattatatatatgtattctttgtac
gatattttttcacttctaaacgtgattatatctttatcaggttgtactgcaaaaccggggtcgt
caatcgtcacattatttattgcaacaaggccttcttttataaaatttttaatttcttttcttgt
gccataacccatatttgataaaagcttatctattctcatttttgacatcttaaattcctcctaa
acaatatgactgtgcttcttagtaaattatatcccaaaaatataaaatttgtagcaaaaatgtg
atatatatcatattttttgcgttttcctgatgatacaattaagatgatgtttcaagataataaa
tttttctgaagtgtatacagtatattgactacaaagaacaaaatactgcaggtcgataaaccca
gcgaaccatttgaggtgataggtaagattataccgaggtatgaaaacgagaattggacctttac
agaattactctatgaagcgccatatttaaaaagctaccaagacgaagaggatgaagaggatgag
gaggcagattgccttgaatatattgacaatactgataagataatatatcttttatatagaagat
atcgccgtatgtaaggatttcagggggcaaggcataggcagcgcgcttatcaatatatctatag
aatgggcaaagcataaaaacttgcatggactaatgcttgaaacccaggacaataaccttatagc
ttgtaaattctatcataattgtggtttcaaaatcggctccgtcgatactatgttatacgccaac
tttcaaaacaactttgaaaaagctgttttctggtatttaaggttttagaatgcaaggaacagtg
aattggagttcgtcttgttataattagcttcttggggtatctttaaatactgtagaaaagagga
aggaaataataaatggctaaaatgagaatatcaccggaattgaaaaaactgatcgaaaaatacc
gctgcgtaaaagatacggaaggaatgtctcctgctaaggtatataagctggtgggagaaaatga
aaacctatatttaaaaatgacggacagccggtataaagggaccacctatgatgtggaacgggaa
aaggacatgatgctatggctggaaggaaagctgcctgttccaaaggtcctgcactttgaacggc
atgatggctggagcaatctgctcatgagtgaggccgatggcgtcctttgctcggaagagtatga
agatgaacaaagccctgaaaagattatcgagctgtatgcggagtgcatcaggctctttcactcc
atcgacatatcggattgtccctatacgaatagcttagacagccgcttagccgaattggattact
tactgaataacgatctggccgatgtggattgcgaaaactgggaagaagacactccatttaaaga
tccgcgcgagctgtatgattttttaaagacggaaaagcccgaagaggaacttgtcttttcccac
ggcgacctgggagacagcaacatctttgtgaaagatggcaaagtaagtggctttattgatcttg
ggagaagcggcagggcggacaagtggtatgacattgccttctgcgtccggtcgatcagggagga
tatcggggaagaacagtatgtcgagctattttttgacttactggggatcaagcctgattgggag
aaaataaaatattatattttactggatgaattgttttagtacctagatttagatgtctaaaaag
ctttttagacatctaatcttttctgaagtacatccgcaactgtccatactctgatgttttatat
cttttctaaaagttcgctagataggggtcccgagcgcctacgaggaatttgtatcgactctaga
ggatccccgggtaccgaaaaggtgattgtcatggttatggggaagatacattcaatagagacat
gtggtactgtagatgggcctggcataaggtacgtagtctttatgcaaggttgtcctttaaggtg
cgcttattgccataaccctgacacatggaattataacggtggtaaagaagtatcaacagatgag
atatttaacgatgcaaaaagatatataccgtacatgaaatcatcaggcggcggcgtgacgctga
caggtggagagcctacattacagcctgaattttgcgaagatctatttaaaaagcttaaagcgtc
tggcatacacactgcattagacacatcgggatatgtgaatatagataaagtaaaagaacttgta
aaacacactgatctttttttgcttgatataaagcacattgatgatgaaagccataaaaagctta
caggagtgtcgaatagaaagactttggagtttgcaagatacctttccgatgaaggcaagaaaat
gtggataaggcatgtgatagtacctggaataacggatgatatggaagagataaggaaattggct
gattttgtctcatcattgaaaaatgtagatagagttgagatacttccgtatcataaaatgggtg
tgtataaatatgaggcacttgggataccatatagattgaagggaataaatcctcctgacacatc
aaaaattaaagagataaaagaagagtttaggaaaagagatataaaagtggtctaaaagcctcat
gattcgtatcatggggcttttcctttgaattaatttgataaagggtgtaaaattatcatgtgat
gatgtgattttggaggtaatcgcatgaatttaaataagataaatagaaacacgtactacataga
taatcctacgaatattggcgtttatgcctataaaaataaaaattgtctattagtagatactggt
ataaacgcaagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctca
caattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgag
ctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccag
ctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgctt
cctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaa
ggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggc
cagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgccccc
ctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaag
ataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttacc
ggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggt
atctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcc
cgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcg
ccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagt
tcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgct
gaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggt
agcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatc
ctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggt
catgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatca
atctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcaccta
tctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactac
gatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccg
gctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaa
ctttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagt
taatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggt
atggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgca
aaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatc
actcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttct
gtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctctt
gcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattgg
aaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaa
cccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaa
aaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcat
actcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacata
tttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccac
ctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggcc
ctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacg
gtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtg
ttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcacca
tatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgcca
ttcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctgg
cgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacg
ttgtaaaacgacggcca
序列49:C.therm.pfl KO单交换质粒
gtttatgatgtatatactcccgaaatgagaaaagccaaaaaagccgggattattacaggacttc
ccgacgcatacggcagaggaagaataattggcgattacagaagggttgcactttatggcgttga
caggctgattgctgaaaaagagaaagaaatggcaagtcttgaaagagattacattgactatgag
actgttcgagacagagaagaaataagcgagcagattaaatctttaaaacaacttaaagaaatgg
ctttaagttacggttttgacatatcttgtcctgcaaaggatgccagagaagcctttcaatggtt
gtattttgcatatcttgcagcagtcaaggaacagaacggcgcggcaatgagtattggaagaatt
tcgactttccttgacatatacattgaaagggatctcaaagaaggaaaactcacggaggagttgg
ctcaggaactggttgaccagctggttataaagctgagaattgtgagatttttgagaactcctga
gtatgaaaagctcttcagcggagaccccacttgggtaaccgaaagtatcggaggtatggcgctg
gatggaagaacgctggttacaaaatcttcgttcaggtttttgcacactcttttcaacctgggac
atgcaccggagcccaaccttacagtactttggtccgtcaatcttcccgaaggctttaaaaagta
ctgtgcaaaggtatcaattcattcaagctccatccagtatgaaagcgacgacataatgaggaaa
cactggggagacgattatggaatagcagatggattttctattattgcaatgtggaattgggaac
ggaaaaattattttattaaagagtagttcaacaaacgggattgacttttaaaaaaggattgatt
ctaatgaagaaagcagacaagtaagcctcctaaattcactttagataaaaatttaggaggcata
tcaaatgaactttaataaaattgatttagacaattggaagagaaaagagatatttaatcattat
ttgaaccaacaaacgacttttagtataaccacagaaattgatattagtgttttataccgaaaca
taaaacaagaaggatataaattttaccctgcatttattttcttagtgacaagggtgataaactc
aaatacagcttttagaactggttacaatagcgacggagagttaggttattgggataagttagag
ccactttatacaatttttgatggtgtatctaaaacattctctggtatttggactcctgtaaaga
atgacttcaaagagttttatgatttatacctttctgatgtagagaaatataatggttcggggaa
attgtttcccaaaacacctatacctgaaaatgctttttctctttctattattccatggacttca
tttactgggtttaacttaaatatcaataataatagtaattaccttctacccattattacagcag
gaaaattcattaataaaggtaattcaatatatttaccgctatctttacaggtacatcattctgt
ttgtgatggttatcatgcaggattgtttatgaactctattcaggaattgtcagataggcctaat
gactggcttttataatatgagataatgccgactgtactttttacagtcggttttctaatgtcac
tagggctcgcctttgggaagtttgaagggctggcacgacaggtttcccgactggaaagcgggca
gtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttat
gcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctat
gaccatgattacgccaagcttgcatgcctgcaggtcgactctagaggatccgcaagcttggcgt
aatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacg
agccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcg
ttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggcc
aacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgct
gcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatcc
acagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaacc
gtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaa
tcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccct
ggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttc
tcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggt
cgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatcc
ggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactg
gtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaa
ctacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcgga
aaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgttt
gcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggg
gtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaagg
atcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagt
aaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatt
tcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttacca
tctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaa
taaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatcca
gtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgtt
gttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccg
gttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctcctt
cggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagca
ctgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaa
ccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacggga
taataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcga
aaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaact
gatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgc
cgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatat
tattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaa
ataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccat
tattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttc
ggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaag
cggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctg
gcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccg
cacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgtt
gggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgc
aaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggcca
序列50:C.therm.pfl KO双交换质粒
cccgcaataatggaagtaaagagcatgaaagtgggaatgctggcttacaccgatatggcggaaa
ttgtgtacaagggcaatccgaactacaagtttgcggccggagaggacaagccgggggttgcacc
aagacctttgaaatttgacgattccataaaaaaagacatagaagagttacggagcaaggtggat
attttaattgtttcacttcactggggagtggaggaaagctttgaagttctgcctgaacagaggg
aatttgcccacagtcttatagataacggagtggatgtaatattgggacaccatccccaccagtt
ccaaggtatagaaatctacaagggcaaacctgttttctacagtctgggtaattttatttttgat
cagaacgatcccgaaaaccaggagtcctttattgtgacacttgattacaaaggcagcagactga
caggaatagaggctgtacccgtgagaacaatcggaaaaatacaggtagttcctcaaaaaggaga
tgaagcaaaacctattttggaaagagagaaaaatttatgtaataggcttgatacaaactgcatt
ataaaagatgacaaattatattttgaaattggaaaataatgataatataattaagttggacgta
ttttgacaaaataaaatcataaagtggttgcatttgtcgagatttgtgatatcattggatagta
aattatattttaggttaaaaatggaaaaatagttttttatttaaactttatttttaaactttat
ttaaaatatcaaaataattgcctttgtattttacttattgtacaatatatttgtacaatatatt
aaggaaaaaaatacttttgtagcgacttaaaagtcaattgaatggaccaataaaggaccttttc
aaatttgtcaaggtattttaggacaattttttttattttggatattgttcttgtttattgggta
aataagatggattttctattattgcaatgtggaattgggaacggaaaaattattttattaaaga
gtagttcaacaaacgggattgacttttaaaaaaggattgattctaatgaagaaagcagacaagt
aagcctcctaaattcactttagataaaaatttaggaggcatatcaaatgaactttaataaaatt
gatttagacaattggaagagaaaagagatatttaatcattatttgaaccaacaaacgactttta
gtataaccacagaaattgatattagtgttttataccgaaacataaaacaagaaggatataaatt
ttaccctgcatttattttcttagtgacaagggtgataaactcaaatacagcttttagaactggt
tacaatagcgacggagagttaggttattgggataagttagagccactttatacaatttttgatg
gtgtatctaaaacattctctggtatttggactcctgtaaagaatgacttcaaagagttttatga
tttatacctttctgatgtagagaaatataatggttcggggaaattgtttcccaaaacacctata
cctgaaaatgctttttctctttctattattccatggacttcatttactgggtttaacttaaata
tcaataataatagtaattaccttctacccattattacagcaggaaaattcattaataaaggtaa
ttcaatatatttaccgctatctttacaggtacatcattctgtttgtgatggttatcatgcagga
ttgtttatgaactctattcaggaattgtcagataggcctaatgactggcttttataatatgaga
taatgccgactgtactttttacagtcggttttctaatgtcactagggctcgcctttgggaagtt
tgaagggctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgt
gagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgt
ggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcttg
catgcctgcaggtcgactctagaggatcccattaaagggcaggatacactcatttgaatctttt
gggacactggacggaccgggtataagatttgtggttttcatgcagggctgtcccttgcgttgta
tatattgccacaacagggatacctgggatgttaatgcggggagtgagtacactccccggcaagt
aattgatgaaatgatgaaatacatagactatataaaggtctccggaggcggaataactgttacc
ggcggggagcctgttctccaggccgattttgtggccgaggtgttcagacttgcaaaagagcagg
gagtgcatacggcgctggataccaatggatttgctgacatagagaaggttgaaaggcttataaa
atacaccgatcttgtattgctggatataaagcatgcccgggaggataaacataagataattacc
ggtgtgtccaacgaaaaaatcaagcgttttgcgctgtatctttcggaccagggagtgcctatct
ggataagatatgtccttgtccccggatataccgacgatgaagatgaccttaaaatggcggctga
tttcataaaaaagcttaaaacggtggaaaaaatcgaagttcttccttatcacaacatgggagca
tacaaatgggaaaaacttggtcagaaatacatgcttgaaggagtaaaggggccgagtgcgcaag
aggtggaaaaagcaaagaggattctgtcaggcaaataataaaagcttttttcttttattatttg
cttttttctattaccaatttgctttgcttaagtttaggtttggttttgatgagttttttaatgt
ttcttttatatttatcttttatatgaacagtgttgtaaacttccaaatccagtttgtcaaatat
tgatttaaaaatctttgccgtatactgggcgtcagttaatgcccggtgaagattttcgtctatt
tcaacgcaagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcac
aattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagc
taactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagc
tgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttc
ctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaag
gcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggcc
agcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccc
tgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaaga
taccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccg
gatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggta
tctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagccc
gaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgc
cactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagtt
cttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctg
aagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggta
gcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcc
tttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtc
atgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaa
tctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctat
ctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacg
atacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccgg
ctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaac
tttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagtt
aatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggta
tggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaa
aaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatca
ctcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctg
tgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttg
cccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattgga
aaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaac
ccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaa
aacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcata
ctcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatat
ttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacc
tgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccc
tttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacgg
tcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgt
tggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccat
atgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccat
tcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggc
gaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgt
tgtaaaacgacggcca
序列51:C.phyto.pfl KO单交换质粒
tttggtggtattcgtatggcacagagtgcttgccatgaatatggatatgaggtagacgaagagg
tagcacgtatttttacagactaccgcaagacacataatcaaggtgtatttgatgcatacactga
cgaaatgaagctcgctagaaaatcagcaatcattactggtttgcctgatgcttatggtagaggt
agaattattggcgattaccgtcgagtggcactttacggtactgatttacttattgaagacaaga
aagaacaacttacaacttccttaaagagaatgactagtgataatattcgcttaagagaagaatt
agcagaacaaattcgtgcattaaaagaattagcgaagcttggtgaaatctatggttacgatatt
acgaagccagcaataaatgcaaaggaagcaattcagtggctttactttggatatcttgcagcgg
taaaagagcaaaacggtgctgcaatgagcttaggccgtacttctacattccttgatatttatat
ccagagagatttagataatggtgttatcacagaaaaagaagcacaagagtatatcgatcatttt
attatgaaacttcgtctagtgaagtttgcaagaactccagaatacaatgccttattctccggtg
accctacttgggtaacagaaagtatcgctgggattggtacagatggacgccatatggtaacaaa
gacatccttccgttaccttcatacgttagacaaccttggaactgctccagaaccaaacatgaca
gttctatggtcaactagattaccaagattatttaaagagtactgtgctaagatgtcaattaagt
catcctctattcaatacgaaaatgatgatatcatgcgtccaactcatggtgatgattatgcaat
tgctagatggattttctattattgcaatgtggaattgggaacggaaaaattattttattaaaga
gtagttcaacaaacgggattgacttttaaaaaaggattgattctaatgaagaaagcagacaagt
aagcctcctaaattcactttagataaaaatttaggaggcatatcaaatgaactttaataaaatt
gatttagacaattggaagagaaaagagatatttaatcattatttgaaccaacaaacgactttta
gtataaccacagaaattgatattagtgttttataccgaaacataaaacaagaaggatataaatt
ttaccctgcatttattttcttagtgacaagggtgataaactcaaatacagcttttagaactggt
tacaatagcgacggagagttaggttattgggataagttagagccactttatacaatttttgatg
gtgtatctaaaacattctctggtatttggactcctgtaaagaatgacttcaaagagttttatga
tttatacctttctgatgtagagaaatataatggttcggggaaattgtttcccaaaacacctata
cctgaaaatgctttttctctttctattattccatggacttcatttactgggtttaacttaaata
tcaataataatagtaattaccttctacccattattacagcaggaaaattcattaataaaggtaa
ttcaatatatttaccgctatctttacaggtacatcattctgtttgtgatggttatcatgcagga
ttgtttatgaactctattcaggaattgtcagataggcctaatgactggcttttataatatgaga
taatgccgactgtactttttacagtcggttttctaatgtcactagggctcgcctttgggaagtt
tgaagggctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgt
gagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgt
ggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcttg
catgcctgcaggtcgactctagaggatccgcaagcttggcgtaatcatggtcatagctgtttcc
tgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaa
gcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttcc
agtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggttt
gcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcgg
cgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcag
gaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggc
gtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtgg
cgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctc
ctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgct
ttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgt
gtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtcca
acccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgag
gtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggaca
gtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgat
ccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcag
aaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaa
aactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaa
attaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttacca
atgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctga
ctccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatga
taccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggc
cgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaa
gctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcg
tggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagt
tacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcaga
agtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtca
tgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtg
tatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcaga
actttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgc
tgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttacttt
caccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcg
acacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggtt
attgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcg
cacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctat
aaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctct
gacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagc
ccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagag
cagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaat
accgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggc
ctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacg
ccagggttttcccagtcacgacgttgtaaaacgacggcca
序列52:C.phyto.pfl KO双交换质粒
tcacccagcagcagccatgattataaacaccggagttccggagcttagtagcgaagcatctaaa
agcggaaagccttatatttacggcggaacgggaaatggaccagtctttattgaacgtaccgctg
atgtaagaaaagcggtagaggatatcattgcaagccgcacctttgattacggaatcgtgtctgc
ggcagaacaatatatggtagtagacagtcttattgcagctgaagtaaaagctgagatgttaaga
aacggtgcctacttcatgaacgaggaagaggagaaaaagctaatagacctcctaaaccttacga
gtggaaaggcagatacagaaattatgggaagaccagccgaagaacttgccaaacgagcaggatt
tatggtacctaataccacgactgtgctggtttccgaacagaaatatatttccgacaggaaccca
tttgcaaaagagcttctttgtcctgtattggcttactacatcgaaaatgactggatgcatgctt
gtgagaagtgcatgagtcttttagtaaacgaaagccatggacataccctggtgattcattccag
ggatgaagaagtaataggccagttcgccttaaagaaaccagtaggcagagtacttgtaaatacc
cccgctaccctgggtagtatgggtgcaaccacaaacttgtttccggctatgaccctaggaagca
ttacagcaggcgccggaatcacagcggacaatgtttctcctatgaatttcatatacattcgtaa
agtaggatatggagttcggggagtacaagaatttcttggttcggttgagaaaacctcaagcgga
tacgcgaaagctcctgaaacaatcaggaacaatgcccttgaaacaaacaaggtcaatgcctttg
aaacaagcaaaggcatggaagatgctagagatcttttgaaacagattttacaagccttgtccaa
agaactagatggattttctattattgcaatgtggaattgggaacggaaaaattattttattaaa
gagtagttcaacaaacgggattgacttttaaaaaaggattgattctaatgaagaaagcagacaa
gtaagcctcctaaattcactttagataaaaatttaggaggcatatcaaatgaactttaataaaa
ttgatttagacaattggaagagaaaagagatatttaatcattatttgaaccaacaaacgacttt
tagtataaccacagaaattgatattagtgttttataccgaaacataaaacaagaaggatataaa
ttttaccctgcatttattttcttagtgacaagggtgataaactcaaatacagcttttagaactg
gttacaatagcgacggagagttaggttattgggataagttagagccactttatacaatttttga
tggtgtatctaaaacattctctggtatttggactcctgtaaagaatgacttcaaagagttttat
gatttatacctttctgatgtagagaaatataatggttcggggaaattgtttcccaaaacaccta
tacctgaaaatgctttttctctttctattattccatggacttcatttactgggtttaacttaaa
tatcaataataatagtaattaccttctacccattattacagcaggaaaattcattaataaaggt
aattcaatatatttaccgctatctttacaggtacatcattctgtttgtgatggttatcatgcag
gattgtttatgaactctattcaggaattgtcagataggcctaatgactggcttttataatatga
gataatgccgactgtactttttacagtcggttttctaatgtcactagggctcgcctttgggaag
tttgaagggctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaat
gtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgt
gtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagct
tgcatgcctgcaggtcgactctagaggatccaacggagagtaatcaaaatggaccatggaaaat
caggagagattgaacgtaaggcgtttatatttaacgtgcagaagtacaacatgtatgacgggcc
gggaatcagaaccttggtattctttaaaggctgtcctcttcggtgtaaatggtgctccaatccg
gaaggtctggaacgaaaatttcaggtaatgtataagcaaagtttttgtacaaactgcggggcgt
gcgctgatgtgtgccccgtaggaatccacgtgatgtcgaacggaacacatgaaattgttcggga
aaaggaatgcatcggctgcatgaagtgtaaaaacatctgcccaaagtcggcgcttaccattgca
ggagaggtaaagaccatttcagaactgcttaagattgtggaagaggacgctgctttttatgata
tgtccggaggtggcgtgacccttgggggtggtgaagtaaccgcacaaccagaagcggccttaaa
tcttttgatggcttgtaaacaggagggaatcaacacagcaattgaaacttgcggttattcgaat
acagagaacattttaaaaattgcggaatatgtggatcttttcctgtttgatatcaaacatatgg
atccagtacgtcacaacgagttaacaggtgtgaacaatgaacagattcttactaaccttgagga
actgcttcaccgccgctataacgtaaaagtccgtatgccaatgttaaaaggaattaatgacagc
agggaagaaattgatgcggttatcaagtttttaatgccataccgtactgataagaactttaagg
gaattgacttacttccataccataagctcggagttaataaatacaatcagcttgataaggtata
tccgattgacggcgatcctagcttaagtgctgaggatttagaccgaattgaaggttggatgaaa
gaatacgattttccggttaacgtggtaaaacactaagaaaggggaaggacgccatggaagaagg
caagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattcc
acacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactc
acattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcatt
aatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgct
cactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggta
atacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaa
aggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacga
gcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccag
gcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacc
tgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcag
ttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgc
tgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactgg
cagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaa
gtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagcca
gttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtg
gtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgat
cttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgaga
ttatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaa
gtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagc
gatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgg
gagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccag
atttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatc
cgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagt
ttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggctt
cattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagc
ggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatg
gttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactg
gtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggc
gtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgt
tcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactc
gtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacagg
aaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttc
ctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaat
gtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgt
ctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgt
ctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacag
cttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgg
gtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcgg
tgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggc
tgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagg
gggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaa
acgacggcca
序列53:T.sacch.#59L-ldh单交换
tctatcagctgtccctcctgttcagctactgacggggtggtgcgtaacggcaaaagcaccgccg
gacatcagcgctagcggagtgtatactggcttactatgttggcactgatgagggtgtcagtgaa
gtgcttcatgtggcaggagaaaaaaggctgcaccggtgcgtcagcagaatatgtgatacaggat
atattccgcttcctcgctcactgactcgctacgctcggtcgttcgactgcggcgagcggaaatg
gcttacgaacggggcggagatttcctggaagatgccaggaagatacttaacagggaagtgagag
ggccgcggcaaagccgtttttccataggctccgcccccctgacaagcatcacgaaatctgacgc
tcaaatcagtggtggcgaaacccgacaggactataaagataccaggcgtttccccctggcggct
ccctcgtgcgctctcctgttcctgcctttcggtttaccggtgtcattccgctgttatggccgcg
tttgtctcattccacgcctgacactcagttccgggtaggcagttcgctccaagctggactgtat
gcacgaaccccccgttcagtccgaccgctgcgccttatccggtaactatcgtcttgagtccaac
ccggaaagacatgcaaaagcaccactggcagcagccactggtaattgatttagaggagttagtc
ttgaagtcatgcgccggttaaggctaaactgaaaggacaagttttggtgactgcgctcctccaa
gccagttacctcggttcaaagagttggtagctcagagaaccttcgaaaaaccgccctgcaaggc
ggttttttcgttttcagagcaagagattacgcgcagaccaaaacgatctcaagaagatcatctt
attaatcagataaaatatttctagatttcagtgcaatttatctcttcaaatgtagcacctgaag
tcagccccatacgatataagttgtaattctcatgtttgacagcttatcatcgataagctttaat
gcggtagtttatcacagttaaattgctaacgcagtcaggcacctatacatgcatttacttataa
tacagttttttagttttgctggccgcatcttctcaaatatgcttcccagcctgcttttctgtaa
cgttcaccctctaccttagcatcccttccctttgcaaatagtcctcttccaacaataataatgt
cagatcctgtagagaccacatcatccacggttctatactgttgacccaatgcgtctcccttgtc
atctaaacccacaccgggtgtcataatcaaccaatcgtaaccttcatctcttccacccatgtct
ctttgagcaataaagccgataacaaaatctttgtcgctcttcgcaatgtcaacagtacccttag
tatattctccagtagatagggagcccttgcatgacaattctgctaacatcaaaaggcctctagg
ttcctttgttacttcttctgccgcctgcttcaaaccgctaacaatacctgggcccaccacaccg
tgtgcattcgtaatgtctgcccattctgctattctgtatacacccgcagagtactgcaatttga
ctgtattaccaatgtcagcaaattttctgtcttcgaagagtaaaaaattgtacttggcggataa
tgcctttagcggcttaactgtgccctccatggaaaaatcagtcaagatatccacatgtgttttt
agtaaacaaattttgggacctaatgcttcaactaactccagtaattccttggtggtacgaacat
ccaatgaagcacacaagtttgtttgcttttcgtgcatgatattaaatagcttggcagcaacagg
actaggatgagtagcagcacgttccttatatgtagctttcgacatgatttatcttcgtttcctg
caggtttttgttctgtgcagttgggttaagaatactgggcaatttcatgtttcttcaacactac
atatgcgtatatataccaatctaagtctgtgctccttccttcgttcttccttctgttcggagat
taccgaatcaaaaaaatttcaaagaaaccgaaatcaaaaaaaagaataaaaaaaaaatgatgaa
ttgaattgaaaagctagcttatcgatgggtccttttcatcacgtgctataaaaataattataat
ttaaattttttaatataaatatataaattaaaaatagaaagtaaaaaaagaaattaaagaaaaa
atagtttttgttttccgaagatgtaaaagactctagggggatcgccaacaaatactacctttta
tcttgctcttcctgctctcaggtattaatgccgaattgtttcatcttgtctgtgtagaagacca
cacacgaaaatcctgtgattttacattttacttatcgttaatcgaatgtatatctatttaatct
gcttttcttgtctaataaatatatatgtaaagtacgctttttgttgaaattttttaaacctttg
tttatttttttttcttcattccgtaactcttctaccttctttatttactttctaaaatccaaat
acaaaacataaaaataaataaacacagagtaaattcccaaattattccatcattaaaagatacg
aggcgcgtgtaagttacaggcaagcgatctctaagaaaccattattatcatgacattaacctat
aaaaaaggcctctcgagctagagtcgatcttcgccagcagggcgaggatcgtggcatcaccgaa
ccgcgccgtgcgcgggtcgtcggtgagccagagtttcagcaggccgcccaggcggcccaggtcg
ccattgatgcgggccagctcgcggacgtgctcatagtccacgacgcccgtgattttgtagccct
ggccgacggccagcaggtaggccgacaggctcatgccggccgccgccgccttttcctcaatcgc
tcttcgttcgtctggaaggcagtacaccttgataggtgggctgcccttcctggttggcttggtt
tcatcagccatccgcttgccctcatctgttacgccggcggtagccggccagcctcgcagagcag
gattcccgttgagcaccgccaggtgcgaataagggacagtgaagaaggaacacccgctcgcggg
tgggcctacttcacctatcctgcccggctgacgccgttggatacaccaaggaaagtctacacga
accctttggcaaaatcctgtatatcgtgcgaaaaaggatggatataccgaaaaaatcgctataa
tgaccccgaagcagggttatgcagcggaaaagcgctgcttccctgctgttttgtggaatatcta
ccgactggaaacaggcaaatgcaggaaattactgaactgaggggacaggcgagagacgatgcca
aagagctacaccgacgagctggccgagtgggttgaatcccgcgcggccaagaagcgccggcgtg
atgaggctgcggttgcgttcctggcggtgagggcggatgtcgatatgcgtaaggagaaaatacc
gcatcaggcgcatatttgaatgtatttagaaaaataaacaaaaagagtttgtagaaacgcaaaa
aggccatccgtcaggatggccttctgcttaatttgatgcctggcagtttatggcgggcgtcctg
cccgccaccctccgggccgttgcttcgcaacgttcaaatccgctcccggcggatttgtcctact
caggagagcgttcaccgacaaacaacagataaaacgaaaggcccagtctttcgactgagccttt
cgttttatttgatgcctggaaacccagcgaaccatttgaggtgataggtaagattataccgagg
tatgaaaacgagaattggacctttacagaattactctatgaagcgccatatttaaaaagctacc
aagacgaagaggatgaagaggatgaggaggcagattgccttgaatatattgacaatactgataa
gataatatatcttttatatagaagatatcgccgtatgtaaggatttcagggggcaaggcatagg
cagcgcgcttatcaatatatctatagaatgggcaaagcataaaaacttgcatggactaatgctt
gaaacccaggacaataaccttatagcttgtaaattctatcataattgtggtttcaaaatcggct
ccgtcgatactatgttatacgccaactttcaaaacaactttgaaaaagctgttttctggtattt
aaggttttagaatgcaaggaacagtgaattggagttcgtcttgttataattagcttcttggggt
atctttaaatactgtagaaaagaggaaggaaataataaatggctaaaatgagaatatcaccgga
attgaaaaaactgatcgaaaaataccgctgcgtaaaagatacggaaggaatgtctcctgctaag
gtatataagctggtgggagaaaatgaaaacctatatttaaaaatgacggacagccggtataaag
ggaccacctatgatgtggaacgggaaaaggacatgatgctatggctggaaggaaagctgcctgt
tccaaaggtcctgcactttgaacggcatgatggctggagcaatctgctcatgagtgaggccgat
ggcgtcctttgctcggaagagtatgaagatgaacaaagccctgaaaagattatcgagctgtatg
cggagtgcatcaggctctttcactccatcgacatatcggattgtccctatacgaatagcttaga
cagccgcttagccgaattggattacttactgaataacgatctggccgatgtggattgcgaaaac
tgggaagaagacactccatttaaagatccgcgcgagctgtatgattttttaaagacggaaaagc
ccgaagaggaacttgtcttttcccacggcgacctgggagacagcaacatctttgtgaaagatgg
caaagtaagtggctttattgatcttgggagaagcggcagggcggacaagtggtatgacattgcc
ttctgcgtccggtcgatcagggaggatatcggggaagaacagtatgtcgagctattttttgact
tactggggatcaagcctgattgggagaaaataaaatattatattttactggatgaattgtttta
gtacctagatttagatgtctaaaaagctttttagacatctaatcttttctgaagtacatccgca
actgtccatactctgatgttttatatcttttctaaaagttcgctagataggggtcccgagcgcc
tacgaggaatttgtatcgtacaaggatatcgagggcgcagatgtagtagttgtaacagcaggtg
cggctcaaaagccaggagagtctaggctggaccttgtaaaaaagaatacatctatattcaagtc
catgatacctgaacttttaaaatacaatgataaagctatatacctgattgtaacaaatcctgtt
gatatattaacgtatgttacatacaaaatagcgaaacttccgtgggggcgtgtattcggttcag
gtactgtccttgacagttcccgatttaggtatcttttaagtaaacattgcaatattgatcctag
aaatgtacatggaaggataattggagaacacggcgatacagaatttgcggcgtggagcataaca
aatatttcaggaatatcatttaatgagtactgcaatttgtgcggacgagtttgtaatacaaatt
tcagaaaggaagtggaagatgaagttgtcaatgcggcttacaaaattattgataaaaagggtgc
cacgtattacgctgtggctg
序列54:T.sacch.#59L-ldh双交换
gcacatattgatagagaacatggctggataattactataactccaagaaaacgaatagtaaaag
aatggaggcgaattaatgagtaatgtcncaatacaattaatagaaatttgtcggcaatatgtaa
ataataacttaaacataaatgantntatcgaagatttccaagtgctttatgaacaaaagcaaga
tttattaacagatgaagaaatgagtttgtttgatgatatttatatggcttgtgaatattatgaa
caggatgaaaatataagaaatgaatatcacttgtatattggagaaaatgaattaagacaaaaag
tgcaaaaacttgtaaaaaagttagcagcataataaaccgctaaggcatgatagctaaagcggta
tttttatgcaattaaaaggaaaaatgatatctgataaaccgcggaaaagtattttagaaaacaa
ctataaagataatatttcaaagcaagaaggataaaataagattaaactattagacacttttatt
agaaaatgttataatattattaagagaaaatttatattatttaggaggtaattttatgagtaaa
gtggccataataggttcaggatttgtaggtgctacatctgcatttacattggctctaagtggga
ctgtgacagacattgttttagtagatttaaacaaggacaaggcgataggcgatgcactggatat
tagccaaaacccagcgaaccatttgaggtgataggtaagattataccgaggtatgaaaacgaga
attggacctttacagaattactctatgaagcgccatatttaaaaagctaccaagacgaagagga
tgaagaggatgaggaggcagattgccttgaatatattgacaatactgataagataatatatctt
ttatatagaagatatcgccgtatgtaaggatttcagggggcaaggcataggcagcgcgcttatc
aatatatctatagaatgggcaaagcataaaaacttgcatggactaatgcttgaaacccaggaca
ataaccttatagcttgtaaattctatcataattgtggtttcaaaatcggctccgtcgatactat
gttatacgccaactttcaaaacaactttgaaaaagctgttttctggtatttaaggttttagaat
gcaaggaacagtgaattggagttcgtcttgttataattagcttcttggggtatctttaaatact
gtagaaaagaggaaggaaataataaatggctaaaatgagaatatcaccggaattgaaaaaactg
atcgaaaaataccgctgcgtaaaagatacggaaggaatgtctcctgctaaggtatataagctgg
tgggagaaaatgaaaacctatatttaaaaatgacggacagccggtataaagggaccacctatga
tgtggaacgggaaaaggacatgatgctatggctggaaggaaagctgcctgttccaaaggtcctg
cactttgaacggcatgatggctggagcaatctgctcatgagtgaggccgatggcgtcctttgct
cggaagagtatgaagatgaacaaagccctgaaaagattatcgagctgtatgcggagtgcatcag
gctctttcactccatcgacatatcggattgtccctatacgaatagcttagacagccgcttagcc
gaattggattacttactgaataacgatctggccgatgtggattgcgaaaactgggaagaagaca
ctccatttaaagatccgcgcgagctgtatgattttttaaagacggaaaagcccgaagaggaact
tgtcttttcccacggcgacctgggagacagcaacatctttgtgaaagatggcaaagtaagtggc
tttattgatcttgggagaagcggcagggcggacaagtggtatgacattgccttctgcgtccggt
cgatcagggaggatatcggggaagaacagtatgtcgagctattttttgacttactggggatcaa
gcctgattgggagaaaataaaatattatattttactggatgaattgttttagtacctagattta
gatgtctaaaaagctttttagacatctaatcttttctgaagtacatccgcaactgtccatactc
tgatgttttatatcttttctaaaagttcgctagataggggtcccgagcgcctacgaggaatttg
tatcgacgtattacgctgtggctgtagcagtaagaagaatagttgagtgtatcataagggatga
aaattcaattcttacagtttcatctccattaaatggtcaatacggtgtaagagatgtatcttta
agcttgccatcaattgtgggcaaaaatggtgttgcaagggttctggatttgcctttggctgatg
acgaagttgagaagtttaaacattcggcaagcgttatggctgatgttataaaacagttggacat
ataaaataaatcattgtataaggtttataagacggcttttatcatgtatggtaaaggccgcttt
tttatgaatataaaaatacaaagtggaaaatctaaataaaggtgatgcaatatgcagaatatga
gtcctcaagaaattatatcgagtgcctttatgaaggcaaaaaaatctgagaatattatacatgc
taaggctatagattatgggaaaaatatatcagataaccagatgcaagcgatattgaagcaaata
gagataacggctttaaaccatgtggacaaaatagtgacagctgagaagacgatgcatctatcag
ctgtccctcctgttcagctactgacggggtggtgcgtaacggcaaaagcaccgccggacatcag
cgctagcggagtgtatactggcttactatgttggcactgatgagggtgtcagtgaagtgcttca
tgtggcaggagaaaaaaggctgcaccggtgcgtcagcagaatatgtgatacaggatatattccg
cttcctcgctcactgactcgctacgctcggtcgttcgactgcggcgagcggaaatggcttacga
acggggcggagatttcctggaagatgccaggaagatacttaacagggaagtgagagggccgcgg
caaagccgtttttccataggctccgcccccctgacaagcatcacgaaatctgacgctcaaatca
gtggtggcgaaacccgacaggactataaagataccaggcgtttccccctggcggctccctcgtg
cgctctcctgttcctgcctttcggtttaccggtgtcattccgctgttatggccgcgtttgtctc
attccacgcctgacactcagttccgggtaggcagttcgctccaagctggactgtatgcacgaac
cccccgttcagtccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggaaag
acatgcaaaagcaccactggcagcagccactggtaattgatttagaggagttagtcttgaagtc
atgcgccggttaaggctaaactgaaaggacaagttttggtgactgcgctcctccaagccagtta
cctcggttcaaagagttggtagctcagagaaccttcgaaaaaccgccctgcaaggcggtttttt
cgttttcagagcaagagattacgcgcagaccaaaacgatctcaagaagatcatcttattaatca
gataaaatatttctagatttcagtgcaatttatctcttcaaatgtagcacctgaagtcagcccc
atacgatataagttgtaattctcatgtttgacagcttatcatcgataagctttaatgcggtagt
ttatcacagttaaattgctaacgcagtcaggcacctatacatgcatttacttataatacagttt
tttagttttgctggccgcatcttctcaaatatgcttcccagcctgcttttctgtaacgttcacc
ctctaccttagcatcccttccctttgcaaatagtcctcttccaacaataataatgtcagatcct
gtagagaccacatcatccacggttctatactgttgacccaatgcgtctcccttgtcatctaaac
ccacaccgggtgtcataatcaaccaatcgtaaccttcatctcttccacccatgtctctttgagc
aataaagccgataacaaaatctttgtcgctcttcgcaatgtcaacagtacccttagtatattct
ccagtagatagggagcccttgcatgacaattctgctaacatcaaaaggcctctaggttcctttg
ttacttcttctgccgcctgcttcaaaccgctaacaatacctgggcccaccacaccgtgtgcatt
cgtaatgtctgcccattctgctattctgtatacacccgcagagtactgcaatttgactgtatta
ccaatgtcagcaaattttctgtcttcgaagagtaaaaaattgtacttggcggataatgccttta
gcggcttaactgtgccctccatggaaaaatcagtcaagatatccacatgtgtttttagtaaaca
aattttgggacctaatgcttcaactaactccagtaattccttggtggtacgaacatccaatgaa
gcaGacaagtttgtttgcttttcgtgcatgatattaaatagcttggcagcaacaggactaggat
gagtagcagcacgttccttatatgtagctttcgacatgatttatcttcgtttcctgcaggtttt
tgttctgtgcagttgggttaagaatactgggcaatttcatgtttcttcaacactacatatgcgt
atatataccaatctaagtctgtgctccttccttcgttcttccttctgttcggagattaccgaat
caaaaaaatttcaaagaaaccgaaatcaaaaaaaagaataaaaaaaaaatgatgaattgaattg
aaaagctagcttatcgatgggtccttttcatcacgtgctataaaaataattataatttaaattt
tttaatataaatatataaattaaaaatagaaagtaaaaaaagaaattaaagaaaaaatagtttt
tgttttccgaagatgtaaaagactctagggggatcgccaacaaatactaccttttatcttgctc
ttcctgctctcaggtattaatgccgaattgtttcatcttgtctgtgtagaagaccacacacgaa
aatcctgtgattttacattttacttatcgttaatcgaatgtatatctatttaatctgcttttct
tgtctaataaatatatatgtaaagtacgctttttgttgaaattttttaaacctttgtttatttt
tttttcttcattccgtaactcttctaccttctttatttactttctaaaatccaaatacaaaaca
taaaaataaataaacacagagtaaattcccaaattattccatcattaaaagatacgaggcgcgt
gtaagttacaggcaagcgatctctaagaaaccattattatcatgacattaacctataaaaaagg
cctctcgagctagagtcgatcttcgccagcagggcgaggatcgtggcatcaccgaaccgcgccg
tgcgcgggtcgtcggtgagccagagtttcagcaggccgcccaggcggcccaggtcgccattgat
gcgggccagctcgcggacgtgctcatagtccacgacgcccgtgattttgtagccctggccgacg
gccagcaggtaggccgacaggctcatgccggccgccgccgccttttcctcaatcgctcttcgtt
cgtctggaaggcagtacaccttgataggtgggctgcccttcctggttggcttggtttcatcagc
catccgcttgccctcatctgttacgccggcggtagccggccagcctcgcagagcaggattcccg
ttgagcaccgccaggtgcgaataagggacagtgaagaaggaacacccgctcgcgggtgggccta
cttcacctatcctgcccggctgacgccgttggatacaccaaggaaagtctacacgaaccctttg
gcaaaatcctgtatatcgtgcgaaaaaggatggatataccgaaaaaatcgctataatgaccccg
aagcagggttatgcagcggaaaagcgctgcttccctgctgttttgtggaatatctaccgactgg
aaacaggcaaatgcaggaaattactgaactgaggggacaggcgagagacgatgccaaagagcta
caccgacgagctggccgagtgggttgaatcccgcgcggccaagaagcgccggcgtgatgaggct
gcggttgcgttcctggcggtgagggcggatgtcgatatgcgtaaggagaaaataccgcatcagg
cgcatatttgaatgtatttagaaaaataaacaaaaagagtttgtagaaacgcaaaaaggccatc
cgtcaggatggccttctgcttaatttgatgcctggcagtttatggcgggcgtcctgcccgccac
cctccgggccgttgcttcgcaacgttcaaatccgctcccggcggatttgtcctactcaggagag
cgttcaccgacaaacaacagataaaacgaaaggcccagtctttcgactgagcctttcgttttat
ttgatgcctgg
序列55:T.sacch.#59pta/ack敲除单交换
tctatcagctgtccctcctgttcagctactgacggggtggtgcgtaacggcaaaagcaccgccg
gacatcagcgctagcggagtgtatactggcttactatgttggcactgatgagggtgtcagtgaa
gtgcttcatgtggcaggagaaaaaaggctgcaccggtgcgtcagcagaatatgtgatacaggat
atattccgcttcctcgctcactgactcgctacgctcggtcgttcgactgcggcgagcggaaatg
gcttacgaacggggcggagatttcctggaagatgccaggaagatacttaacagggaagtgagag
ggccgcggcaaagccgtttttccataggctccgcccccctgacaagcatcacgaaatctgacgc
tcaaatcagtggtggcgaaacccgacaggactataaagataccaggcgtttccccctggcggct
ccctcgtgcgctctcctgttcctgcctttcggtttaccggtgtcattccgctgttatggccgcg
tttgtctcattccacgcctgacactcagttccgggtaggcagttcgctccaagctggactgtat
gcacgaaccccccgttcagtccgaccgctgcgccttatccggtaactatcgtcttgagtccaac
ccggaaagacatgcaaaagcaccactggcagcagccactggtaattgatttagaggagttagtc
ttgaagtcatgcgccggttaaggctaaactgaaaggacaagttttggtgactgcgctcctccaa
gccagttacctcggttcaaagagttggtagctcagagaaccttcgaaaaaccgccctgcaaggc
ggttttttcgttttcagagcaagagattacgcgcagaccaaaacgatctcaagaagatcatctt
attaatcagataaaatatttctagatttcagtgcaatttatctcttcaaatgtagcacctgaag
tcagccccatacgatataagttgtaattctcatgtttgacagcttatcatcgataagctttaat
gcggtagtttatcacagttaaattgctaacgcagtcaggcacctatacatgcatttacttataa
tacagttttttagttttgctggccgcatcttctcaaatatgcttcccagcctgcttttctgtaa
cgttcaccctctaccttagcatcccttccctttgcaaatagtcctcttccaacaataataatgt
cagatcctgtagagaccacatcatccacggttctatactgttgacccaatgcgtctcccttgtc
atctaaacccacaccgggtgtcataatcaaccaatcgtaaccttcatctcttccacccatgtct
ctttgagcaataaagccgataacaaaatctttgtcgctcttcgcaatgtcaacagtacccttag
tatattctccagtagatagggagcccttgcatgacaattctgctaacatcaaaaggcctctagg
ttcctttgttacttcttctgccgcctgcttcaaaccgctaacaatacctgggcccaccacaccg
tgtgcattcgtaatgtctgcccattctgctattctgtatacacccgcagagtactgcaatttga
ctgtattaccaatgtcagcaaattttctgtcttcgaagagtaaaaaattgtacttggcggataa
tgcctttagcggcttaactgtgccctccatggaaaaatcagtcaagatatccacatgtgttttt
agtaaacaaattttgggacctaatgcttcaactaactccagtaattccttggtggtacgaacat
ccaatgaagcacacaagtttgtttgcttttcgtgcatgatattaaatagcttggcagcaacagg
actaggatgagtagcagcacgttccttatatgtagctttcgacatgatttatcttcgtttcctg
caggtttttgttctgtgcagttgggttaagaatactgggcaatttcatgtttcttcaacactac
atatgcgtatatataccaatctaagtctgtgctccttccttcgttcttccttctgttcggagat
taccgaatcaaaaaaatttcaaagaaaccgaaatcaaaaaaaagaataaaaaaaaaatgatgaa
ttgaattgaaaagctagcttatcgatgggtccttttcatcacgtgctataaaaataattataat
ttaaattttttaatataaatatataaattaaaaatagaaagtaaaaaaagaaattaaagaaaaa
atagtttttgttttccgaagatgtaaaagactctagggggatcgccaacaaatactacctttta
tcttgctcttcctgctctcaggtattaatgccgaattgtttcatcttgtctgtgtagaagacca
cacacgaaaatcctgtgattttacattttacttatcgttaatcgaatgtatatctatttaatct
gcttttcttgtctaataaatatatatgtaaagtacgctttttgttgaaattttttaaacctttg
tttatttttttttcttcattccgtaactcttctaccttctttatttactttctaaaatccaaat
acaaaacataaaaataaataaacacagagtaaattcccaaattattccatcattaaaagatacg
aggcgcgtgtaagttacaggcaagcgatctctaagaaaccattattatcatgacattaacctat
aaaaaaggcctctcgagctagagtcgatcttcgccagcagggcgaggatcgtggcatcaccgaa
ccgcgccgtgcgcgggtcgtcggtgagccagagtttcagcaggccgcccaggcggcccaggtcg
ccattgatgcgggccagctcgcggacgtgctcatagtccacgacgcccgtgattttgtagccct
ggccgacggccagcaggtaggccgacaggctcatgccggccgccgccgccttttcctcaatcgc
tcttcgttcgtctggaaggcagtacaccttgataggtgggctgcccttcctggttggcttggtt
tcatcagccatccgcttgccctcatctgttacgccggcggtagccggccagcctcgcagagcag
gattcccgttgagcaccgccaggtgcgaataagggacagtgaagaaggaacacccgctcgcggg
tgggcctacttcacctatcctgcccggctgacgccgttggatacaccaaggaaagtctacacga
accctttggcaaaatcctgtatatcgtgcgaaaaaggatggatataccgaaaaaatcgctataa
tgaccccgaagcagggttatgcagcggaaaagcgctgcttccctgctgttttgtggaatatcta
ccgactggaaacaggcaaatgcaggaaattactgaactgaggggacaggcgagagacgatgcca
aagagctacaccgacgagctggccgagtgggttgaatcccgcgcggccaagaagcgccggcgtg
atgaggctgcggttgcgttcctggcggtgagggcggatgtcgatatgcgtaaggagaaaatacc
gcatcaggcgcatatttgaatgtatttagaaaaataaacaaaaagagtttgtagaaacgcaaaa
aggccatccgtcaggatggccttctgcttaatttgatgcctggcagtttatggcgggcgtcctg
cccgccaccctccgggccgttgcttcgcaacgttcaaatccgctcccggcggatttgtcctact
caggagagcgttcaccgacaaacaacagataaaacgaaaggcccagtctttcgactgagccttt
cgttttatttgatgcctggaaacccagcgaaccatttgaggtgataggtaagattataccgagg
tatgaaaacgagaattggacctttacagaattactctatgaagcgccatatttaaaaagctacc
aagacgaagaggatgaagaggatgaggaggcagattgccttgaatatattgacaatactgataa
gataatatatcttttatatagaagatatcgccgtatgtaaggatttcagggggcaaggcatagg
cagcgcgcttatcaatatatctatagaatgggcaaagcataaaaacttgcatggactaatgctt
gaaacccaggacaataaccttatagcttgtaaattctatcataattgtggtttcaaaatcggct
ccgtcgatactatgttatacgccaactttcaaaacaactttgaaaaagctgttttctggtattt
aaggttttagaatgcaaggaacagtgaattggagttcgtcttgttataattagcttcttggggt
atctttaaatactgtagaaaagaggaaggaaataataaatggctaaaatgagaatatcaccgga
attgaaaaaactgatcgaaaaataccgctgcgtaaaagatacggaaggaatgtctcctgctaag
gtatataagctggtgggagaaaatgaaaacctatatttaaaaatgacggacagccggtataaag
ggaccacctatgatgtggaacgggaaaaggacatgatgctatggctggaaggaaagctgcctgt
tccaaaggtcctgcactttgaacggcatgatggctggagcaatctgctcatgagtgaggccgat
ggcgtcctttgctcggaagagtatgaagatgaacaaagccctgaaaagattatcgagctgtatg
cggagtgcatcaggctctttcactccatcgacatatcggattgtccctatacgaatagcttaga
cagccgcttagccgaattggattacttactgaataacgatctggccgatgtggattgcgaaaac
tgggaagaagacactccatttaaagatccgcgcgagctgtatgattttttaaagacggaaaagc
ccgaagaggaacttgtcttttcccacggcgacctgggagacagcaacatctttgtgaaagatgg
caaagtaagtggctttattgatcttgggagaagcggcagggcggacaagtggtatgacattgcc
ttctgcgtccggtcgatcagggaggatatcggggaagaacagtatgtcgagctattttttgact
tactggggatcaagcctgattgggagaaaataaaatattatattttactggatgaattgtttta
gtacctagatttagatgtctaaaaagctttttagacatctaatcttttctgaagtacatccgca
actgtccatactctgatgttttatatcttttctaaaagttcgctagataggggtcccgagcgcc
tacgaggaatttgtatcggctgtacaatctgctaatactgcaaagaatttgttgggctttgaac
caaaagttgctatgctatcattttccacaaaaggtagtgcatcacatgaattagtagataaagt
aagaaaagcgacagaaatagcaaaagaattgatgccagatgttgctatcgacggtgaattgcaa
ttggatgctgctcttgtcaaagaagttgcagagctaaaagcgccaggaagcaaagttgcgggat
gtgcaaatgtgcttatattccctgatttacaagctggtaatataggatataagcttgtacagag
attagctagcaaatgcaattggacctataacacaggaatgggtgcaccggttaatgatttatca
agaggatgcagctatagagatattgttgacgtaatagcacacagctgtacaggctcataaatgt
aaagtatggaggatgaaattatgaaaatactggtatatgcgaagtctcactaaatatcactgat
gatcatgatgaaatgtgctggcaaaggccttgctgagagaatcggcataaatgatccctgttga
cacatatgctaacggagaaaaatcaagataaaaaaagacatgaaagatcacaaagacgcaataa
aattgttttagatgctttggtaagcagtgactacggcgttataaaggatatgtctgagatagat
gctgtaggacatagagttgttcacggaggagaatcttttacatcatcagttctcataaatgatg
atgtgttaaaagcgataacagattgcatagaattagctccactgcacaatcctgccaatataga
aggaat
序列56:T.sacch.#59pta/ack双交换
taacagggccatgaccttctgcaaagcacttttttaaaatttcaagagaatattgtggcatata
tttttctaatgcagaatagtttttatcatttataacctttctaagatatgtagcacttgcaaaa
gattctactgtttccagagaattatatcctggaccaaatctttttaatgtcaaaggcttaatat
tgctgtttaacttgattaagctttttatgtattcaattccaagtatgttatttggatttccgat
aatattgttaatggtccttccgaaatattttgataatgctgcttcacgtgcctttgcatatgtg
atgccgctttttaaataggttttcaaagagctttataatcttctggttcgtctaaaagaaattt
tgatatttggcataattcgtcaatagagccatgttcgcttccaaacgatatgtaatcaatgaca
tttaatgaatccagcagctttactgctccatatgcgaaattttctgcggtagaaactgcatata
tagtgggcaattctattactaagtcaatacctgatagcaatgcagcttctgtccttgaccactt
gtcaataatagacggtattcctcgctgaacaaagttaccactcataattgcaatgacaaaatct
gcacctgttgtttcaattgatttttttatatggtatatgtgtccgttatggagagggttatatt
ctacaattactcccaatatactcattattaaaaacctttctaaaaaattattaattgtacttat
tattttataaaaaatatgttaaaatgtaaaatgtgtatacaatatatttcttctttttagtaag
aggaatgtataaaaataaatattttaaaggaagggacgatcttatgagcattattcaaaacatc
attgaaaaagctaaaagtgataaaaagaaaattgttctgccggaaggtgcagaaacccagcgaa
ccatttgaggtgataggtaagattataccgaggtatgaaaacgagaattggacctttacagaat
tactctatgaagcgccatatttaaaaagctaccaagacgaagaggatgaagaggatgaggaggc
agattgccttgaatatattgacaatactgataagataatatatcttttatatagaagatatcgc
cgtatgtaaggatttcagggggcaaggcataggcagcgcgcttatcaatatatctatagaatgg
gcaaagcataaaaacttgcatggactaatgcttgaaacccaggacaataaccttatagcttgta
aattctatcataattgtggtttcaaaatcggctccgtcgatactatgttatacgccaactttca
aaacaactttgaaaaagctgttttctggtatttaaggttttagaatgcaaggaacagtgaattg
gagttcgtcttgttataattagcttcttggggtatctttaaatactgtagaaaagaggaaggaa
ataataaatggctaaaatgagaatatcaccggaattgaaaaaactgatcgaaaaataccgctgc
gtaaaagatacggaaggaatgtctcctgctaaggtatataagctggtgggagaaaatgaaaacc
tatatttaaaaatgacggacagccggtataaagggaccacctatgatgtggaacgggaaaagga
catgatgctatggctggaaggaaagctgcctgttccaaaggtcctgcactttgaacggcatgat
ggctggagcaatctgctcatgagtgaggccgatggcgtcctttgctcggaagagtatgaagatg
aacaaagccctgaaaagattatcgagctgtatgcggagtgcatcaggctctttcactccatcga
catatcggattgtccctatacgaatagcttagacagccgcttagccgaattggattacttactg
aataacgatctggccgatgtggattgcgaaaactgggaagaagacactccatttaaagatccgc
gcgagctgtatgattttttaaagacggaaaagcccgaagaggaacttgtcttttcccacggcga
cctgggagacagcaacatctttgtgaaagatggcaaagtaagtggctttattgatcttgggaga
agcggcagggcggacaagtggtatgacattgccttctgcgtccggtcgatcagggaggatatcg
gggaagaacagtatgtcgagctattttttgacttactggggatcaagcctgattgggagaaaat
aaaatattatattttactggatgaattgttttagtacctagatttagatgtctaaaaagctttt
tagacatctaatcttttctgaagtacatccgcaactgtccatactctgatgttttatatctttt
ctaaaagttcgctagataggggtcccgagcgcctacgaggaatttgtatcgaaagttagcgtga
tggttgtgcccactaatgaagaatacatgattgctaaagatactgaaaagattgtaaagagtat
aaaatagcattcttgacaaatgtttaccccattagtataattaattttggcaattatattgggg
tgagaaaatgaaaattgatttatcaaaattaagggacataggggccgcagcatcgaagtcaact
acgtagaaaatctgagtgttcttgaggcaaatagcaatagatacgtagttataaagcctattag
cgtaactggaagcataacatacgatagtgaaggaatagttttaaaacttttggcacgcggggct
attaaagtaacatgcgataggtgccttgacgaatttgagtatgagttcgtaatacctattgacg
aaatagtaaacgagtctgatgatgaattttcaggtgaagtggaagatgaaaagcttgatttgac
gaaaattgtgattgaaaatgtggaactttctcttccgatgaagttcatttgctcgaatgattgc
aagggtctatgttctacttgcggtaaaaatcttaatcatgaaaaatgcgattgccaaataaaag
aaattgatccacgcctttcagttttgaataaattactgcagaagatgtaggaggtgtataatat
gccagttccaaagcgtagaacatctaaggcaagaagagataaaagaaggcatagccatagttta
gctgtacctgcttatgttttgtgcccacaatgtcatgaaccaaaattgccccacagagtttgtt
taagctgtggttattacgacggtaaagaggtattgaaagtggaagaaaagtaatggagttttct
ctattacttttcttttttatttcttgacttttatgtatggcgtaatttataattatgagtaagt
cataaaaacaacctatatttggagctgataatgtggccacgaagcttagtaaaagagatagatt
aaaaaagttaaaaattgaaatcgaaaaatatccattttacactgatgatgagttagctgatttg
ttttcggttagcgttcagacgataaggctggattctatcagctgtccctcctgttcagctactg
acggggtggtgcgtaacggcaaaagcaccgccggacatcagcgctagcggagtgtatactggct
tactatgttggcactgatgagggtgtcagtgaagtgcttcatgtggcaggagaaaaaaggctgc
accggtgcgtcagcagaatatgtgatacaggatatattccgcttcctcgctcactgactcgcta
cgctcggtcgttcgactgcggcgagcggaaatggcttacgaacggggcggagatttcctggaag
atgccaggaagatacttaacagggaagtgagagggccgcggcaaagccgtttttccataggctc
cgcccccctgacaagcatcacgaaatctgacgctcaaatcagtggtggcgaaacccgacaggac
tataaagataccaggcgtttccccctggcggctccctcgtgcgctctcctgttcctgcctttcg
gtttaccggtgtcattccgctgttatggccgcgtttgtctcattccacgcctgacactcagttc
cgggtaggcagttcgctccaagctggactgtatgcacgaaccccccgttcagtccgaccgctgc
gccttatccggtaactatcgtcttgagtccaacccggaaagacatgcaaaagcaccactggcag
cagccactggtaattgatttagaggagttagtcttgaagtcatgcgccggttaaggctaaactg
aaaggacaagttttggtgactgcgctcctccaagccagttacctcggttcaaagagttggtagc
tcagagaaccttcgaaaaaccgccctgcaaggcggttttttcgttttcagagcaagagattacg
cgcagaccaaaacgatctcaagaagatcatcttattaatcagataaaatatttctagatttcag
tgcaatttatctcttcaaatgtagcacctgaagtcagccccatacgatataagttgtaattctc
atgtttgacagcttatcatcgataagctttaatgcggtagtttatcacagttaaattgctaacg
cagtcaggcacctatacatgcatttacttataatacagttttttagttttgctggccgcatctt
ctcaaatatgcttcccagcctgcttttctgtaacgttcaccctctaccttagcatcccttccct
ttgcaaatagtcctcttccaacaataataatgtcagatcctgtagagaccacatcatccacggt
tctatactgttgacccaatgcgtctcccttgtcatctaaacccacaccgggtgtcataatcaac
caatcgtaaccttcatctcttccacccatgtctctttgagcaataaagccgataacaaaatctt
tgtcgctcttcgcaatgtcaacagtacccttagtatattctccagtagatagggagcccttgca
tgacaattctgctaacatcaaaaggcctctaggttcctttgttacttcttctgccgcctgcttc
aaaccgctaacaatacctgggcccaccacaccgtgtgcattcgtaatgtctgcccattctgcta
ttctgtatacacccgcagagtactgcaatttgactgtattaccaatgtcagcaaattttctgtc
ttcgaagagtaaaaaattgtacttggcggataatgcctttagcggcttaactgtgccctccatg
gaaaaatcagtcaagatatccacatgtgtttttagtaaacaaattttgggacctaatgcttcaa
ctaactccagtaattccttggtggtacgaacatccaatgaagcacacaagtttgtttgcttttc
gtgcatgatattaaatagcttggcagcaacaggactaggatgagtagcagcacgttccttatat
gtagctttcgacatgatttatcttcgtttcctgcaggtttttgttctgtgcagttgggttaaga
atactgggcaatttcatgtttcttcaacactacatatgcgtatatataccaatctaagtctgtg
ctccttccttcgttcttccttctgttcggagattaccgaatcaaaaaaatttcaaagaaaccga
aatcaaaaaaaagaataaaaaaaaaatgatgaattgaattgaaaagctagcttatcgatgggtc
cttttcatcacgtgctataaaaataattataatttaaattttttaatataaatatataaattaa
aaatagaaagtaaaaaaagaaattaaagaaaaaatagtttttgttttccgaagatgtaaaagac
tctagggggatcgccaacaaatactaccttttatcttgctcttcctgctctcaggtattaatgc
cgaattgtttcatcttgtctgtgtagaagaccacacacgaaaatcctgtgattttacattttac
ttatcgttaatcgaatgtatatctatttaatctgcttttcttgtctaataaatatatatgtaaa
gtacgctttttgttgaaattttttaaacctttgtttatttttttttcttcattccgtaactctt
ctaccttctttatttactttctaaaatccaaatacaaaacataaaaataaataaacacagagta
aattcccaaattattccatcattaaaagatacgaggcgcgtgtaagttacaggcaagcgatctc
taagaaaccattattatcatgacattaacctataaaaaaggcctctcgagctagagtcgatctt
cgccagcagggcgaggatcgtggcatcaccgaaccgcgccgtgcgcgggtcgtcggtgagccag
agtttcagcaggccgcccaggcggcccaggtcgccattgatgcgggccagctcgcggacgtgct
catagtccacgacgcccgtgattttgtagccctggccgacggccagcaggtaggccgacaggct
catgccggccgccgccgccttttcctcaatcgctcttcgttcgtctggaaggcagtacaccttg
ataggtgggctgcccttcctggttggcttggtttcatcagccatccgcttgccctcatctgtta
cgccggcggtagccggccagcctcgcagagcaggattcccgttgagcaccgccaggtgcgaata
agggacagtgaagaaggaacacccgctcgcgggtgggcctacttcacctatcctgcccggctga
cgccgttggatacaccaaggaaagtctacacgaaccctttggcaaaatcctgtatatcgtgcga
aaaaggatggatataccgaaaaaatcgctataatgaccccgaagcagggttatgcagcggaaaa
gcgctgcttccctgctgttttgtggaatatctaccgactggaaacaggcaaatgcaggaaatta
ctgaactgaggggacaggcgagagacgatgccaaagagctacaccgacgagctggccgagtggg
ttgaatcccgcgcggccaagaagcgccggcgtgatgaggctgcggttgcgttcctggcggtgag
ggcggatgtcgatatgcgtaaggagaaaataccgcatcaggcgcatatttgaatgtatttagaa
aaataaacaaaaagagtttgtagaaacgcaaaaaggccatccgtcaggatggccttctgcttaa
tttgatgcctggcagtttatggcgggcgtcctgcccgccaccctccgggccgttgcttcgcaac
gttcaaatccgctcccggcggatttgtcctactcaggagagcgttcaccgacaaacaacagata
aaacgaaaggcccagtctttcgactgagcctttcgttttatttgatgcctgg
序列57:T.pseudoethanolicus Lldh KO单交换
cagcacaaaaaccgggagaaaccagacttgacttagtgaagagaaatacgatgatttttaaaga
catagtggcaaaacttattaaagtaaatgacacagcaatataccttatagttacaaatccagta
gatattcttacatacgttacctataaaatatctggcttgccatacggaagagtattggggtctg
gcacagttctcgacagtgcgagattcagatatcttttaagcaaacattgtaacatagatccgag
gaatatacacggatatataattggggagcatggcgattctgagcttgcagcttggagcattacg
aacatagcaggcataccaattgataattactgcaatttatgtggaaaagcatgtgaaaaagatt
ttagagaggagatttttaataatgttgtaagagctgcctatacgataatagaaaaaaagggtgc
gacatattatgcggttgctctcgcagtaagaagaatcgtagaagctattaaacccagcgaacca
tttgaggtgataggtaagattataccgaggtatgaaaacgagaattggacctttacagaattac
tctatgaagcgccatatttaaaaagctaccaagacgaagaggatgaagaggatgaggaggcaga
ttgccttgaatatattgacaatactgataagataatatatcttttatatagaagatatcgccgt
atgtaaggatttcagggggcaaggcataggcagcgcgcttatcaatatatctatagaatgggca
aagcataaaaacttgcatggactaatgcttgaaacccaggacaataaccttatagcttgtaaat
tctatcataattgtggtttcaaaatcggctccgtcgatactatgttatacgccaactttcaaaa
caactttgaaaaagctgttttctggtatttaaggttttagaatgcaaggaacagtgaattggag
ttcgtcttgttataattagcttcttggggtatctttaaatactgtagaaaagaggaaggaaata
ataaatggctaaaatgagaatatcaccggaattgaaaaaactgatcgaaaaataccgctgcgta
aaagatacggaaggaatgtctcctgctaaggtatataagctggtgggagaaaatgaaaacctat
atttaaaaatgacggacagccggtataaagggaccacctatgatgtggaacgggaaaaggacat
gatgctatggctggaaggaaagctgcctgttccaaaggtcctgcactttgaacggcatgatggc
tggagcaatctgctcatgagtgaggccgatggcgtcctttgctcggaagagtatgaagatgaac
aaagccctgaaaagattatcgagctgtatgcggagtgcatcaggctctttcactccatcgacat
atcggattgtccctatacgaatagcttagacagccgcttagccgaattggattacttactgaat
aacgatctggccgatgtggattgcgaaaactgggaagaagacactccatttaaagatccgcgcg
agctgtatgattttttaaagacggaaaagcccgaagaggaacttgtcttttcccacggcgacct
gggagacagcaacatctttgtgaaagatggcaaagtaagtggctttattgatcttgggagaagc
ggcagggcggacaagtggtatgacattgccttctgcgtccggtcgatcagggaggatatcgggg
aagaacagtatgtcgagctattttttgacttactggggatcaagcctgattgggagaaaataaa
atattatattttactggatgaattgttttagtacctagatttagatgtctaaaaagctttttag
acatctaatcttttctgaagtacatccgcaactgtccatactctgatgttttatatcttttcta
aaagttcgctagataggggtcccgagcgcctacgaggaatttgtatcgctgcaggcatgcaagc
ttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacaca
acatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacatt
aattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatga
atcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactg
actcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacg
gttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggcc
aggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatc
acaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtt
tccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtcc
gcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcgg
tgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgc
cttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagca
gccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggt
ggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttac
cttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttt
tttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatctttt
ctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatc
aaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtata
tatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatct
gtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggaggg
cttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagattta
tcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcct
ccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcg
caacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattc
agctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggtta
gctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttat
ggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgag
tactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaa
tacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttc
ggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgca
cccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggc
aaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttt
tcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatt
tagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaag
aaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgc
gcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgt
ctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtc
ggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtga
aataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgc
aactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggat
gtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgac
ggccagtgaattc
序列58:T.pseudoethanolicus Lldh KO双交换
aggtttatcgccgccttgtacagttttaaattgcatagaaatcatgagacccattgcaataaat
acaagtatcaaagataaagtttgcaatgcctttcctttcaatttctccacatcctttctctata
taaaaagacatcttcgtcttgcttttggtttcagcttatatgcacttttataaataactatgat
actctataaatactataacatggaaaatgttaaaatttattaagaaattattaagtttttatta
caaaaaagttacaaaacctctgacatttttcatatcagaggttgtcattttttattttattttc
tatagaattttttagtgacaatatttcttctaattctttattgtatttatctattttcaacatg
gtacttctatataggcgtatatcttcttcgtttttttgtatacattttttaagggagtttttta
cagtttcaaaaagcgtatcataagtaatgtaattatgcatttcaaggtcggagattggaactgc
gattaattcctccccttctattttatagtgataaaaaatgttgtcgggttttaaaactattttt
tcatctatccttttatcgtaaattattataccttctcctatttcaaaagtttttccgctataac
ttctaagttttatattttcgactcctatgtatttttttattgccaaaagtatgttttttatttc
tgaaatggattttacaagcacttcttgcatttttttatttgccatctctttatctttttcactt
tttatgagttcttccatcaaagactttatttcatgtatatctcccataaaatatcacctctttc
ttaatattccacagaggaatcattttaaacgttgaatattttaaattattagagaaaaaataga
cttgactattttttgaaatttgatagactattattaatagaaaattaatattgaaaaggagaag
atattatgaacaaaatatctataataggttctggatttgtcggaaacccagcgaaccatttgag
gtgataggtaagattataccgaggtatgaaaacgagaattggacctttacagaattactctatg
aagcgccatatttaaaaagctaccaagacgaagaggatgaagaggatgaggaggcagattgcct
tgaatatattgacaatactgataagataatatatcttttatatagaagatatcgccgtatgtaa
ggatttcagggggcaaggcataggcagcgcgcttatcaatatatctatagaatgggcaaagcat
aaaaacttgcatggactaatgcttgaaacccaggacaataaccttatagcttgtaaattctatc
ataattgtggtttcaaaatcggctccgtcgatactatgttatacgccaactttcaaaacaactt
tgaaaaagctgttttctggtatttaaggttttagaatgcaaggaacagtgaattggagttcgtc
ttgttataattagcttcttggggtatctttaaatactgtagaaaagaggaaggaaataataaat
ggctaaaatgagaatatcaccggaattgaaaaaactgatcgaaaaataccgctgcgtaaaagat
acggaaggaatgtctcctgctaaggtatataagctggtgggagaaaatgaaaacctatatttaa
aaatgacggacagccggtataaagggaccacctatgatgtggaacgggaaaaggacatgatgct
atggctggaaggaaagctgcctgttccaaaggtcctgcactttgaacggcatgatggctggagc
aatctgctcatgagtgaggccgatggcgtcctttgctcggaagagtatgaagatgaacaaagcc
ctgaaaagattatcgagctgtatgcggagtgcatcaggctctttcactccatcgacatatcgga
ttgtccctatacgaatagcttagacagccgcttagccgaattggattacttactgaataacgat
ctggccgatgtggattgcgaaaactgggaagaagacactccatttaaagatccgcgcgagctgt
atgattttttaaagacggaaaagcccgaagaggaacttgtcttttcccacggcgacctgggaga
cagcaacatctttgtgaaagatggcaaagtaagtggctttattgatcttgggagaagcggcagg
gcggacaagtggtatgacattgccttctgcgtccggtcgatcagggaggatatcggggaagaac
agtatgtcgagctattttttgacttactggggatcaagcctgattgggagaaaataaaatatta
tattttactggatgaattgttttagtacctagatttagatgtctaaaaagctttttagacatct
aatcttttctgaagtacatccgcaactgtccatactctgatgttttatatcttttctaaaagtt
cgctagataggggtcccgagcgcctacgaggaatttgtatcgaagatcagccgaagttatcaaa
agtgtaatacaagagcttgatatataagaggggaaaccctctttttttgtatataaaaagtcac
agcgtgaaaatataataattaaaataatgattttttagggtgtgatagtcgtgcagaaaataac
tcagcaggagattattttaagtgcctttgttgaagcacaaaatttagaaaagatactgttggat
aaagtaagagaatatgggaaagaatcagtagataatcaaataaaagcattgttaaagcaaattg
aaataatgataaaaaatcataaagaagacataaaaaaggcacaaaagactatgcatattaattc
ccttgtcaaaaaaaatatgtctcaagagcctttagacatgcttcaagatttattaaaaaattta
gttaatattcaagccttttataatgaaactgttgtgaatattactaatccttacgttagacagt
tgtttactcaaatgagggatgatgttatgagatttatttctattcttcaaatggagattgaaag
tctggaatcgaaaccttctattccaaataacacagttttaaatacaccggagatgagttaatat
gaaagtggctattattggtgctggtgtttcagggctggctgcggcaattacttttcaaaggtat
ggcattacaccagatatttttgaaaaaaagtgcaaaataggtgaattatttaaccatgttgcgg
ggttattaaaagtgataaataggcctataaaggatccgcttcatcatcttaaaaatgtttatgg
aatagaagttaaaccaattaacaccattgacaaaatagtaatgaaggggccaactgtaacagct
tctgttactgggagtaatcttgggtatatgattttaagaggacaggacgcaaactctcttgaaa
atcaattgtataataagttagaaataccagttaatttcaatatagaagctgattataagaagtt
aaaaaataattacgattatgtctgcaggcatgcaagcttggcgtaatcatggtcatagctgttt
cctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgta
aagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgcttt
ccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggt
ttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgc
ggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgc
aggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctg
gcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggt
ggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctc
tcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcg
ctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggct
gtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtc
caacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcg
aggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagga
cagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttg
atccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgc
agaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacg
aaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatcctttt
aaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttac
caatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcct
gactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaat
gataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagg
gccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccggg
aagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcat
cgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcga
gttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtca
gaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgt
catgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatag
tgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagca
gaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttacc
gctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttact
ttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataaggg
cgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcaggg
ttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccg
cgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacct
ataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacct
ctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaa
gcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcag
agcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaa
ataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgg
gcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaa
cgccagggttttcccagtcacgacgttgtaaaacgacggccagtgaattc
序列59:T.pseudoethanolicus ack KO单交换
tattgattgacgatgaggtaatcaaaaaattagaagcatgtattgaccttgcacctttgcacaa
tcctgctaatattgagggaataaaagcttgtcggcagataatgccaggggtgccaatggtagca
gtttttgatacggctttccatcaaacaatgccagattatgcgtatatttatcccattccttatg
aatactacgaaaaatatagaataagaagatatggattccatgggacttctcataaatatgtatc
tttaagagctgctgaaatattaaagaggcctattgaagagttaaaaattattacttgccattta
gggaatgggtctagtattgctgcggttaaaggcggtaagtcgatagatacaagtatgggattta
ctccattagaagggctggctatgggtacaaggtccggaaatgttgatccttcaattataacttt
cttaatggaaaaagaaggattgactgcagaacaggttatagatatacttaataagaaatcaggt
gtatacggaatttcaggaataagtaatgactttagagatatagaaaacccagcgaaccatttga
ggtgataggtaagattataccgaggtatgaaaacgagaattggacctttacagaattactctat
gaagcgccatatttaaaaagctaccaagacgaagaggatgaagaggatgaggaggcagattgcc
ttgaatatattgacaatactgataagataatatatcttttatatagaagatatcgccgtatgta
aggatttcagggggcaaggcataggcagcgcgcttatcaatatatctatagaatgggcaaagca
taaaaacttgcatggactaatgcttgaaacccaggacaataaccttatagcttgtaaattctat
cataattgtggtttcaaaatcggctccgtcgatactatgttatacgccaactttcaaaacaact
ttgaaaaagctgttttctggtatttaaggttttagaatgcaaggaacagtgaattggagttcgt
cttgttataattagcttcttggggtatctttaaatactgtagaaaagaggaaggaaataataaa
tggctaaaatgagaatatcaccggaattgaaaaaactgatcgaaaaataccgctgcgtaaaaga
tacggaaggaatgtctcctgctaaggtatataagctggtgggagaaaatgaaaacctatattta
aaaatgacggacagccggtataaagggaccacctatgatgtggaacgggaaaaggacatgatgc
tatggctggaaggaaagctgcctgttccaaaggtcctgcactttgaacggcatgatggctggag
caatctgctcatgagtgaggccgatggcgtcctttgctcggaagagtatgaagatgaacaaagc
cctgaaaagattatcgagctgtatgcggagtgcatcaggctctttcactccatcgacatatcgg
attgtccctatacgaatagcttagacagccgcttagccgaattggattacttactgaataacga
tctggccgatgtggattgcgaaaactgggaagaagacactccatttaaagatccgcgcgagctg
tatgattttttaaagacggaaaagcccgaagaggaacttgtcttttcccacggcgacctgggag
acagcaacatctttgtgaaagatggcaaagtaagtggctttattgatcttgggagaagcggcag
ggcggacaagtggtatgacattgccttctgcgtccggtcgatcagggaggatatcggggaagaa
cagtatgtcgagctattttttgacttactggggatcaagcctgattgggagaaaataaaatatt
atattttactggatgaattgttttagtacctagatttagatgtctaaaaagctttttagacatc
taatcttttctgaagtacatccgcaactgtccatactctgatgttttatatcttttctaaaagt
tcgctagataggggtcccgagcgcctacgaggaatttgtatcgctgcaggcatgcaagcttggc
gtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacata
cgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattg
cgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcgg
ccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcg
ctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttat
ccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaa
ccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaa
aatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttcccc
ctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctt
tctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtag
gtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttat
ccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccac
tggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcct
aactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcg
gaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgt
ttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacg
gggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaa
ggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatga
gtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtcta
tttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttac
catctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagc
aataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatc
cagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacg
ttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctc
cggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctcc
ttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcag
cactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactc
aaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgg
gataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggc
gaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaa
ctgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaat
gccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaat
attattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaa
aaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaacc
attattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtt
tcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgta
agcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggc
tggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaatac
cgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactg
ttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgct
gcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggcca
gtgaattc
序列60:T.pseudoethanolicus pta/ack KO双交换
gttttataagaagctttgtaaattttattttctaagccttcagagacatcaaatatgtgattga
gaggaatgtgatttctcaacagataaattaaaatattagaaaattcctctaaagatacgggacc
ttgttttttttcaaaagaactttgcaaaatttcttttgtaaagtccgggatatatttttctaag
ccttctagtcccttttcaaaaatatgctttcttatagaagaggcactggcaaattctcctttta
gctctaaagaggtgtacaaagagccttttctctttatagtaaaaggtgtaatagaactacctat
tttctttaaagatttaaggtattctattgccaatatgttattggatgtttgtaaaatcttttct
atttcattattatttataactttttgtaatgctaattcccgtgcttttgcaaaggttatgccgc
tttttaaatattcttttaatgcttttctataataaattggctcttctaaaagtatttcagcaat
ttttgtgagttcgtttaaatcgcctttttcactcccaaaggaaaaacaatctactatttttaaa
gagtctaatagtttcaccgctccataagcgaaattttcagctgtagaggtagcataaactactg
gtaactcgattaccaaatctataccggcttttaatgccatttgagttcgtttccatttgtctac
aattgctggttctcctctttgcacgaagtttccactcattactgctatagtataatcgcatttg
gttaattcttttgaagtttgcagatggtaaaggtggccattgtgaaaaggattatattcgacaa
taattcctaaaattcccatacaacttcttaccctttcaaaaaattttttaagatatacttatta
ttttacataaaatatgataaaatgtaaaagggacatcgtgtatacaatattatagtgataaaat
taaaaaaggaagggagattttaaatggcagtaatggatagtaaaacccagcgaaccatttgagg
tgataggtaagattataccgaggtatgaaaacgagaattggacctttacagaattactctatga
agcgccatatttaaaaagctaccaagacgaagaggatgaagaggatgaggaggcagattgcctt
gaatatattgacaatactgataagataatatatcttttatatagaagatatcgccgtatgtaag
gatttcagggggcaaggcataggcagcgcgcttatcaatatatctatagaatgggcaaagcata
aaaacttgcatggactaatgcttgaaacccaggacaataaccttatagcttgtaaattctatca
taattgtggtttcaaaatcggctccgtcgatactatgttatacgccaactttcaaaacaacttt
gaaaaagctgttttctggtatttaaggttttagaatgcaaggaacagtgaattggagttcgtct
tgttataattagcttcttggggtatctttaaatactgtagaaaagaggaaggaaataataaatg
gctaaaatgagaatatcaccggaattgaaaaaactgatcgaaaaataccgctgcgtaaaagata
cggaaggaatgtctcctgctaaggtatataagctggtgggagaaaatgaaaacctatatttaaa
aatgacggacagccggtataaagggaccacctatgatgtggaacgggaaaaggacatgatgcta
tggctggaaggaaagctgcctgttccaaaggtcctgcactttgaacggcatgatggctggagca
atctgctcatgagtgaggccgatggcgtcctttgctcggaagagtatgaagatgaacaaagccc
tgaaaagattatcgagctgtatgcggagtgcatcaggctctttcactccatcgacatatcggat
tgtccctatacgaatagcttagacagccgcttagccgaattggattacttactgaataacgatc
tggccgatgtggattgcgaaaactgggaagaagacactccatttaaagatccgcgcgagctgta
tgattttttaaagacggaaaagcccgaagaggaacttgtcttttcccacggcgacctgggagac
agcaacatctttgtgaaagatggcaaagtaagtggctttattgatcttgggagaagcggcaggg
cggacaagtggtatgacattgccttctgcgtccggtcgatcagggaggatatcggggaagaaca
gtatgtcgagctattttttgacttactggggatcaagcctgattgggagaaaataaaatattat
attttactggatgaattgttttagtacctagatttagatgtctaaaaagctttttagacatcta
atcttttctgaagtacatccgcaactgtccatactctgatgttttatatcttttctaaaagttc
gctagataggggtcccgagcgcctacgaggaatttgtatcgtgactttagagatatagaaaatg
cagcttttaaagaagggcataaaagggctatgttggcattaaaagttttcgcttatagggtgaa
aaagacaataggttcttatacagctgctatgggtggggttgatgtaattgtgtttactgctgga
gttggagaaaatggaccagaaatgagagagtttattttagaggatctagagtttttaggcttta
aactggacaaagagaagaataaggtaagaggaaaagaggaaattatatctacagaagattcaaa
agttaaagttatggttattcctacaaatgaagaatatatgattgctaaagatactgaaaaattg
gtaaaaggtttaaagtagataatcttgacaacgggttgtggggttagtataataggtgatgtca
attattttaaggtgtgagaagaaaaatgaaaatcgatctattaaaaatcaaaggacagcttggc
cgcagcataaatatagactatgtagaggacatagagaacattgaatttaaaggggaagaataca
aactgcaggcatgcaagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatc
cgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatg
agtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcg
tgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctctt
ccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctca
ctcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagca
aaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctcc
gcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggact
ataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccg
cttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgct
gtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgt
tcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgac
ttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgcta
cagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgc
tctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccacc
gctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaag
aagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggat
tttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagtttt
aaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgagg
cacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagat
aactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgc
tcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtc
ctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttc
gccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcg
tttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgt
tgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagt
gttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgc
ttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagtt
gctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcat
cattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcg
atgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggt
gagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaat
actcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcgga
tacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaag
tgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcac
gaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccg
gagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcag
cgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagt
gcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccat
tcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgcc
agctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtc
acgacgttgtaaaacgacggccagtgaattc
序列64:pMU360的DNA序列
aattcgagctcggtacccggggatcctctagagtcgacctgcaggcatgcaagggaagatatgc
ctgcttgacattattgtccgtcatttttcggtttatcctggtaaaaaaagttttaatcctctca
aggctttcttcgtgtaaaacaagctttttccccaaaacttccgcaacagtctcctttgtaaggt
catcctgcgtggggccgagtcctccggtcataataacaaggtcgcacctttccaaagctgcaag
aagacattttttcagccgaacggaattgtcccccaccacactgtgataatacacattcacacca
atgtcattgagccttttggatatatactgggcattggtatttgctatctgccccattaaaagct
cggttccaaccgctaatatctccgcattcatattgaaagaccccttaaatttaaactttttgta
acttattatatcaattagtgttataaaataaaagggaaaaagaattaaaatcaaaggtttcaag
agcagccgtatcacccgtaaaagtttcagccgattcaacctttttacacataaaactttcaaaa
attgatgacttacaattatcaagtaggatataatattactaatgctaaacagttattgataaag
gaggaaggaatatgaacaataacaaagtaattaaaaaagtaacccggactttctgagaagctga
atttcttcatcgttgaaaggcacgttcaatatttcctcaataccgtttacacccacgattgtcg
gaacacttaagcatacatcgctaagtccgtactgtccttccaaaaggcttgaaacggtaaggat
ggagttttcatttcttacaatggcttcaacgattcttcttacggcaagggctacggcataatag
gttgcacctttgttcctgatgatttcataagctgcatttttaacactttcatatattttattcc
gggaaatctgctcctcgcactgatggcattcgtcacagtagcgatccatgggaattcccgcaat
atttgcaagactccaggccgcaacttcggtgtcaccgtgttcgccaataatataagcatgtaca
tttcgtgcatccacttttacatgttcgcttaaaagataacggaacctggctgtgtccaaaaccg
ttccggaacctattactttgtttttcgggaatccggatagtttgtaagttacataggttaaaat
atccaccggatttgtgactaccagaagaatacaatcgttgttgtactttacaatttcatttatg
atatttttgaatacttccgtgtttcttttaacaagatctattctcgtttcgccttctttttggt
tggcaccggcggtaatgattactatgtcggatccggcacagtctttgtagtcaccacgataaat
ttcaacgggccttacaaaaggcatgccgtgatttaagtccatgacttctccgtcggcttttttt
gcatttatgtctatcagtacaatttcagatataagtccgctgagcatcaatgtataagctgtgg
tggaacctacaaagcctgcaccaactacgaaacactctaaaagaaataataaaaacactagata
tatgaaagttctccttttcttttatgaaaaggagaactttcattattgataaatatataaacta
gtatataattttaatataaaacctattttacataatggaaattatctatcgggggaggaaatat
gaacaattcagtggaaattttaaataaaatcgtgtcaaatattgaaaaagtcattgttggaaaa
aagaaagctatcgagttgatattaatatcacttatttgcgatggacatgttttgattgaagatg
tccccggtgtcggaaaaaccagtacttggcgtaatcatggtcatagctgtttcctgtgtgaaat
tgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtg
cctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaa
cctgtcgtgccagcccttcaaacttcccaaaggcgagccctagtgacattagaaaaccgactgt
aaaaagtacagtcggcattatctcatattataaaagccagtcattaggcctatctgacaattcc
tgaatagagttcataaacaatcctgcatgataaccatcacaaacagaatgatgtacctgtaaag
atagcggtaaatatattgaattacctttattaatgaattttcctgctgtaataatgggtagaag
gtaattactattattattgatatttaagttaaacccagtaaatgaagtccatggaataatagaa
agagaaaaagcattttcaggtataggtgttttgggaaacaatttccccgaaccattatatttct
ctacatcagaaaggtataaatcataaaactctttgaagtcattctttacaggagtccaaatacc
agagaatgttttagatacaccatcaaaaattgtataaagtggctctaacttatcccaataacct
aactctccgtcgctattgtaaccagttctaaaagctgtatttgagtttatcacccttgtcacta
agaaaataaatgcagggtaaaatttatatccttcttgttttatgtttcggtataaaacactaat
atcaatttctgtggttatactaaaagtcgtttgttggttcaaataatgattaaatatctctttt
ctcttccaattgtctaaatcaattttattaaagttcatttgatatgcctcctaaatttttatct
aaagtgaatttaggaggcttacttgtctgctttcttcattagaatcaatccttttttaaaagtc
aatcccgtttgttgaactactctttaataaaataatttttccgttcccaattccacattgcaat
aatagaaaatccatcttcatcggctttttcgtcatcatctgtatgaatcaaatcgccttcttct
gtgtcatcaaggtttaattttttatgtatttcttttaacaaaccaccataggagattaaccttt
tacggtgtaaaccttcctccaaatcagacaaacgtttcaaattcttttcttcatcatcggtcat
aaaatccgtatcctttacaggatattttgcagtttcgtcaattgccgattgtatatccgattta
tatttatttttcggtcgaatcatttgaacttttacatttggatcatagtctaatttcattgcct
ttttccaaaattgaatccattgtttttgattcacgtagttttctgtattcttaaaataagttgg
ttccacacataccaatacatgcatgtgctgattataagaattatctttattatttattgtcact
tccgttgcacgcataaaaccaacaagatttttattaatttttttatattgcatcattcggcgaa
atccttgagccatatctgacaaactcttatttaattcttcgccatcataaacatttttaactgt
taatgtgagaaacaaccaacgaactgttggcttttgtttaataacttcagcaacaaccttttgt
gactgaatgccatgtttcattgctctcctccagttgcacattggacaaagcctggatttacaaa
accacactcgatacaactttctttcgcctgtttcacgattttgtttatactctaatatttcagc
acaatcttttactctttcagcctttttaaattcaagaatatgcagaagttcaaagtaatcaaca
ttagcgattttcttttctctccatggtctcacttttccactttttgtcttgtccactaaaaccc
ttgatttttcatctgaataaatgctactattaggacacataatattaaaagaaacccccatcta
tttagttatttgtttggtcacttataactttaacagatggggtttttctgtgcaaccaatttta
agggttttcaatactttaaaacacatacataccaacacttcaacgcacctttcagcaactaaaa
taaaaatgacgttatttctatatgtatcaagaatagaaagaactcgtttttcgctacgctcaaa
acgcaaaaaaagcactcattcgagtgctttttcttatcgctccaaatcatgcgattttttcctc
tttgcttttctttgctcacgaagttctcgatcacgctgcaaaacatcttgaagcgaaaaagtat
tcttcttttcttccgatcgctcatgctgacgcacgaaaagccctctaggcgcataggaacaact
cctaaatgcatgtgaggggttttctcgtccatgtgaacagtcgcatacgcaatattttgtttcc
catactgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttcc
gcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcact
caaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaa
aggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgc
ccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactat
aaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgct
taccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgt
aggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttc
agcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgactt
atcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctaca
gagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctc
tgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgc
tggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaa
gatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattt
tggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaa
atcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggca
cctatctcagcgatctgtc
序列62
ATTTACCTGGCTGGGAATACTGAGACATAT-intron-GTCATTGAGGCCGTA
序列63
AAAAAAGCTTATAATTATCCTTAATTTCCTACTACGTGCGCCCAGATAGGGTG
序列64
CAGATTGTACAAATGTGGTGATAACAGATAAGTCTACTACTGTAACTTACCTTTCTTTGT
序列65
TGAACGCAAGTTTCTAATTTCGGTTGAAATCCGATAGAGGAAAGTGTCT
序列66
TTAAATGTTGATAAGGAAGCTCTTTTCAAT-intron-GAAGTTAAGGTAGCA
序列67
AAAAAAGCTTATAATTATCCTTAGCTCTCTTCAATGTGCGCCCAGATAGGGTG
序列68
CAGATTGTACAAATGTGGTGATAACAGATAAGTCTTCAATGATAACTTACCTTTCTTTGT
序列69
TGAACGCAAGTTTCTAATTTCGATTAGAGCTCGATAGAGGAAAGTGTCT
序列70:C.kristjanssonii木酮糖激酶基因
ATGTATTTTATTGGAATTGACGTTGGAACATCTGGAACAAAGACAATCCTGATTGACTCAAAAG
GTAAGATTCTGGCTTCTGCAACCTTTGAATATCCTCTTTATCAGCCTCAGATTGGCTGGGCTGA
GCAAAATCCCGAAGACTGGTGGGATGCAAGCGTAAAAGGAATTAAAGCTGTGCTTGAAAAGTCA
AAAGTAGACCCCAAGGAAGTTAAGGCTGTGGGACTTACCGGGCAGATGCACGGGCTTGTGCTGC
TTGACAAAAACTACAACGTTATAAGACCATCAATCATCTGGTGTGACCAGAGAACGGCAAAAGA
ATGTGATGAAATAACAGAAAAGGTTGGCAAGGAAAAGCTTGTGGAGATTACAGCAAACCCTGCA
CTGACAGGTTTTACAGCGTCCAAGATTCTGTGGGTGAGAAACAACGAGCCCCAAAACTATGAGA
AGGTCTACAAAATTTTGCTTCCCAAAGACTATATAAGGTTTAAACTTACAGGCGAGTTTGCAAC
AGATGTGTCGGACGCCTCGGGTATGCAGCTTTTGGACATTAAAAACAGGTGCTGGTCTGATGAG
GTACTTGAAAAGCTTGAGATAGACAAAGGGCTTCTTGGAAAAGTCTATGAGTCGCCAGAGGTAA
CGGGAAAAGTTAGCGGGCAAGCAAGCGAACTTACAGGTCTTTGTGAAGGTACGCTTGTTGTTGC
AGGTGGAGGAGACCAGGCAGCAGGTGCAGTTGGAAATGGCATAGTAAAGACGGGTGTGATTTCA
TCTACAATTGGTTCGTCTGGCGTTGTTTTTGCCCATCTTGACGAGTTTAAGATTGACCCACAGG
GAAGGGTTCACACATTTTGTCATGCAGTGCCGGGAAAATGGCATGTGATGGGTGTAACACAAGG
TGCCGGACTTTCTCTCAAGTGGTTTAGAGACAACTTTGCACACATCGAAAAGGCTGCGTTTGAG
TTTATTGACAAAGACCCATACATTTTGATGGACCAGGAGGCAGAACTTGCAAACCCAGGCGCAG
ACGGACTTGtTTTCCTGCCATATTTGATGGGGGAAAGAACGCCCATTTTGGACCCATACGCCAA
AGGAATATTCTTTGGAATAACAGCAAAGCATACACGAAGAGAGTTCATTAGAGCTGTCATGGAA
GGTGTTGTATTTTCACTTAAAAACTGTCTTGATATTTTGTATGAGATGGGCATCGAGGTGAAGG
AGGTAAGAGTTTCAGGCGGTGGTGCAAAGAGCAAGCTCTGGAGACAGATGCAGGCAGACATATT
TGAGATGGATGTATGGACACTGAATTCCAAAGAAGGACCTGCGTTTGGTGCAGCTATCCTGGCA
GCAGTTGGTGCAGGAGAATATCAGAAGGTTGAAGAAGCCTGTGATACTATGATTCAAAAGGTAG
ATAACTGCAGCCCAAATGAAAAACTATTTGAAATATATAGAAAAACTTATAAACTTTACAACAG
TATATATCCAAGAGTTAAGGACTTATTCAACATGTAA
序列71:C.kristjanssonii木糖异构酶基因
ATGAAATACTTCAAAGACATTCCAGAAGTAAAATATGAAGGACCACAGTCAGACAATCCATTTG
CTTTCAAGTATTACAATCCTGACGAGGTTATTGATGGCAAGCCTTTAAAAGACCACCTTCGTTT
TGCAATTGCTTACTGGCACACGTTCTGTGCAACCGGTAGCGACCCTTTTGGACAACCTACAATT
AATCGTCCATGGGACAGGTTCTCAAACCCAATGGACAATGCAAAAGCAAGAGTTGAAGCTGCAT
TTGAATTTTTTGAAAAGCTAAATGTTCCATTTTTCTGCTTCCACGACAGAGACATCGCACCTGA
AGGAGAAAATTTAAGAGAAACAAACAAGAATTTGGATGAGATAGTCTCTATGATAAAAGAATAT
TTAAAGACAAGCAAAACAAGAGTTTTGTGGGGAACAGCAAACCTATTTTCACATCCGCGATATG
TTCATGGTGCTGCAACATCCTGCAATGCCGATGTTTTTGCGTATGCAGCAGCGCAGGTGAAAAA
GGCGTTAGAGGTTACAAAAGAGCTTGGCGGCGAAAACTATGTGTTCTGGGGCGGAAGAGAAGGT
TATGAGACACTTTTGAACACCGACATGGAGCTTGAGCTTGACAACTTGGCAAGATTTTTGCACA
TGGCAGTTGACTATGCAAAAGAGATAGGGTTTGACGGTCAGTTTTTGATTGAACCAAAGCCAAA
AGAACCAACTAAGCATCAGTACGATTTTGATGCCGCTCATGTTTATGGATTTTtGAAAAAaTAT
GACCTTGACAAGTACTTCAAGCTCAACATAGAGGTAAACCATGCGACTTTGGCAGGACATGATT
TCCACCATGAGTTGAGATTTGCACGAATAAACAACATGCTTGGCTCAATTGATGCTAACATGGG
CGACTTGCTTTTGGGCTGGGATACAGACCAGTTCCCAACAGATGTAAGACTTACCACGCTTGCT
ATGTATGAGGTTATTAAAGCTGGTGGCTTTGACAAAGGCGGGCTCAACTTTGACGCAAAGGTAA
GAAGAGGTTCTTTTGAGCTTGAAGACTTGGTCATTGGTCACATTGCAGGGATGGACGCTTTTGC
AAAAGGATTTAAGATAGCATATAAGCTTGTCAAAGACGGTGTATTTGATAAGTTTATTGAAGAA
AGGTATAGAAGCTACAAAGAAGGAATTGGAGCTAAGATTGTAAGTGGTCAGGCAGATTTTAAGA
CGTTAGAAGAATATGCTTTGAATCTTTCAAAGATAGAAAACAAATCTGGCAAGCAAGAGCTTCT
TGAGATGATTTTGAACAAATATATGTTCAGTGAATAA
序列72:C.straminsolvens木酮糖激酶基因
TTGTCATATTTACTGGGAGTAGATATAGGTACATCAGGCACGAAAACTGTTTTATATGATGAAC
TGGGAAATACCGTAGCAAGCAGCCTTGAGGAATATCCATTGTACCAGCCCCATATTGGGTGGGC
AGAGCAGGAACCGGAAGACTGGTGGAGGGCAACATGCCTATCTATCAAACATGTTATTTCCAAA
AGAGGAATTGATGCTTCCTCTATTAAGGGAATCGGACTTTCAGGACAGATGCACGGGGCTGTTC
TTCTGGACAAAGACGGCAAAGTGCTAAGAAAAGCAATTATATGGTGTGACCAGAGAAGTTTTGC
CGAGTGCGAGCAGATTACTTCAATTATAGGGAAGGAAAGGCTCGTTGAGATAACTGCCAACCCT
GCACTGACGGGATTTACAGCATCAAAGGTTATGTGGGTTAAAAATAATGAACCTGAAATTTTTG
AGAAGATTTATAAGATACTTCTCCCTAAAGACTATATAAGATATAAATTAACGGGAGAATTTGC
TACAGAGGTATCTGATGCCAGTGGAATGCAGTTTATGGATATACCGGGGAGAAAATGGAGCGAC
GAAGTCATAAGTAAACTCGGACTTGATAAAAGCATGCTGGGAGAACTCTATGAGTCTCAGGAAG
TTAGCGGGAAAGTGAATAAGTATGCTGCTTCATTAACCGGACTTAAGGAAGGAACTCCTGTCGT
GGGTGGAGCAGGAGACCAGGCAGCAGGAGCTGTCGGTAATGGAATTGTGAGACCCGGGGTGGTT
TCATCCACTATAGGAACTTCAGGAGTAGTATTTGCATTCTCTGAAAAGGTTACTATTGATCCAA
AGGGTAGAGTTCATACTTTTTGTCATGCGGTACCAAATACCTGGCACATTATGGGGGTTACACA
AGGGGCCGGGCTGTCTCTTAAGTGGTTCCGTGACAATTTCTGTATAGAAGAAAAGAGAACTGCA
GAGCTAATGAAAATAGACCCGTACATAATTATGGATAAAGAAGCTGAAAAAGTGGCTCCGGGCT
GTAACGGTTTAATCTATTTACCTTATCTGATGGGAGAAAGAACGCCACATCTTGACCCTAATGC
CAAGGGTGTCTTTTTCGGATTAACAGCAAAGCATGAAAAACAGGATATCTTAAGGTCGATTATG
GAAGGTGTTGTATATAGCCTTAGAGATTGCCTTGAAATTATTGAGGAAATGGGTGTTAACGTTT
CTGAAGTAAGAGCTTCCGGTGGAGGCGGTAAAAGTGAATTGTGGAGAAAAATGCAGGCGGATAT
ATTCGGCACTGATATTACAACCGTAAAGTCAAGTGAGGGACCGGCACTTGGGGTAGCACTTCTT
GCCGGAGTAGGAACGGGTGTGTACAACAACATTAATGAAGCATGTGAAGCAGTAATAAAAGAAA
ATACCCGGCAGGCTTCGGACCCGGAGCTATATGTAAAATACACGAAGTTTTATGATATTTATAA
ACGTCTGTATAACTCTTTGAAAAAGGAATTTGCAGACCTTTCGGCTATGCTGCAAAGTTTATAG
序列73:C.straminsolvens木糖异构酶基因
ATGGCAGAGTATTTTAAAAATGTACCGAAAATCAAATATGAAGGAAAGGATTCGGACAATCCTT
TAGCGTTTAAGTACTATAATCCCGATGAGGTCATTGGCGGTAAAACAATGAAAGAGCATCTAAG
GTTTGCTGTTGCATATTGGCATACATATCAGGGAACGGGTGCAGACCCATTTGGGCCGGGTACT
GCTGTAAGACCGTGGGATGACATATCGGACCCAATGGATCTTGCAAAGGCCAAAGTGGCCGCAA
ATTTCGAGCTGTGTGAAAAATTGGGAGTACCATTTTTCTGCTTCCATGACAGAGATATTGCGCC
TGAAGCTTCAACTTTAAGAGAGACCAATAAAAGACTTGATGAGATTGTTGCACTGATAAAGGAC
TATATGAAAACAAGTAGTGTAAAACTACTCTGGGGTACAACAAATGCTTTTAGCCACCCAAGGT
TTGTCCATGGTGCATCTACTTCTCCGAATGCAGATGTATTTGCATATGCAGCAGCTCAGGTTAA
AAAGGCTATGGAAATTACCCTGGAACTTGGCGGTCAGAACTATGTGTTCTGGGGTGGAAGAGAA
GGCTATGAAACCTTACTTAATACTGATATGAAATTGGAGCTTGACAATATGGCAAGGTTCCTTG
GAATGGCAGTTGACTATGCAAAAGAGATTGGTTTTAAAGGGCAGCTCTTGATTGAACCTAAGCC
AAAAGAACCGACAAAGCACCAGTATGACTTTGATACAGCTACAGTTATCGGTTTCTTAAGGACT
TATGGTCTTGAGAATTACTTCAAAATGAATATTGAAGCAAATCACGCTACACTTGCAGCTCATA
CTTTCCAGCATGAACTTAGGGTTTCAAGAATTAACGGTGTGCTAGGAAGTATCGATGCAAACCA
GGGTGATCTTCTTTTAGGATGGGACACTGACCAATTCCCGACAAATATCTACGATACTACCCTT
GCTATGTATGAAGTAATTAAGGCAGGCGGATTTACAACGGGAGGTCTGAATTTTGATTCTAAAG
TCAGAAGAGGATCATTTGAGCCTGTGGACCTGTTCTATGCACATATTGCAGGTATGGACGCTTT
TGCAAAAGGATTTAAAATAGCATATAAAATGGTTTCCGACGGTAAGTTTGACAAATTTATTGAT
GAAAGATATGAAAGCTATAAGAGCGGTATTGGAAAAGATATTGTAGATGGAAAAGTAGGGTTTA
AAGAGCTTGAAAAATATGCTTTAGAGCTTGATGGTATCAAGAATGTGTCGGGAAGACAGGAAGT
TCTCGAAGCTATGTTAAACAAATATATTCTTGAGGACTAG
序列74:pMU340
catgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatccagtattct
gacatgggtgtatcaataacccatgcgtttccgtattgtatcggaatggtttcggacagggcgg
tgggaatagacatggaaaagatttttttgcccgaggatgcattgataaagtatttcttttccga
aagagaggaaaagattctaaagagttttggaaatactgatgaatattgtgtgcagagtacaatt
ctatggacaagaaaagaggctttgtcaaaactttttcgtctgggaatgaggatggattttaaaa
agctggatactttggaggacgaggtggtttttcaggaaacaaacagggcgcgtctgttttcttt
tatatgcaataattactgtatctctctggcattgccaggttttaataaagattaaaattattga
ctagaaataaaaaaattgtccataatattaatggacaaaaaaacaaagaattacatcaaaggaa
gataaaaatactttgttaaaaaattaattattttttatctaaactattgaaaatgaaaataaaa
taatataaaatgaatcatagtgcaagagatacttgccagaggatgaatattttactgcattcat
gctttatggcagctaatagaggcattaaattaaattttaatttacaataggaggcgatattaat
gaataaatattttgagaacgtatctaaaataaaatatgaaggaccaaaatcaaacaatccttat
tcttttaaattttacaatccagaagaagtaatcgatggcaagacgatggaggagcatctacgct
tttctatagcttactggcacacttttactgctgatggaacagatcaatttggcaaagctaccat
gcaaagaccatggaaccactacacagatcctatggacatagcaaaggcaagggtagaagcagca
tttgagttttttgataagataaatgcacctttcttctgcttccatgacagggatattgcacctg
aaggagacactcttagagagacaaacaaaaacttagatacaatagttgccatgataaaggatta
cttgaagaccagcaagacgaaagttttgtggggcaccgcaaatcttttctccaatccgagattt
gtacatggtgcatcaacatcctgcaatgctgatgttttcgcatattctgcagctcaagttaaaa
aagctcttgagattactaaggagcttggcggcgaaaactacgtattctggggtggcagagaagg
atatgaaacacttctcaatacagacatggagtttgagcttgacaactttgcaagatttttgcac
atggctgttgactacgcgaaggaaatcggctttgaaggccagttcttgattgagccgaagccaa
aggagcctacgaaacaccaatacgactttgacgtggcaaatgtattggcattcttgagaaaata
cggccttgacaaatatttcaaagtgaatatcgaggcaaaccatgcgacattggcattccacgac
ttccaacatgagctaagatacgccagaataaacggtgtattaggatcaattgacgcaaatacag
gcgatatgcttttaggatgggatacagaccagttccctacagatatacgcatgacaacgcttgc
tatgtatgaagtcataaagatgggtggatttgacaaaggcggccttaacttcgatgcaaaagta
agacgtgcttcatttgaaccagaagatcttttcttaggtcatatagccggaatggatgcctttg
caaaaggcttcaaagttgcttacaaacttgtgaaagatggcgtatttgacaagttcatcgagga
aagatacgcaagctacaaagacggcattggcgctgacattgtaagcgggaaagctgacttcaag
agccttgaaaagtacgcattagagcacagccagattgtcaacaaatcaggcaggcaagagctgt
tagaatcaatcctaaatcagtatttgtttgcagaataatgaaacatgagggcggcttcatgctt
cattaaagctgccctcaacaaaaatcatggaggtaaatgtatgtattttttagggatagattta
gggacatcatcagttaagataatactgatgaatgaaagcggcaatgtggtatcaagcgtttcaa
aagaatatcctgtgtactatccagagccaggctgggctgagcaaaatccagaagattggtggaa
tggcacaagggatggaataagagagattattgcgaaaagcggcgtaaatggcgatgaaataaag
ggtgttggcttaagcgggcagatgcatggactggtgcttttagacaaagacaataacgttttaa
cgccagccatactttggtgtgaccagaggacacaggaagaatgcgactacatcacagagaaaat
aggaaaagaaggccttttgaagtacacagggaataaagcattgacaggttttactgcaccaaag
atattatgggtaaagaagcaccttaaagacgtatatgaaagaatcgctcatatccttttgccaa
aagattatataaggtttaaattgacaggtgagtacgctacagaagtttcagatgcatcaggtac
acttcttttcgatgtggaaaatagaagatggtcaaaggaaatgatagacatatttgaaataccg
gaaaaagcccttcctaagtgctacgaatcaacagatgtcacagggtatgtcaccaaagaggcag
cagatttgacagggcttcatgaagggactattgtcgtaggcggtggtggtgaccaagccagcgg
cgctgtaggcactggcacggtgaaaagcggcatagtgtccatcgcattaggaacttcaggcgtc
gtatttgcatcacaggacaagtacgcagcagatgatgagcttaggcttcactcattctgccatg
caaacggcaaatggcatgtgatgggtgtcatgctttcggctgcatcatgtcttaaatggtgggt
agatgatgtaaataattacaagaccgatgttatgacatttgatggactcttagaagaagcagag
aaggtgaagccaggcagtgatggattgatattcttgccatacctgatgggtgaaaggacccctt
acagcgatccttatgcgagaggcagctttgtaggtttaacaattacacacaatagaagccacat
gacaagatctatattagaaggcgtcgcatttggacttagggattcgctggagcttataaaggct
ttaaatatacctgtaaatgaagccagggtaagtggtggtggtgctaaaagcaggctttggaggc
aaatacttgccgatgtattcaatgtaaggatagacatgataaatgctacagaaggaccttcatt
tggtgcagcaataatggcgtctgtgggatatggcctttacaaaaatgtagatgatgcatgcaat
agtttaataaaagttacagacagcgtatatccaatcaaagaaaacgtcgaaaagtacaacaaac
tgtatccaatctacgtgagcttgtattcaaggcttaaaggcgcctttgaagaaattgggaagtt
ggatttgtaaaataaattcatttggaaataaatttatgacagtacaagggacattgattaacaa
agcttcaggttaataatagtaaagttaatatttgctatgaaatgaaagcataataatctgttcc
ttgtactttgctttatcatgtttatttaagatactaattaataaaagtcaatttagccaataat
aaaatcctatatatagtaaatatttacaataaaatcactacaaaataaaaaactttatttaatc
tcttaaaaatatctacataagggggtgttagatgaaaaaggccgtaatcatggtcatagctgtt
tcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgt
aaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctt
tccagtcgggaaacctgtcgtgccagcccttcaaacttcccaaaggcgagccctagtgacatta
gaaaaccgactgtaaaaagtacagtcggcattatctcatattataaaagccagtcattaggcct
atctgacaattcctgaatagagttcataaacaatcctgcatgataaccatcacaaacagaatga
tgtacctgtaaagatagcggtaaatatattgaattacctttattaatgaattttcctgctgtaa
taatgggtagaaggtaattactattattattgatatttaagttaaacccagtaaatgaagtcca
tggaataatagaaagagaaaaagcattttcaggtataggtgttttgggaaacaatttccccgaa
ccattatatttctctacatcagaaaggtataaatcataaaactctttgaagtcattctttacag
gagtccaaataccagagaatgttttagatacaccatcaaaaattgtataaagtggctctaactt
atcccaataacctaactctccgtcgctattgtaaccagttctaaaagctgtatttgagtttatc
acccttgtcactaagaaaataaatgcagggtaaaatttatatccttcttgttttatgtttcggt
ataaaacactaatatcaatttctgtggttatactaaaagtcgtttgttggttcaaataatgatt
aaatatctcttttctcttccaattgtctaaatcaattttattaaagttcatttgatatgcctcc
taaatttttatctaaagtgaatttaggaggcttacttgtctgctttcttcattagaatcaatcc
ttttttaaaagtcaatcccgtttgttgaactactctttaataaaataatttttccgttcccaat
tccacattgcaataatagaaaatccatcttcatcggctttttcgtcatcatctgtatgaatcaa
atcgccttcttctgtgtcatcaaggtttaattttttatgtatttcttttaacaaaccaccatag
gagattaaccttttacggtgtaaaccttcctccaaatcagacaaacgtttcaaattcttttctt
catcatcggtcataaaatccgtatcctttacaggatattttgcagtttcgtcaattgccgattg
tatatccgatttatatttatttttcggtcgaatcatttgaacttttacatttggatcatagtct
aatttcattgcctttttccaaaattgaatccattgtttttgattcacgtagttttctgtattct
taaaataagttggttccacacataccaatacatgcatgtgctgattataagaattatctttatt
atttattgtcacttccgttgcacgcataaaaccaacaagatttttattaatttttttatattgc
atcattcggcgaaatccttgagccatatctgacaaactcttatttaattcttcgccatcataaa
catttttaactgttaatgtgagaaacaaccaacgaactgttggcttttgtttaataacttcagc
aacaaccttttgtgactgaatgccatgtttcattgctctcctccagttgcacattggacaaagc
ctggatttacaaaaccacactcgatacaactttctttcgcctgtttcacgattttgtttatact
ctaatatttcagcacaatcttttactctttcagcctttttaaattcaagaatatgcagaagttc
aaagtaatcaacattagcgattttcttttctctccatggtctcacttttccactttttgtcttg
tccactaaaacccttgatttttcatctgaataaatgctactattaggacacataatattaaaag
aaacccccatctatttagttatttgtttggtcacttataactttaacagatggggtttttctgt
gcaaccaattttaagggttttcaatactttaaaacacatacataccaacacttcaacgcacctt
tcagcaactaaaataaaaatgacgttatttctatatgtatcaagaatagaaagaactcgttttt
cgctacgctcaaaacgcaaaaaaagcactcattcgagtgctttttcttatcgctccaaatcatg
cgattttttcctctttgcttttctttgctcacgaagttctcgatcacgctgcaaaacatcttga
agcgaaaaagtattcttcttttcttccgatcgctcatgctgacgcacgaaaagccctctaggcg
cataggaacaactcctaaatgcatgtgaggggttttctcgtccatgtgaacagtcgcatacgca
atattttgtttcccatactgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtat
tgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcg
gtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaaga
acatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgttttt
ccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaac
ccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttc
cgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctca
tagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcac
gaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccgg
taagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgt
aggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtattt
ggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggca
aacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaa
aggatctcaagaagatcctttgatcttttctacggggatcgcttgcctgtaacttacacgcgcc
tcgtatcttttaatgatggaataatttgggaatttactctgtgtttatttatttttatgttttg
tatttggattttagaaagtaaataaagaaggtagaagagttacggaatgaagaaaaaaaaataa
acaaaggtttaaaaaatttcaacaaaaagcgtactttacatatatatttattagacaagaaaag
cagattaaatagatatacattcgattaacgataagtaaaatgtaaaatcacaggattttcgtgt
gtggtcttctacacagacaagatgaaacaattcggcattaatacctgagagcaggaagagcaag
ataaaaggtagtatttgttggcgatccccctagagtcttttacatcttcggaaaacaaaaacta
ttttttctttaatttctttttttactttctatttttaatttatatatttatattaaaaaattta
aattataattatttttatagcacgtgatgaaaaggacccatcgataagctagcttttcaattca
attcatcattttttttttattcttttttttgatttcggtttctttgaaatttttttgattcggt
aatctccgaacagaaggaagaacgaaggaaggagcacagacttagattggtatatatacgcata
tgtagtgttgaagaaacatgaaattgcccagtattcttaacccaactgcacagaacaaaaacct
gcaggaaacgaagataaatcatgtcgaaagctacatataaggaacgtgctgctactcatcctag
tcctgttgctgccaagctatttaatatcatgcacgaaaagcaaacaaacttgtgtgcttcattg
gatgttcgtaccaccaaggaattactggagttagttgaagcattaggtcccaaaatttgtttac
taaaaacacatgtggatatcttgactgatttttccatggagggcacagttaagccgctaaaggc
attatccgccaagtacaattttttactcttcgaagacagaaaatttgctgacattggtaataca
gtcaaattgcagtactctgcgggtgtatacagaatagcagaatgggcagacattacgaatgcac
acggtgtggtgggcccaggtattgttagcggtttgaagcaggcggcagaagaagtaacaaagga
acctagaggccttttgatgttagcagaattgtcatgcaagggctccctatctactggagaatat
actaagggtactgttgacattgcgaagagcgacaaagattttgttatcggctttattgctcaaa
gagacatgggtggaagagatgaaggttacgattggttgattatgacacccggtgtgggtttaga
tgacaagggagacgcattgggtcaacagtatagaaccgtggatgatgtggtctctacaggatct
gacattattattgttggaagaggactatttgcaaagggaagggatgctaaggtagagggtgaac
gttacagaaaagcaggctgggaagcatatttgagaagatgcggccagcaaaactaaaaaactgt
attataagtaaatgcatgtatactaaactcacaaattagagcttcaatttaattatatcagtta
ttacccacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaa
atatgtatccgct
序列75:pMU102-Z.mobilis PDC-ADH
gacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatct
tcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaac
ttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtcaattcgag
ctcggtacccggggatccttaagaagactaataaaaagtttctaaaagcatgaaatatcctgta
ttttggagttacttgccgttatttatggctaaagccgaaaaaaagataaggagggtgttgtata
caaaaaacattttgtatataattagagttgcttggaacccagtaaaatagctgtttatagtgag
taggtattattacctgatgagttatactgtcggtacctatttagcggagcggcttgtccagatt
ggtctcaagcatcacttcgcagtcgcgggcgactacaacctcgtccttcttgacaacctgcttt
tgaacaaaaacatggagcaggtttattgctgtaacgaactgaactgcggtttcagtgcagaagg
ttatgctcgtgccaaaggcgcagcagcagccgtcgttacctacagcgtcggtgcgctttccgca
tttgatgctatcggtggcgcctatgcagaaaaccttccggttatcctgatctccggtgctccga
acaacaatgatcacgctgctggtcacgtgttgcatcacgctcttggcaaaaccgactatcacta
tcagttggaaatggccaagaacatcacggccgccgctgaagcgatttacaccccggaagaagct
ccggctaaaatcgatcacgtgattaaaactgctcttcgtgagaagaagccggtttatctcgaaa
tcgcttgcaacattgcttccatgccctgcgccgctcctggaccggcaagcgcattgttcaatga
cgaagccagcgacgaagcttctttgaatgcagcggttgaagaaaccctgaaattcatcgccaac
cgcgacaaagttgccgtcctcgtcggcagcaagctgcgcgcagctggtgctgaagaagctgctg
tcaaatttgctgatgctctcggtggcgcagttgctaccatggctgctgcaaaaagcttcttccc
agaagaaaacccgcattacatcggcacctcatggggtgaagtcagctatccgggcgttgaaaag
acgatgaaagaagccgatgcggttatcgctctggctcctgtcttcaacgactactccaccactg
gttggacggatattcctgatcctaagaaactggttctcgctgaaccgcgttctgtcgtcgttaa
cggcattcgcttccccagcgtccatctgaaagactatctgacccgtttggctcagaaagtttcc
aagaaaaccggtgcattggacttcttcaaatccctcaatgcaggtgaactgaagaaagccgctc
cggctgatccgagtgctccgttggtcaacgcagaaatcgcccgtcaggtcgaagctcttctgac
cccgaacacgacggttattgctgaaaccggtgactcttggttcaatgctcagcgcatgaagctc
ccgaacggtgctcgcgttgaatatgaaatgcagtggggtcacattggttggtccgttcctgccg
ccttcggttatgccgtcggtgctccggaacgtcgcaacatcctcatggttggtgatggttcctt
ccagctgacggctcaggaagtcgctcagatggttcgcctgaaactgccggttatcatcttcttg
atcaataactatggttacaccatcgaagttatgatccatgatggtccgtacaacaacatcaaga
actgggattatgccggtctgatggaagtgttcaacggtaacggtggttatgacagcggtgctgg
taaaggcctgaaggctaaaaccggtggcgaactggcagaagctatcaaggttgctctggcaaac
accgacggcccaaccctgatcgaatgcttcatcggtcgtgaagactgcactgaagaattggtca
aatggggtaagcgcgttgctgccgccaacagccgtaagcctgttaacaagctcctctagattct
gttaaaaccggacattgaagaaggtgttgcgcagcgtttaataaaaacatctgtttatcgaagt
ttaggaataggaaaattaaaaaaaacaagacgggagtgagtttttgaaatggcttcttcaactt
tttatattcctttcgtcaacgaaatgggcgaaggttcgcttgaaaaagcaatcaaggatcttaa
cggcagcggctttaaaaatgcgctgatcgtttctgatgctttcatgaacaaatccggtgttgtg
aagcaggttgctgacctgttgaaagcacagggtattaattctgctgtttatgatggcgttatgc
cgaacccgactgttaccgcagttctggaaggccttaagatcctgaaggataacaattcagactt
cgtcatctccctcggtggtggttctccccatgactgcgccaaagccatcgctctggtcgcaacc
aatggtggtgaagtcaaagactacgaaggtatcgacaaatctaagaaacctgccctgcctttga
tgtcaatcaacacgacggctggtacggcttctgaaatgacgcgtttctgcatcatcactgatga
agtccgtcacgttaagatggccattgttgaccgtcacgttaccccgatggtttccgtcaacgat
cctctgttgatggttggtatgccaaaaggcctgaccgccgccaccggtatggatgctctgaccc
acgcatttgaagcttattcttcaacggcagctactccgatcaccgatgcttgcgctttgaaagc
agcttccatgatcgctaagaatctgaagaccgcttgcgacaacggtaaggatatgccggctcgt
gaagctatggcttatgcccaattcctcgctggtatggccttcaacaacgcttcgcttggttatg
tccatgctatggctcaccagttgggcggttactacaacctgccgcatggtgtctgcaacgctgt
tctgcttccgcatgttctggcttataacgcctctgtcgttgctggtcgtctgaaagacgttggt
gttgctatgggtctcgatatcgccaatctcggtgataaagaaggcgcagaagccaccattcagg
ctgttcgcgatctggctgcttccattggtattccagcaaacctgaccgagctgggtgctaagaa
agaagatgtgccgcttcttgctgaccacgctctgaaagatgcttgtgctctgaccaacccgcgt
cagggtgatcagaaagaagttgaagaactcttcctgagcgctttctaaaagatgcgttataatt
ttacaagcctgtttttttaggaaacgggcttataaaatttttttatttttttgccgggtttttt
cttgtattaatatgtggaatatgttaataatattaagaagaaattccgaatttaactaaacaaa
attatttttgttatttaagccaatctgtcatataattcttgacatgagggttattagttagtat
aatagtccttgtcggttttaagagggatcctctagagtcgacctgcaggcatgcaagcttggcg
taatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatac
gagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgc
gttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagcccttcaaacttcccaaa
ggcgagccctagtgacattagaaaaccgactgtaaaaagtacagtcggcattatctcatattat
aaaagccagtcattaggcctatctgacaattcctgaatagagttcataaacaatcctgcatgat
aaccatcacaaacagaatgatgtacctgtaaagatagcggtaaatatattgaattacctttatt
aatgaattttcctgctgtaataatgggtagaaggtaattactattattattgatatttaagtta
aacccagtaaatgaagtccatggaataatagaaagagaaaaagcattttcaggtataggtgttt
tgggaaacaatttccccgaaccattatatttctctacatcagaaaggtataaatcataaaactc
tttgaagtcattctttacaggagtccaaataccagagaatgttttagatacaccatcaaaaatt
gtataaagtggctctaacttatcccaataacctaactctccgtcgctattgtaaccagttctaa
aagctgtatttgagtttatcacccttgtcactaagaaaataaatgcagggtaaaatttatatcc
ttcttgttttatgtttcggtataaaacactaatatcaatttctgtggttatactaaaagtcgtt
tgttggttcaaataatgattaaatatctcttttctcttccaattgtctaaatcaattttattaa
agttcatttgatatgcctcctaaatttttatctaaagtgaatttaggaggcttacttgtctgct
ttcttcattagaatcaatccttttttaaaagtcaatcccgtttgttgaactactctttaataaa
ataatttttccgttcccaattccacattgcaataatagaaaatccatcttcatcggctttttcg
tcatcatctgtatgaatcaaatcgccttcttctgtgtcatcaaggtttaattttttatgtattt
cttttaacaaaccaccataggagattaaccttttacggtgtaaaccttcctccaaatcagacaa
acgtttcaaattcttttcttcatcatcggtcataaaatccgtatcctttacaggatattttgca
gtttcgtcaattgccgattgtatatccgatttatatttatttttcggtcgaatcatttgaactt
ttacatttggatcatagtctaatttcattgcctttttccaaaattgaatccattgtttttgatt
cacgtagttttctgtattcttaaaataagttggttccacacataccaatacatgcatgtgctga
ttataagaattatctttattatttattgtcacttccgttgcacgcataaaaccaacaagatttt
tattaatttttttatattgcatcattcggcgaaatccttgagccatatctgacaaactcttatt
taattcttcgccatcataaacatttttaactgttaatgtgagaaacaaccaacgaactgttggc
ttttgtttaataacttcagcaacaaccttttgtgactgaatgccatgtttcattgctctcctcc
agttgcacattggacaaagcctggatttacaaaaccacactcgatacaactttctttcgcctgt
ttcacgattttgtttatactctaatatttcagcacaatcttttactctttcagcctttttaaat
tcaagaatatgcagaagttcaaagtaatcaacattagcgattttcttttctctccatggtctca
cttttccactttttgtcttgtccactaaaacccttgatttttcatctgaataaatgctactatt
aggacacataatattaaaagaaacccccatctatttagttatttgtttggtcacttataacttt
aacagatggggtttttctgtgcaaccaattttaagggttttcaatactttaaaacacatacata
ccaacacttcaacgcacctttcagcaactaaaataaaaatgacgttatttctatatgtatcaag
aatagaaagaactcgtttttcgctacgctcaaaacgcaaaaaaagcactcattcgagtgctttt
tcttatcgctccaaatcatgcgattttttcctctttgcttttctttgctcacgaagttctcgat
cacgctgcaaaacatcttgaagcgaaaaagtattcttcttttcttccgatcgctcatgctgacg
cacgaaaagccctctaggcgcataggaacaactcctaaatgcatgtgaggggttttctcgtcca
tgtgaacagtcgcatacgcaatattttgtttcccatactgcattaatgaatcggccaacgcgcg
gggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcgg
tcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatc
aggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaag
gccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgct
caagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctc
cctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcg
ggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgct
ccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaacta
tcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacagg
attagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggct
acactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagt
tggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcag
cagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtct
序列76:pMU102-Z.palmae PDC,Z.mobilis ADH.
gacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatct
tcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaac
ttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtcaattcgag
ctcggtacccggggatccttaagaagactaataaaaagtttctaaaagcatgaaatatcctgta
ttttggagttacttgccgttatttatggctaaagccgaaaaaaagataaggagggtgttgtata
caaaaaacattttgtatataattagagttgcttggaacccagtaaaatagctgtttatagtgag
taggtattattacctgatgtataccgttggtatgtacttggcagaacgcctagcccagatcggc
ctgaaacaccactttgccgtggccggtgactacaacctggtgttgcttgatcagctcctgctga
acaaagacatggagcaggtctactgctgtaacgaacttaactgcggctttagcgccgaaggtta
cgctcgtgcacgtggtgccgccgctgccatcgtcacgttcagcgtaggtgctatctctgcaatg
aacgccatcggtggcgcctatgcagaaaacctgccggtcatcctgatctctggctcaccgaaca
ccaatgactacggcacaggccacatcctgcaccacaccattggtactactgactataactatca
gctggaaatggtaaaacacgttacctgcgcacgtgaaagcatcgtttctgccgaagaagcaccg
gcaaaaatcgaccacgtcatccgtacggctctacgtgaacgcaaaccggcttatctggaaatcg
catgcaacgtcgctggcgctgaatgtgttcgtccgggcccgatcaatagcctgctgcgtgaact
cgaagttgaccagaccagtgtcactgccgctgtagatgccgccgtagaatggctgcaggaccgc
cagaacgtcgtcatgctggtcggtagcaaactgcgtgccgctgccgctgaaaaacaggctgttg
ccctagcggaccgcctgggctgcgctgtcacgatcatggctgccgaaaaaggcttcttcccgga
agatcatccgaacttccgcggcctgtactggggtgaagtcagctccgaaggtgcacaggaactg
gttgaaaacgccgatgccatcctgtgtctggcaccggtattcaacgactatgctaccgttggct
ggaactcctggccgaaaggcgacaatgtcatggtcatggacaccgaccgcgtcactttcgcagg
acagtccttcgaaggtctgtcattgagcaccttcgccgcagcactggctgagaaagcaccttct
cgcccggcaacgactcaaggcactcaagcaccggtactgggtattgaggccgcagagcccaatg
caccgctgaccaatgacgaaatgacgcgtcagatccagtcgctgatcacttccgacactactct
gacagcagaaacaggtgactcttggttcaacgcttctcgcatgccgattcctggcggtgctcgt
gtcgaactggaaatgcaatggggtcatatcggttggtccgtaccttctgcattcggtaacgccg
ttggttctccggagcgtcgccacatcatgatggtcggtgatggctctttccagctgactgctca
agaagttgctcagatgatccgctatgaaatcccggtcatcatcttcctgatcaacaaccgcggt
tacgtcatcgaaatcgctatccatgacggcccttacaactacatcaaaaactggaactacgctg
gcctgatcgacgtcttcaatgacgaagatggtcatggcctgggtctgaaagcttctactggtgc
agaactagaaggcgctatcaagaaagcactcgacaatcgtcgcggtccgacgctgatcgaatgt
aacatcgctcaggacgactgcactgaaaccctgattgcttggggtaaacgtgtagcagctacca
actctcgcaaaccacaagcgtaaattctgttaaaaccggacattgaagaaggtgttgcgcagcg
tttaataaaaacatctgtttatcgaagtttaggaataggaaaattaaaaaaaacaagacgggag
tgagtttttgaaatggcttcttcaactttttatattcctttcgtcaacgaaatgggcgaaggtt
cgcttgaaaaagcaatcaaggatcttaacggcagcggctttaaaaatgcgctgatcgtttctga
tgctttcatgaacaaatccggtgttgtgaagcaggttgctgacctgttgaaagcacagggtatt
aattctgctgtttatgatggcgttatgccgaacccgactgttaccgcagttctggaaggcctta
agatcctgaaggataacaattcagacttcgtcatctccctcggtggtggttctccccatgactg
cgccaaagccatcgctctggtcgcaaccaatggtggtgaagtcaaagactacgaaggtatcgac
aaatctaagaaacctgccctgcctttgatgtcaatcaacacgacggctggtacggcttctgaaa
tgacgcgtttctgcatcatcactgatgaagtccgtcacgttaagatggccattgttgaccgtca
cgttaccccgatggtttccgtcaacgatcctctgttgatggttggtatgccaaaaggcctgacc
gccgccaccggtatggatgctctgacccacgcatttgaagcttattcttcaacggcagctactc
cgatcaccgatgcttgcgctttgaaagcagcttccatgatcgctaagaatctgaagaccgcttg
cgacaacggtaaggatatgccggctcgtgaagctatggcttatgcccaattcctcgctggtatg
gccttcaacaacgcttcgcttggttatgtccatgctatggctcaccagttgggcggttactaca
acctgccgcatggtgtctgcaacgctgttctgcttccgcatgttctggcttataacgcctctgt
cgttgctggtcgtctgaaagacgttggtgttgctatgggtctcgatatcgccaatctcggtgat
aaagaaggcgcagaagccaccattcaggctgttcgcgatctggctgcttccattggtattccag
caaacctgaccgagctgggtgctaagaaagaagatgtgccgcttcttgctgaccacgctctgaa
agatgcttgtgctctgaccaacccgcgtcagggtgatcagaaagaagttgaagaactcttcctg
agcgctttctaaaagatgcgttataattttacaagcctgtttttttaggaaacgggcttataaa
atttttttatttttttgccgggttttttcttgtattaatatgtggaatatgttaataatattaa
gaagaaattccgaatttaactaaacaaaattatttttgttatttaagccaatctgtcatataat
tcttgacatgagggttattagttagtataatagtccttgtcggttttaagagggatcctctaga
gtcgacctgcaggcatgcaagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgt
tatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcct
aatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacct
gtcgtgccagcccttcaaacttcccaaaggcgagccctagtgacattagaaaaccgactgtaaa
aagtacagtcggcattatctcatattataaaagccagtcattaggcctatctgacaattcctga
atagagttcataaacaatcctgcatgataaccatcacaaacagaatgatgtacctgtaaagata
gcggtaaatatattgaattacctttattaatgaattttcctgctgtaataatgggtagaaggta
attactattattattgatatttaagttaaacccagtaaatgaagtccatggaataatagaaaga
gaaaaagcattttcaggtataggtgttttgggaaacaatttccccgaaccattatatttctcta
catcagaaaggtataaatcataaaactctttgaagtcattctttacaggagtccaaataccaga
gaatgttttagatacaccatcaaaaattgtataaagtggctctaacttatcccaataacctaac
tctccgtcgctattgtaaccagttctaaaagctgtatttgagtttatcacccttgtcactaaga
aaataaatgcagggtaaaatttatatccttcttgttttatgtttcggtataaaacactaatatc
aatttctgtggttatactaaaagtcgtttgttggttcaaataatgattaaatatctcttttctc
ttccaattgtctaaatcaattttattaaagttcatttgatatgcctcctaaatttttatctaaa
gtgaatttaggaggcttacttgtctgctttcttcattagaatcaatccttttttaaaagtcaat
cccgtttgttgaactactctttaataaaataatttttccgttcccaattccacattgcaataat
agaaaatccatcttcatcggctttttcgtcatcatctgtatgaatcaaatcgccttcttctctg
tcatcaaggtttaattttttatgtatttcttttaacaaaccaccataggagattaaccttttac
ggtgtaaaccttcctccaaatcagacaaacgtttcaaattcttttcttcatcatcggtcataaa
atccgtatcctttacaggatattttgcagtttcgtcaattgccgattgtatatccgatttatat
ttatttttcggtcgaatcatttgaacttttacatttggatcatagtctaatttcattgcctttt
tccaaaattgaatccattgtttttgattcacgtagttttctgtattcttaaaataagttggttc
cacacataccaatacatgcatgtgctgattataagaattatctttattatttattgtcacttcc
gttgcacgcataaaaccaacaagatttttattaatttttttatattgcatcattcggcgaaatc
cttgagccatatctgacaaactcttatttaattcttcgccatcataaacatttttaactgttaa
tgtgagaaacaaccaacgaactgttggcttttgtttaataacttcagcaacaaccttttgtgac
tgaatgccatgtttcattgctctcctccagttgcacattggacaaagcctggatttacaaaacc
acactcgatacaactttctttcgcctgtttcacgattttgtttatactctaatatttcagcaca
atcttttactctttcagcctttttaaattcaagaatatgcagaagttcaaagtaatcaacatta
gcgattttcttttctctccatggtctcacttttccactttttgtcttgtccactaaaacccttg
atttttcatctgaataaatgctactattaggacacataatattaaaagaaacccccatctattt
agttatttgtttggtcacttataactttaacagatggggtttttctgtgcaaccaattttaagg
gttttcaatactttaaaacacatacataccaacacttcaacgcacctttcagcaactaaaataa
aaatgacgttatttctatatgtatcaagaatagaaagaactcgtttttcgctacgctcaaaacg
caaaaaaagcactcattcgagtgctttttcttatcgctccaaatcatgcgattttttcctcttt
gcttttctttgctcacgaagttctcgatcacgctgcaaaacatcttgaagcgaaaaagtattct
tcttttcttccgatcgctcatgctgacgcacgaaaagccctctaggcgcataggaacaactcct
aaatgcatgtgaggggttttctcgtccatgtgaacagtcgcatacgcaatattttgtttcccat
actgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgct
tcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaa
aggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaagg
ccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccc
cctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaa
gataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttac
cggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtagg
tatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagc
ccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatc
gccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagag
ttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgc
tgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctgg
tagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagat
cctttgatcttttctacggggtct
Claims (92)
1.分离的核酸分子,其包含SEQ ID NOS:1-5、30-31和47-61的任一个的核苷酸序列或其互补序列。
2.分离的核酸分子,其包含核苷酸序列,所述核苷酸序列与SEQ IDNOS:1-5、30-31和47-61的任一个的核苷酸序列或其互补序列具有至少80%的同一性。
3.权利要求2的核酸分子,其具有与SEQ ID NOS:1-5、30-31和47-61的任一个的核苷酸序列或其互补序列具有至少大约95%的序列同一性。
4.基因构建体,其包含与可在嗜热或嗜温细菌中表达的启动子可操作连接的SEQ ID NOS:1-5、30-31和47-61的任一个。
5.重组嗜热或嗜温细菌,其包含权利要求4的基因构建体。
6.载体,其包含权利要求1至3的任一项的核酸分子。
7.宿主细胞,其包含权利要求1至3的任一项的核酸分子。
8.权利要求7的宿主细胞,其中所述宿主细胞是嗜热或嗜温细菌细胞。
9.经遗传修饰的嗜热或嗜温微生物,其中第一天然基因被部分、基本上或完全缺失、沉默、失活或下调,所述第一天然基因编码参与有机酸或其盐的代谢产生的第一天然酶,从而提高所述嗜热或嗜温微生物以发酵产物的形式产生乙醇的天然能力。
10.根据权利要求9的经遗传修饰的微生物,其中所述微生物是革兰氏阴性细菌或***。
11.根据权利要求9的经遗传修饰的微生物,其中所述微生物是好热厌氧杆菌属、热厌氧杆菌属、梭菌属、地芽孢杆菌属、糖球菌属、类芽孢杆菌属、杆菌属、热解纤维素菌属、厌氧细菌属或厌氧芽孢杆菌属的种。
12.根据权利要求9的经遗传修饰的微生物,其中所述微生物是选自下列的细菌:热硫化氢好热厌氧杆菌、新西兰好热厌氧杆菌、解多糖好热厌氧杆菌、玉米好热厌氧杆菌、解木聚糖好热厌氧杆菌、解糖好热厌氧杆菌、布氏好热厌氧杆菌、热解糖嗜热厌氧杆菌、热硫化氢嗜热厌氧杆菌、嗜热厌氧乙醇杆菌、布氏嗜热厌氧杆菌、热解纤维梭菌、解纤维梭菌、Clostridium phytofermentans、Clostridium straminosolvens、热葡糖苷酶地芽孢杆菌、嗜热脂肪地芽孢杆菌、Saccharococcuscaldoxylosilyticus、喜温链球菌、坎皮纳斯类芽孢杆菌、黄热芽孢杆菌、勘察加无氧芽孢杆菌、Anoxybacillus gonensis、产乙酸热解纤维素菌、极度嗜热菌、克里斯托热解纤维素菌、欧文湖热解纤维素菌、产乳酸乙酸热解纤维素菌和Anaerocellum thermophilum。
13.根据权利要求9的经遗传修饰的微生物,其中所述微生物是解糖好热厌氧杆菌。
14.根据权利要求9的经遗传修饰的微生物,其中所述微生物选自:(a)具有代谢己糖的天然能力的嗜热或嗜温微生物;(b)具有代谢戊糖的天然能力的嗜热或嗜温微生物;和(c)具有代谢己糖和戊糖的天然能力的嗜热或嗜温微生物。
15.根据权利要求9的经遗传修饰的微生物,其中所述微生物具有代谢己糖的天然能力。
16.根据权利要求15的经遗传修饰的微生物,其中所述微生物是Clostridium straminisolvens或热解纤维梭菌。
17.根据权利要求9的经遗传修饰的微生物,其中所述微生物具有代谢己糖和戊糖的天然能力。
18.根据权利要求17的经遗传修饰的微生物,其中所述微生物是解纤维梭菌,Clostridium kristjanssonii或粪堆梭菌leptosaprartum亚种。
19.权利要求15的经遗传修饰的微生物,其中***了第一非天然基因,所述第一非天然基因编码提供代谢戊糖的能力的第一非天然酶,从而使得所述嗜热或嗜温微生物能够从戊糖以发酵产物的形式产生乙醇。
20.根据权利要求9的经遗传修饰的微生物,其中所述微生物具有代谢戊糖的天然能力。
21.根据权利要求20的经遗传修饰的微生物,其中所述微生物选自解糖好热厌氧杆菌、解木聚糖好热厌氧杆菌、解多糖好热厌氧杆菌和解糖嗜热厌氧杆菌。
22.权利要求20的经遗传修饰的微生物,其中***了第一非天然基因,所述第一非天然基因编码提供代谢己糖的能力的第一非天然酶,从而使得所述嗜热或嗜温微生物能够从己糖以发酵产物的形式产生乙醇。
23.根据权利要求9至22的任一项的经遗传修饰的微生物,其中所述有机酸选自乳酸和乙酸。
24.根据权利要求9至22的任一项的经遗传修饰的微生物,其中所述有机酸是乳酸。
25.根据权利要求9至22的任一项的经遗传修饰的微生物,其中所述有机酸是乙酸。
26.根据权利要求9至22的任一项的经遗传修饰的微生物,其中所述第一天然酶选自乳酸脱氢酶、乙酸激酶和磷酸转乙酰酶。
27.根据权利要求9至22的任一项的经遗传修饰的微生物,其中所述第一天然酶是乳酸脱氢酶。
28.根据权利要求9至22的任一项的经遗传修饰的微生物,其中所述第一天然酶是乙酸激酶。
29.根据权利要求9至22的任一项的经遗传修饰的微生物,其中所述第一天然酶是磷酸转乙酰酶。
30.权利要求9至29的任一项的经遗传修饰的微生物,其中第二天然基因被部分、基本上或完全缺失、沉默、失活或下调,所述第二天然基因编码参与有机酸或其盐的代谢产生的第二天然酶。
31.根据权利要求30的经遗传修饰的微生物,其中所述第二天然酶是乙酸激酶或磷酸转乙酰酶。
32.根据权利要求30的经遗传修饰的微生物,其中所述第二天然酶是乳酸脱氢酶。
33.经遗传修饰的嗜热或嗜温微生物,其中(a)第一天然基因被部分、基本上或完全缺失、沉默、失活或下调,所述第一天然基因编码参与有机酸或其盐的代谢产生的第一天然酶,和(b)***了第一非天然基因,所述第一非天然基因编码参与乙醇的代谢产生的第一非天然酶,从而使得所述嗜热或嗜温微生物能够以发酵产物的形式产生乙醇。
34.权利要求33的经遗传修饰的微生物,其中所述第一非天然基因编码提供代谢己糖的能力的第一非天然酶,从而使得所述嗜热或嗜温微生物能够代谢己糖。
35.权利要求33的经遗传修饰的微生物,其中所述第一非天然基因编码提供代谢戊糖的能力的第一非天然酶,从而使得所述嗜热或嗜温微生物能够代谢戊糖。
36.权利要求33的经遗传修饰的微生物,其中所述第一非天然基因编码提供代谢己糖的能力的第一非天然酶;和***了第二非天然基因,所述第二非天然基因编码提供代谢戊糖的能力的第二非天然酶,从而使得所述嗜热或嗜温微生物能够代谢己糖和戊糖。
37.根据权利要求33至36的任一项的经遗传修饰的微生物,其中所述有机酸是乳酸。
38.根据权利要求33至36的任一项的经遗传修饰的微生物,其中的所述有机酸是乙酸。
39.根据权利要求33、34或36的经遗传修饰的微生物,其中所述第一非天然酶是丙酮酸脱羧酶(PDC)或乙醇脱氢酶(ADH)。
40.根据权利要求33或35的经遗传修饰的微生物,其中所述第二非天然酶是木糖异构酶。
41.根据权利要求40的经遗传修饰的微生物,其中所述第一非天然基因相应于SEQ ID NOS:6、10或14。
42.根据权利要求33、35或36的经遗传修饰的微生物,其中所述非天然酶是木酮糖激酶。
43.根据权利要求42的经遗传修饰的微生物,其中所述非天然基因相应于SEQ ID NOS:7、11或15。
44.根据权利要求33、35或36的经遗传修饰的微生物,其中所述非天然酶是L-***糖异构酶.
45.根据权利要求44的经遗传修饰的微生物,其中所述非天然基因相应于SEQ ID NOS:8或12。
46.根据权利要求33、35或36的经遗传修饰的微生物,其中所述非天然酶是L-核酮糖-5-磷酸4-表异构酶。
47.根据权利要求46的经遗传修饰的微生物,其中所述非天然基因相应于SEQ ID NO:9或13。
48.根据权利要求9至47的任一项的经遗传修饰的微生物,其中所述微生物能够将至少60%的来自被代谢的生物质的碳转化成乙醇。
49.根据权利要求9的经遗传修饰的微生物,其中所述微生物选自:(a)具有水解纤维素的天然能力的嗜热或嗜温微生物;(b)具有水解木聚糖的天然能力的嗜热或嗜温微生物;和(c)具有水解纤维素和木聚糖的天然能力的嗜热或嗜温微生物。
50.根据权利要求9或33的经遗传修饰的微生物,其中所述微生物具有水解纤维素的天然能力。
51.根据权利要求9或33的经遗传修饰的微生物,其中所述微生物具有水解纤维素和木聚糖的天然能力。
52.权利要求50的经遗传修饰的微生物,其中***了第一非天然基因,所述第一非天然基因编码提供水解木聚糖的能力的第一非天然酶。
53.根据权利要求9或33的经遗传修饰的微生物,其中所述微生物具有水解木聚糖的天然能力。
54.权利要求53的经遗传修饰的微生物,其中***了第一非天然基因,所述第一非天然基因编码提供水解纤维素的能力的第一非天然酶。
55.权利要求49至54的任一项的经遗传修饰的微生物,其中所述有机酸选自乳酸和乙酸。
56.根据权利要求49至54的任一项的经遗传修饰的微生物,其中所述有机酸是乳酸。
57.根据权利要求49至54的任一项的经遗传修饰的微生物,其中所述有机酸是乙酸。
58.根据权利要求49至54的任一项的经遗传修饰的微生物,其中所述第一天然酶选自乳酸脱氢酶、乙酸激酶和磷酸转乙酰酶。
59.根据权利要求49至54的任一项的经遗传修饰的微生物,其中所述第一天然酶是乳酸脱氢酶。
60.根据权利要求49至54的任一项的经遗传修饰的微生物,其中所述第一天然酶是乙酸激酶。
61.根据权利要求49至54的任一项的经遗传修饰的微生物,其中所述第一天然酶是磷酸转乙酰酶。
62.根据权利要求50至61的任一项的经遗传修饰的微生物,其中第二天然基因被部分、基本上或完全缺失、沉默、失活或下调,所述第二天然基因编码参与有机酸或其盐的代谢产生的第二天然酶。
63.根据权利要求62的经遗传修饰的微生物,其中所述第二天然酶是乙酸激酶或磷酸转乙酰酶。
64.根据权利要求62的经遗传修饰的微生物,其中所述第二天然酶是乳酸脱氢酶。
65.经遗传修饰的嗜热或嗜温微生物,其中(a)第一天然基因被部分、基本上或完全缺失、沉默、失活或下调,所述第一天然基因编码参与有机酸或其盐的代谢产生的第一天然酶,和(b)***了第一非天然基因,所述第一非天然基因编码参与水解多糖的第一非天然酶,从而使得所述嗜热或嗜温微生物能够以发酵产物的形式产生乙醇。
66.权利要求65的经遗传修饰的微生物,其中所述第一非天然基因编码提供水解纤维素的能力的第一非天然酶,从而使得所述嗜热或嗜温微生物能够水解纤维素。
67.权利要求65的经遗传修饰的微生物,其中所述第一非天然基因编码提供水解木聚糖的能力的第一非天然酶,从而使得所述嗜热或嗜温微生物能够水解木聚糖。
68.权利要求65的经遗传修饰的微生物,其中所述第一非天然基因编码提供水解纤维素的能力的第一非天然酶;并且***了第二非天然基因,所述第二非天然基因编码提供水解木聚糖的能力的第二非天然酶,从而使得所述嗜热或嗜温微生物能够水解纤维素和木聚糖。
69.根据权利要求65至68的任一项的经遗传修饰的微生物,其中所述有机酸是乳酸。
70.根据权利要求65至68的任一项的经遗传修饰的微生物,其中所述有机酸是乙酸。
71.根据权利要求65、66或68的经遗传修饰的微生物,其中所述第一非天然酶是丙酮酸脱羧酶(PDC)或乙醇脱氢酶(ADH)。
72.根据权利要求49至71的任一项的经遗传修饰的微生物,其中所述微生物能够将至少60%的来自被代谢的生物质的碳转化成乙醇。
73.根据权利要求9、11、12、14、16、18、19、22、33、34、35、36、49、65、66、67和68的任一项的经遗传修饰的微生物,其中所述微生物是嗜温微生物。
74.根据权利要求9、11、12、13、14、19、21、22、33、34、35、36、49、65、66、67和68的任一项的经遗传修饰的微生物,其中所述微生物是嗜热微生物。
75.用于将木质纤维生物质转化成乙醇的方法,其包括将木质纤维生物质与根据权利要求9至48的任一项的经遗传修饰的嗜热或嗜温微生物接触。
76.权利要求75的方法,其中所述木质纤维生物质选自草、柳枝稷、互花米草、黑麦草、草芦、混合大网茅草、芒属植物、糖加工废渣、甘蔗渣、甘蔗秆、农业废料、稻草、稻壳、大麦稿秆、玉米芯、谷类稿秆、麦秸、油菜秆、燕麦秸秆、燕麦皮、玉米纤维、干草、大豆干草、玉米秸秆、森林废弃物、再循环木浆纤维、造纸污泥、锯屑、硬木、软木材和其组合。
77.权利要求75的方法,其中所述木质纤维生物质选自玉米秸秆、甘蔗渣、柳枝稷和杨木材。
78.权利要求75的方法,其中所述木质纤维生物质是玉米秸秆。
79.权利要求75的方法,其中所述木质纤维生物质是甘蔗渣。
80.权利要求75的方法,其中所述木质纤维生物质是玉柳枝稷。
81.权利要求75的方法,其中所述木质纤维生物质是是杨木材。
82.权利要求75的方法,其中所述木质纤维生物质是柳木。
83.权利要求75的方法,其中所述木质纤维生物质是造纸污泥。
84.用于将木质纤维生物质转化成乙醇的方法,其包括将木质纤维生物质与根据权利要求49-74的任一项的经遗传修饰的嗜热或嗜温微生物接触。
85.权利要求84的方法,其中所述木质纤维生物质选自草、柳枝稷、互花米草、黑麦草、草芦、混合大网茅草、芒属植物、糖加工废渣、甘蔗渣、甘蔗秆农业废料、稻草、稻壳、大麦稿秆、玉米芯、谷类稿秆、麦秸、油菜秆、燕麦秸秆、燕麦皮、玉米纤维、干草、大豆干草、玉米秸秆、森林废弃物、再循环木浆纤维、造纸污泥、锯屑、硬木、软木材和其组合。
86.权利要求84的方法,其中所述木质纤维生物质选自玉米秸秆、甘蔗渣、柳枝稷和杨木材。
87.权利要求84的方法,其中所述木质纤维生物质是玉米秸秆。
88.权利要求84的方法,其中所述木质纤维生物质是甘蔗渣。
89.权利要求84的方法,其中所述木质纤维生物质是柳枝稷。
90.权利要求84的方法,其中所述木质纤维生物质是杨木材。
91.权利要求84的方法,其中所述木质纤维生物质是柳木。
92.权利要求84的方法,其中所述木质纤维生物质是造纸污泥。
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CN106893745A (zh) * | 2017-04-12 | 2017-06-27 | 浙江畯和生物科技有限公司 | 一种木质纤维素生物质的高效转化利用方法 |
CN110484466A (zh) * | 2019-08-15 | 2019-11-22 | 华南理工大学 | 一种提高嗜热厌氧杆菌发酵性能的方法 |
CN110484466B (zh) * | 2019-08-15 | 2023-04-21 | 华南理工大学 | 一种提高嗜热厌氧杆菌发酵性能的方法 |
CN111423256A (zh) * | 2020-04-23 | 2020-07-17 | 上海国璨环境科技有限公司 | 一种有机垃圾热解方法 |
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EP2511287A2 (en) | 2012-10-17 |
ZA200908721B (en) | 2010-08-25 |
BRPI0811556A2 (pt) | 2017-05-02 |
EP2511284A2 (en) | 2012-10-17 |
KR20100037031A (ko) | 2010-04-08 |
EP2511285A2 (en) | 2012-10-17 |
RU2009145274A (ru) | 2011-06-20 |
US20150024450A1 (en) | 2015-01-22 |
RU2541785C2 (ru) | 2015-02-20 |
EP2511286A3 (en) | 2012-11-28 |
WO2008141174A3 (en) | 2009-03-12 |
NZ599526A (en) | 2013-07-26 |
EP2511284A3 (en) | 2012-11-28 |
NZ581493A (en) | 2012-05-25 |
JP2010526536A (ja) | 2010-08-05 |
CA2685879A1 (en) | 2008-11-20 |
US20100297721A1 (en) | 2010-11-25 |
AU2008251465B2 (en) | 2013-01-10 |
IL201824A0 (en) | 2010-06-16 |
EP2511286A2 (en) | 2012-10-17 |
EP2064225A2 (en) | 2009-06-03 |
US8435770B2 (en) | 2013-05-07 |
EP2511285A3 (en) | 2012-11-28 |
WO2008141174A2 (en) | 2008-11-20 |
WO2008141174A9 (en) | 2012-07-05 |
AU2008251465A1 (en) | 2008-11-20 |
EP2064225A4 (en) | 2010-03-03 |
EP2511287A3 (en) | 2012-11-28 |
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