CN114292315A - ppc突变体及其在制备L-缬氨酸中的应用 - Google Patents
ppc突变体及其在制备L-缬氨酸中的应用 Download PDFInfo
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- CN114292315A CN114292315A CN202111676686.2A CN202111676686A CN114292315A CN 114292315 A CN114292315 A CN 114292315A CN 202111676686 A CN202111676686 A CN 202111676686A CN 114292315 A CN114292315 A CN 114292315A
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- ppc
- protein
- valine
- gene
- mutant
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Abstract
本发明公开了ppc突变体及其在制备L‑缬氨酸中的应用。本发明公开的ppc突变体是将ppc蛋白质第186位氨基酸残基由甘氨酸突变为精氨酸得到的蛋白质。本发明首先通过对ppc基因进行单点突变得到ppcG556C基因,然后构建过表达ppc基因或ppcG556C基因的重组菌以及ppc基因敲除的重组菌,最后通过对构建的重组菌进行发酵培养发现,ppc基因或ppcG556C基因可以调控细菌L‑缬氨酸产量。本发明首次发现ppc基因参与L‑缬氨酸的生物合成,对于培育符合工业化生产的高产、高质量菌种,以及缬氨酸工业化生产具有重大的应用价值。
Description
技术领域
本发明属于生物技术领域,具体涉及ppc突变体及其在制备L-缬氨酸中的应用。
背景技术
L-缬氨酸(L-valine),化学名称为α-氨基异戊酸,是支链氨基酸之一,人和动物自身不能合成。L-缬氨酸是人体八种必需氨基酸之一,具有促进蛋白合成、抑制蛋白分解的作用,增强机体的免疫防护作用,有助于纠正因手术、创伤、感染等引起的负氮平衡。另外,L-缬氨酸还具有抗中枢疲劳、抗外周疲劳、延缓运动性疲劳和加快运动后机体修复的作用,因此在食品和医药行业具有广泛的应用及商业价值。由L-缬氨酸配制的复合支链氨基酸输液在血脑屏障、肝昏迷、慢性肝硬化以及肾功能衰竭的治疗,先天性代谢缺陷病的膳食治疗,败血症及术后糖尿病患者的治疗,加快外科创伤愈合的治疗和肿瘤患者的营养支持治疗中应用广泛。L-缬氨酸在食品工业上主要用作食品添加剂、营养增补液及风味剂等。L-缬氨酸凝胶具有带正电的端基,是新型低分子量凝胶,不仅可以使纯化和含有无机酸和盐的水溶液成凝胶而且可以使有机溶剂和油成胶状,可以制备形成水凝胶,其在生物医药、组织工程、光化学、电化学、食品工业、化妆品等领域已被广泛应用。L-缬氨酸因其含有特殊的生理功能,市场需求量大,使得L-缬氨酸的生产备受关注。
目前,L-缬氨酸大都采用直接发酵法生产,工业发酵中获得高产的菌种,对于L-缬氨酸的发酵生产来说是至关重要的,是整个L-缬氨酸发酵工业的核心,是决定发酵产品工业价值的重要因素。随着L-缬氨酸的市场需求不断增加,选育高产、稳定的生产菌种,促进L-缬氨酸在微生物体内的积累,进一步提高L-缬氨酸的产量一直是L-缬氨酸发酵工业技术开发和发酵工程化研究的热点,并且也将一直伴随L-缬氨酸发酵工业的发展,对于促进L-缬氨酸产业化的进程具有重要的意义。
发明内容
本发明的目的是如何利用ppc基因构建产缬氨酸工程菌,以进一步提高缬氨酸产量。
为了实现上述目的,本发明首先提供了一种ppc突变体。
本发明提供的ppc突变体是将ppc蛋白质第186位氨基酸残基由甘氨酸突变为其他氨基酸残基得到的蛋白质;
所述ppc蛋白质为如下A1)-A3)中的任一种:
A1)SEQ ID No.2所示的氨基酸序列组成的蛋白质;
A2)将A1)所示的氨基酸序列经过除第186位氨基酸残基以外的一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与细菌产缬氨酸相关的蛋白质;
A3)来源于细菌且与A1)或A2)具有95%以上同一性且与细菌产缬氨酸相关的蛋白质。
上述A2)所述的蛋白质中,所述一个或几个氨基酸残基的取代和/或缺失和/或添加为不超过10个氨基酸残基的取代和/或缺失和/或添加。
上述A3)所述的蛋白质中,这里使用的术语“同一性”指与天然氨基酸序列的序列相似性。“同一性”包括与本发明的SEQ ID No.2所示的氨基酸序列具有95%或更高,或96%或更高,或97%或更高,或98%或更高,或99%或更高同一性的氨基酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件,两个或多个序列之间的同一性可以用百分比(%)表示,其可以用来评价相关序列之间的同一性。
上述A1)或A2)或A3)所述的蛋白质可人工合成,也可先合成其编码基因,再进行生物表达得到。
进一步的,所述ppc突变体是将ppc蛋白质第186位氨基酸残基由甘氨酸突变为精氨酸得到的蛋白质(对应本发明实施例中的ppcG556C蛋白质)。
更进一步的,所述ppc突变体(ppcG556C蛋白质)是SEQ ID No.4所示的氨基酸序列组成的蛋白质。
为了实现上述目的,本发明又提供了与ppc突变体相关的生物材料。
本发明提供的与ppc突变体相关的生物材料为如下B1)至B4)中的任一种:
B1)编码上述ppc突变体的核酸分子;
B2)含有B1)所述核酸分子的表达盒;
B3)含有B1)所述核酸分子的重组载体、或含有B2)所述表达盒的重组载体;
B4)含有B1)所述核酸分子的重组微生物、或含有B2)所述表达盒的重组微生物、或含有B3)所述重组载体的重组微生物。
为了实现上述目的,本发明还提供了上述ppc蛋白质或与上述ppc蛋白质相关的生物材料或上述ppc突变体或与上述ppc突变体相关的生物材料的新用途。
本发明提供了上述ppc蛋白质或与上述ppc蛋白质相关的生物材料或上述ppc突变体或与上述ppc突变体相关的生物材料在如下X1)至X4)中任一种中的应用:
X1)调控细菌缬氨酸产量;
X2)构建产缬氨酸工程菌;
X3)制备缬氨酸;
与ppc蛋白质相关的生物材料为如下D1)至D4)中的任一种:
D1)编码所述ppc蛋白质的核酸分子;
D2)含有D1)所述核酸分子的表达盒;
D3)含有D1)所述核酸分子的重组载体、或含有D2)所述表达盒的重组载体;
D4)含有D1)所述核酸分子的重组微生物、或含有D2)所述表达盒的重组微生物、或含有D3)所述重组载体的重组微生物。
上述生物材料或应用中,B1)所述编码ppc突变体的核酸分子为如下C1)或C2)中的任一种:
C1)核苷酸序列为SEQ ID No.3的DNA分子;
C2)将SEQ ID No.3所示的核苷酸序列经过修饰和/或一个或几个核苷酸的取代和/或缺失和/或添加得到的与C1)所示的DNA分子具有90%以上的同一性,且具有相同功能的DNA分子。
D1)所述编码ppc蛋白质的核酸分子为如下E1)或E2)中的任一种:
E1)核苷酸序列为SEQ ID No.1的DNA分子;
E2)将SEQ ID No.1所示的核苷酸序列经过修饰和/或一个或几个核苷酸的取代和/或缺失和/或添加得到的与E1)所示的DNA分子具有90%以上的同一性,且具有相同功能的DNA分子。
其中,SEQ ID No.1所示的DNA分子为谷氨酸棒杆菌(Corynebacteriumglutamicum)CGMCC No.21260中的ppc基因,其编码的ppc蛋白质的氨基酸序列如SEQ IDNo.2所示。在本发明中通过引入点突变,得到SEQ ID No.3所示的ppcG556C基因,其编码的ppcG556C蛋白质的氨基酸序列如SEQ ID No.4所示。
本领域普通技术人员可以很容易地采用已知的方法,例如定向进化和点突变的方法,对本发明的编码ppc蛋白质或ppc突变体的核苷酸序列进行突变。那些经过人工修饰的,具有编码ppc蛋白质或ppc突变体的核苷酸序列90%或者更高同一性的核苷酸,只要编码ppc蛋白质或ppc突变体且具有相同功能,均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明的编码SEQ ID No.2或SEQ ID No.4所示的氨基酸序列组成的蛋白质的核苷酸序列具有90%或更高,或91%或更高,或92%或更高,或93%或更高,或94%或更高,或95%或更高,或96%或更高,或97%或更高,或98%或更高,或99%或更高同一性的核苷酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件,两个或多个序列之间的同一性可以用百分比(%)表示,其可以用来评价相关序列之间的同一性。
所述严格条件是在2×SSC,0.1%SDS的溶液中,在68℃下杂交并洗膜2次,每次5min;或,0.5×SSC,0.1%SDS的溶液中,在68℃下杂交并洗膜2次,每次15min;或,0.1×SSPE(或0.1×SSC)、0.1%SDS的溶液中,65℃条件下杂交并洗膜。
上述生物材料或应用中,B2)所述含有编码ppc突变体的核酸分子的表达盒是指能够在宿主细胞中表达ppc突变体的DNA,该DNA不但可包括启动ppc突变体基因转录的启动子,还可包括终止ppc突变体基因转录的终止子。进一步,所述表达盒还可包括增强子序列。D2)所述含有编码ppc蛋白质的核酸分子的表达盒是指能够在宿主细胞中表达ppc蛋白质的DNA,该DNA不但可包括启动ppc基因转录的启动子,还可包括终止ppc基因转录的终止子。进一步,所述表达盒还可包括增强子序列。
上述生物材料或应用中,B3)或D3)所述载体可为质粒、黏粒、噬菌体或病毒载体。所述质粒具体可为pK18mobsacB质粒或pXMJ19质粒。
在本发明的一个具体实施例中,所述重组载体为重组载体pK18-ppcG556C。
在本发明的另一个具体实施例中,所述重组载体为重组载体pK18-ppcOE或重组载体pK18-ppcG556COE。
在本发明的又一个具体实施例中,所述重组载体为重组载体pXMJ19-ppc或重组载体pXMJ19-ppcG556C。
上述生物材料中,B4)或D4)所述微生物可为酵母、细菌、藻或真菌。
进一步的,细菌可为任一具有产缬氨酸能力的细菌,如来自短杆菌属(Brevibacterium)、棒杆菌属(Corynebacterium)、埃希氏菌属(Escherichia)、气杆菌属(Aerobacter)、微球菌属(Micrococcus)、黄杆菌属(Flavobacterium)或芽胞杆菌属(Bacillus)的细菌等。
更进一步的,所述细菌包括但不限于谷氨酸棒杆菌(Corynebacteriumglutamicum)、黄色短杆菌(Brevibacterium flavum)、乳酸发酵短杆菌(Brevibacteriumlactofermentum)、产谷氨酸微球菌(Micrococcus glutamicus)、产氨短杆菌(Brevibacterum ammoniagenes)、大肠杆菌(Escherichia coli)、产气气杆菌(Aerobacteraerogenes)。
在本发明的一个具体实施例中,所述微生物为谷氨酸棒杆菌(Corynebacteriumglutamicum)CGMCC No.21260,该菌株名称为YPFV1,已于2020年11月30日保藏于中国微生物菌种保藏管理委员会普通微生物中(简称CGMCC,地址为:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所),保藏登记号为CGMCC No.21260。
上述应用中,调控为正调控。具体体现为当细菌中ppc蛋白质或ppc突变体含量或活性提高时,所述细菌缬氨酸产量提高;当细菌中ppc蛋白质含量或活性降低时,所述细菌缬氨酸产量降低。
为了实现上述目的,本发明还提供了提高ppc蛋白质或ppc突变体含量和/或活性的物质或提高ppc基因或ppc突变体基因表达量的物质的新用途。
本发明提供了提高ppc蛋白质或ppc突变体含量和/或活性的物质或提高ppc基因或ppc突变体基因表达量的物质在如下Y1)至Y4)中任一种中的应用:
Y1)提高细菌缬氨酸产量;
Y2)构建产缬氨酸工程菌;
Y3)制备缬氨酸。
进一步的,所述提高ppc基因表达量的物质可为ppc基因或含有所述ppc基因的重组载体。
所述提高ppc突变体基因表达量的物质可为ppc突变体基因或含有所述ppc突变体基因的重组载体。
更进一步的,含有所述ppc基因的重组载体具体可为重组载体pK18-ppcOE或重组载体pXMJ19-ppc。
含有所述ppc突变体基因的重组载体具体可为重组载体pK18-ppcG556COE或重组载体pXMJ19-ppcG556C。
为了实现上述目的,本发明还提供了一种提高细菌缬氨酸产量的方法。
本发明提供的提高细菌缬氨酸产量的方法为如下M1)或M2):
所述M1)包括如下步骤:将细菌基因组中的ppc基因替换为ppc突变体基因,实现细菌缬氨酸产量的提高;
所述M2)包括如下步骤:提高细菌中ppc蛋白质或ppc突变体含量和/或活性,或提高细菌中ppc基因或ppc突变体基因表达量,实现细菌缬氨酸产量的提高。
为了实现上述目的,本发明还提供了一种产缬氨酸工程菌的构建方法。
本发明提供的产缬氨酸工程菌的构建方法为如下N1)或N2):
所述N1)包括如下步骤:将细菌基因组中的ppc基因替换为ppc突变体基因,得到所述产缬氨酸工程菌;
所述N2)包括如下步骤:提高细菌中ppc蛋白质或ppc突变体含量和/或活性,或提高细菌中ppc基因或ppc突变体基因表达量,得到所述产缬氨酸工程菌;
上述任一所述应用或方法中,所述ppc突变体具体为ppcG556C蛋白质,具体为SEQ IDNo.4所示的氨基酸序列组成的蛋白质。
所述ppc突变体基因具体为ppcG556C基因,具体为SEQ ID No.3所示的DNA分子。
按照上述产缬氨酸工程菌的构建方法构建得到的产缬氨酸工程菌在制备缬氨酸中的应用也属于本发明的保护范围。
为了实现上述目的,本发明最后提供了一种制备缬氨酸的方法。
本发明提供的制备缬氨酸的方法包括如下步骤:发酵培养按照上述产缬氨酸工程菌的构建方法构建得到的产缬氨酸工程菌,得到所述缬氨酸。
所述发酵培养方法可按照现有技术中的常规试验方法进行。也可采用优化和改进后的常规试验方法进行。
所述发酵培养采用的培养基如实施例中的表3所示。
所述发酵培养条件如实施例中的表4所示。
上述任一所述应用或方法中,所述缬氨酸具体为L-缬氨酸。
本发明首先通过对ppc基因进行单点突变得到ppcG556C基因,然后构建过表达ppc基因或ppcG556C基因的重组菌以及ppc基因敲除的重组菌,最后通过对构建的重组菌进行发酵培养发现,ppc基因或ppcG556C基因可以调控细菌L-缬氨酸产量。本发明首次发现ppc基因参与L-缬氨酸的生物合成,对于培育符合工业化生产的高产、高质量菌种,以及缬氨酸工业化生产具有重大的应用价值。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南,并不以任何方式构成对本发明的限制。
下述实施例中的实验方法,如无特殊说明,均为常规方法,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1、构建包含点突变的ppc基因编码区片段的重组载体
依据NCBI公布的谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC14067基因组序列,设计并合成两对扩增ppc基因编码区的引物,以等位基因置换的方式在谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCC No.21260(经测序确认该菌株染色体上保留有野生型的ppc基因)的ppc基因编码区(SEQ ID No.1)中引入点突变,所述点突变为将ppc基因的核苷酸序列(SEQ ID No.1)中的第556位鸟嘌呤(G)突变为胞嘧啶(C)得到SEQ ID No.3所示的DNA分子(突变的ppc基因,名称为ppcG556C)。
其中,SEQ ID No.1所示的DNA分子编码氨基酸序列为SEQ ID No.2的蛋白质(所述蛋白质名称为蛋白质ppc)。
SEQ ID No.3所示的DNA分子编码氨基酸序列为SEQ ID No.4的突变蛋白质(所述突变蛋白质名称为ppcG556C)。所述突变蛋白质ppcG556C氨基酸序列(SEQ ID No.4)中的第186位精氨酸(R)由甘氨酸(G)突变而来。
采用重叠PCR(Overlap PCR)技术进行基因定点突变,引物设计如下(上海invitrogen公司合成),加粗字体的碱基为突变位置:
P1:5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGGAGAATGATCAATGGTCAAC-3';
P4:5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGCCCAGCTAAACACCTGTTT-3'。
构建方法:以谷氨酸棒杆菌ATCC14067为模板,分别采用引物P1/P2和P3/P4进行PCR扩增,获得两条分别带有突变碱基,大小分别为636bp和692bp的ppc基因编码区的DNA片段(ppc Up和ppc Down)。
PCR扩增体系为:10×Ex Taq Buffer 5μL,dNTP Mixture(各2.5mM)4μL,Mg2+(25mM)4μL,引物(10pM)各2μL,Ex Taq(5U/μL)0.25μL,总体积50μL。
PCR扩增反应程序为:94℃预变性5min,(94℃变性30s;52℃退火30s;72℃延伸40s;30个循环),72℃过度延伸10min。
将上述两条DNA片段(ppc Up和ppc Down)经琼脂糖凝胶电泳分离纯化后,对目的条带进行回收,再以上述两条DNA片段为模板,采用引物P1/P4通过Overlap PCR扩增,得到大小为1294bp的DNA片段(名称为ppc Up-Down,序列如SEQ ID No.5所示)。SEQ ID No.5所示的DNA片段中,第37-1171位(1135bp)为含有突变位点的ppcG556C基因片段(即SEQ ID No.3的1-1135位)。
Overlap PCR扩增反应体系为:10×Ex Taq Buffer 5μL,dNTP Mixture(各2.5mM)4μL,Mg2+(25mM)4μL,引物(10pM)各2μL,Ex Taq(5U/μL)0.25μL,总体积50μL。
Overlap PCR扩增反应程序为:94℃预变性5min,(94℃变性30s;52℃退火30s;72℃延伸60s;30个循环),72℃过度延伸10min。
SEQ ID No.5所示的DNA片段(ppc Up-Down)含有突变位点,导致菌株谷氨酸棒杆菌CGMCC No.21260中ppc基因编码区的第556位鸟嘌呤(G)变为胞嘧啶(C),最终导致编码蛋白的第186位甘氨酸(G)变为精氨酸(R)。将此DNA片段(ppc Up-Down)经琼脂糖凝胶电泳分离后进行纯化,与经过酶切(Xbal I/BamH I)后纯化的pK18mobsacB质粒(购自Addgene公司,用Xbal I/BamH I酶切)用NEBuilder酶(购自NEB公司)50℃连接30min,连接产物转化后长出的单克隆经PCR鉴定获得阳性重组载体pK18-ppcG556C,该重组载体上含有卡那霉素抗性标记。将酶切正确的重组载体pK18-ppcG556C送测序公司测序鉴定,并将含有正确点突变(G-C)的重组载体pK18-ppcG556C保存备用。
所述重组载体pK18-ppcG556C是将pK18mobsacB载体的Xbal I和/BamH I识别位点间的片段(小片段)替换为序列表中SEQ ID No.5的第37-1171位所示的DNA片段,且保持pK18mobsacB载体的其他序列不变得到的重组载体。
所述重组载体pK18-ppcG556C含有SEQ ID No.3所示的突变的基因ppcG556C的第1-1135位所示的DNA分子。
实施例2、构建包含基因ppcG556C的工程菌株YPV-049
构建方法:将实施例1中的等位替换质粒(pK18-ppcG556C)通过电击转化入谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCC No.21260后,在培养基中进行培养,培养基成分和培养条件参见表1,对培养产生的单菌落分别通过实施例1中的引物P1和通用引物M13R进行鉴定,能扩增出1301bp大小条带的菌株为阳性菌株。将阳性菌株在含15%蔗糖的培养基上培养,对培养产生的单菌落分别在含有卡那霉素和不含卡那霉素的培养基上培养,选择在不含卡那霉素的培养基上生长,而在含卡那霉素的培养基上不生长的菌株进一步采用引物P5/P6(上海invitrogen公司合成)进行PCR鉴定。引物序列如下:
P5:5'-CACGGTTGATACGTGGAATC-3';
P6:5'-GTTTTTGATGCGCAAAAGTG-3'。
将得到的PCR扩增产物(247bp)通过95℃高温变性10min、冰浴5min后进行SSCP(Single-Strand Conformation Polymorphis)电泳(以质粒pK18-ppcG556C扩增片段为阳性对照,谷氨酸棒杆菌ATCC14067扩增片段为阴性对照,水作为空白对照),SSCP电泳的PAGE的制备及电泳条件参见表2,由于片段结构不同,电泳位置不同,因此片段电泳位置与阴性对照片段位置不一致且与阳性对照片段位置一致的菌株为等位替换成功的菌株。再次通过引物P5和P6 PCR扩增阳性菌株ppc基因片段,并连接到PMD19-T载体进行测序,通过序列比对,碱基序列发生突变(G-C)的菌株为等位替换成功的阳性菌株,并将其命名为YPV-049。
重组菌YPV-049是将谷氨酸棒杆菌(Corynebacterium glutamicum)CGMCCNo.21260基因组中的ppc基因进行单点突变(对应于SEQ ID No.1所示ppc基因的第556位的碱基G突变为碱基C),且保持谷氨酸棒杆菌CGMCC No.21260的基因组中的其它序列不变得到的重组菌。
表1、培养基的组成和培养条件
成分 | 配方 |
蔗糖 | 10g/L |
多聚蛋白胨 | 10g/L |
牛肉膏 | 10g/L |
酵母粉 | 5g/L |
尿素 | 2g/L |
氯化钠 | 2.5g/L |
琼脂粉 | 20g/L |
水 | |
pH | 7.0 |
培养条件 | 32℃ |
表2、SSCP电泳的PAGE的制备及电泳条件
成分 | 用量(丙烯酰胺终浓度为8%) |
40%丙烯酰胺 | 8mL |
ddH<sub>2</sub>O | 26mL |
甘油 | 4mL |
10×TBE | 2mL |
TEMED | 40μL |
10%APS | 600μL |
电泳条件 | 将电泳槽置入冰中,使用1×TBE缓冲液电压120V,电泳时间10h |
实施例3、构建基因组上过表达ppc基因或ppcG556C基因的工程菌株YPV-050和YPV-051
依据NCBI公布的谷氨酸棒杆菌ATCC14067基因组序列,设计并合成三对扩增上下游同源臂片段及ppc或ppcG556C基因编码区及启动子区的引物,以同源重组的方式在谷氨酸棒杆菌CGMCC No.21260中引入ppc或ppcG556C基因。
引物设计如下(上海invitrogen公司合成):
P7:5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGGTAGTGCCGTGCGTACCCCA-3';
P8:5'-CACTGCGCAACTCTGGCTAGCCCAACCCCAATCGCAATGT-3';
P9:5'-ACATTGCGATTGGGGTTGGGCTAGCCAGAGTTGCGCAGTG-3';
P10:5'-GTGCGGGTTGGGGTTTTTGAAGTACAGAGCTTTAAAGCAC-3';
P11:5'-GTGCTTTAAAGCTCTGTACTTCAAAAACCCCAACCCGCAC-3';
P12:5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGTTGGTTTAGCGGAGCTGCA-3'。
构建方法:分别以谷氨酸棒杆菌ATCC14067或YPV-049为模板,分别采用引物P7/P8、P9/P10、P11/P12进行PCR扩增,获得上游同源臂片段795bp(对应于谷氨酸棒杆菌CGMCCNo.21260 CEY17_RS02570基因及其CEY17_RS02575的间隔区,序列如SEQ ID No.6所示),ppc基因及其启动子片段2985bp(序列如SEQ ID No.7所示)或ppcG556C基因及其启动子片段2985bp(序列如SEQ ID No.8所示)及下游同源臂片段769bp(对应于谷氨酸棒杆菌CGMCCNo.21260 CEY17_RS02575基因及其与CEY17_RS02570的间隔区,序列如SEQ ID No.9所示)。PCR反应结束后,对每个模板扩增得到的3个片段采用柱式DNA凝胶回收试剂盒分别进行电泳回收。回收后的3个片段与经过Xbal I/BamH I酶切后纯化的pK18mobsacB质粒(购自Addgene公司)用NEBuilder酶(购自NEB公司)50℃连接30min,连接产物转化后长出的单克隆用M13引物经PCR鉴定获得阳性整合质粒(重组载体),分别为pK18-ppcOE、pK18-ppcG556COE,该阳性整合质粒上含有卡那霉素抗性标记,可以通过卡那霉素筛选获得质粒整合到基因组上的重组子。
PCR反应体系为:10×Ex Taq Buffer 5μL,dNTP Mixture(各2.5mM)4μL,Mg2+(25mM)4μL,引物(10pM)各2μL,Ex Taq(5U/μL)0.25μL,总体积50μL。
PCR反应程序为:94℃预变性5min,94℃变性30s;52℃退火30s;72℃延伸60s(30个循环),72℃过度延伸10min。
将测序正确的整合质粒(pK18-ppcOE、pK18-ppcG556COE)分别电转化入谷氨酸棒杆菌CGMCC No.21260,在培养基中进行培养,培养基成分和培养条件参见表1,对培养产生的单菌落采用P13/P14引物进行PCR鉴定,PCR扩增出大小为3123bp的片段的菌株为阳性菌株,扩增不到片段的菌株为原菌。将阳性菌株在含15%蔗糖的培养基上培养,对培养产生的单菌落进一步采用P15/P16引物进行PCR鉴定,PCR扩增出大小为1668bp的片段的菌株为ppc或ppcG556C基因整合到谷氨酸棒杆菌CGMCC No.21260基因组上同源臂CEY17_02570和下同源臂CEY17_02575的间隔区上的阳性菌株,分别命名为YPV-050(不含突变点)和YPV-051(含突变点)。
PCR鉴定引物序列如下:
P13:5'-CGGTTAGATTTTTTGGCCCC-3'(对应上同源臂CEY17_RS02570的外侧);
P14:5'-TCGCCGCCGCACTGTTTTTG-3'(对应ppc基因内部);
P15:5'-CACGGTTGATACGTGGAATC-3'(对应ppc基因内部);
P16:5'-TCTGGACTGGGTGTTGCGCT-3'(对应下同源臂CEY17_RS02575的外侧)。
重组菌YPV-050含有双拷贝的SEQ ID No.1所示的ppc基因;具体地,重组菌YPV-050是将谷氨酸棒杆菌CGMCC No.21260基因组中上同源臂CEY17_02570和下同源臂CEY17_02575的间隔区替换为ppc基因,且保持谷氨酸棒杆菌CGMCC No.21260的基因组中的其它序列不变得到的重组菌。含有双拷贝ppc基因的重组菌可以显著和稳定地提高ppc基因的表达量。
重组菌YPV-051含有SEQ ID No.3所示的突变的ppcG556C基因;具体地,重组菌YPV-051是将谷氨酸棒杆菌CGMCC No.21260基因组中上同源臂CEY17_02570和下同源臂CEY17_02575的间隔区替换为ppcG556C基因,且保持谷氨酸棒杆菌CGMCC No.21260的基因组中的其它序列不变得到的重组菌。
实施例4、构建质粒上过表达ppc基因或ppcG556C基因的工程菌株YPV-052和YPV-053
依据NCBI公布的谷氨酸棒杆菌ATCC14067基因组序列,设计并合成一对扩增ppc或ppcG556C基因编码区及启动子区的引物,引物设计如下(上海invitrogen公司合成):
P17:5'-GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCCTAGCCAGAGTTGCGCAGTG-3'(带下划线的核苷酸序列为pXMJ19上的序列),
P18:5'-ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACAGTACAGAGCTTTAAAGCAC-3'(带下划线的核苷酸序列为pXMJ19上的序列)。
构建方法:分别以谷氨酸棒杆菌ATCC14067和YPV-049为模板,采用引物P17/P18进行PCR扩增,获得ppc基因及其启动子片段3015bp和ppcG556C基因及其启动子片段3015bp。然后将扩增产物进行电泳并采用柱式DNA凝胶回收试剂盒进行纯化回收,回收的DNA片段与经EcoR I酶切回收的穿梭质粒pXMJ19用NEBuilder酶(购自NEB公司)50℃连接30min,连接产物转化后长出的单克隆用M13引物经PCR鉴定获得阳性过表达质粒pXMJ19-ppc(含有ppc基因)和pXMJ19-ppcG556C(含有ppcG556C基因),再将该质粒送测序。因质粒上含有氯霉素抗性标记,可以通过氯霉素来筛选质粒是否转化到菌株中。
PCR反应体系为:10×Ex Taq Buffer 5μL,dNTP Mixture(各2.5mM)4μL,Mg2+(25mM)4μL,引物(10pM)各2μL,Ex Taq(5U/μL)0.25μL,总体积50μL。
PCR反应程序为:94℃预变性5min,94℃变性30s;52℃退火30s;72℃延伸60s(30个循环),72℃过度延伸10min。
将测序正确的pXMJ19-ppc和pXMJ19-ppcG556C质粒分别电转化入谷氨酸棒杆菌CGMCC No.21260中,在培养基中进行培养,培养基成分和培养条件参见表1,对培养产生的单菌落通过引物M13R(-48)/P18进行PCR鉴定,PCR扩增出大小为3054bp片段的菌株为阳性菌株,其被命名为YPV-052(不含突变点)和YPV-053(含突变点)。
重组菌YPV-052含有SEQ ID No.1所示的ppc基因,是通过质粒过表达ppc基因的重组菌。
重组菌YPV-053含有SEQ ID No.3所示的突变的ppcG556C基因,是通过质粒过表达ppcG556C基因的重组菌。
实施例5、构建基因组上缺失ppc基因的工程菌株YPV-054
根据NCBI公布的谷氨酸棒杆菌ATCC14067的基因组序列,合成两对扩增ppc基因编码区两端片段的引物,作为上下游同源臂片段。引物设计如下(上海invitrogen公司合成):
P19:5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGGGCTTACCAACGCAAAAGCT-3';
P20:5'-AAAGAGTGTTTAAAGTAGTTTCCTGCTGGGTAGGTAGTAC-3';
P21:5'-GTACTACCTACCCAGCAGGAAACTACTTTAAACACTCTTT-3';
P22:5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCCCGTTCCTTTCACTGACATC-3'。
构建方法:以谷氨酸棒杆菌ATCC14067为模板,分别采用引物P19/P20和P21/P22进行PCR扩增,获得ppc的上游同源臂片段849bp及ppc的下游同源臂片段873bp。再采用引物P19/P22进行Overlap PCR得到整个同源臂片段1722bp(序列如SEQ ID No.10所示)。对扩增的产物进行电泳并采用柱式DNA凝胶回收试剂盒进行纯化,回收的DNA片段与经过Xbal I/BamH I酶切后纯化的pK18mobsacB质粒(购自Addgene公司)用NEBuilder酶(购自NEB公司)50℃连接30min,连接产物转化后长出的单克隆用M13引物经PCR鉴定获得阳性敲除载体pK18-Δppc,将该质粒送测序。该质粒上含有卡那霉素抗性作为筛选标记。
Overlap PCR扩增反应体系为:10×Ex Taq Buffer 5μL,dNTP Mixture(各2.5mM)4μL,Mg2+(25mM)4μL,引物(10pM)各2μL,Ex Taq(5U/μL)0.25μL,总体积50μL。
Overlap PCR扩增反应程序为:94℃预变性5min,94℃变性30s;52℃退火30s;72℃延伸90s(30个循环),72℃过度延伸10min。
将测序正确的敲除质粒pK18-Δppc电转化入谷氨酸棒杆菌CGMCC No.21260,在培养基中进行培养,培养基成分和培养条件参见表1,对培养产生的单菌落通过如下引物(上海invitrogen公司合成)进行PCR鉴定:
P23:5'-GGCTTACCAACGCAAAAGCT-3'(对应于谷氨酸棒杆菌CGMCC No.21260ppc基因内部);
P24:5'-CCGTTCCTTTCACTGACATC-3'(对应于谷氨酸棒杆菌CGMCC No.21260ppc基因内部)。
上述PCR同时扩增出大小1722bp及4408bp的条带的菌株为阳性菌株,只扩增出1722bp条带的菌株为原菌。阳性菌株在15%蔗糖培养基上筛选后分别在含有卡那霉素和不含卡那霉素的培养基上培养,选择在不含卡那霉素的培养基上生长,而在含卡那霉素的培养基上不生长的菌株进一步采用P23/P24引物进行PCR鉴定,扩增出大小为1722bp条带的菌株为ppc基因编码区被敲除的阳性菌株ppc。再次采用P23/P24引物PCR扩增阳性菌株ppc片段,并连接到pMD19-T载体进行测序,将测序正确的菌株命名为YPV-054。
重组菌YPV-054是将谷氨酸棒杆菌CGMCC No.21260基因组序列上的ppc基因敲除,且保持谷氨酸棒杆菌CGMCC No.21260基因组中的其它序列不变得到的重组菌。
实施例6、L-缬氨酸发酵实验
将上述实施例构建的菌株(重组菌YPV-049、YPV-050、YPV-051、YPV-052、YPV-053和YPV-054)和原始菌株谷氨酸棒杆菌CGMCC No.21260在BLBIO-5GC-4-H型号的发酵罐(购自上海百仑生物科技有限公司)中以表3所示的培养基和表4所示的控制工艺进行发酵实验。每个菌株重复三次。完成发酵后,收集上清,采用HPLC检测上清中的L-缬氨酸产量。
结果如表5所示,在谷氨酸棒杆菌CGMCC No.21260中对ppc基因编码区进行点突变及过表达ppcG556C,有助于L-缬氨酸产量及转化率的提高,而对基因进行敲除或弱化,不利于L-缬氨酸的积累。
表3、发酵培养基配方(其余为水)
表4、发酵控制工艺
表5、L-缬氨酸发酵实验结果
以上对本发明进行了详述。对于本领域技术人员来说,在不脱离本发明的宗旨和范围,以及无需进行不必要的实验情况下,可在等同参数、浓度和条件下,在较宽范围内实施本发明。虽然本发明给出了特殊的实施例,应该理解为,可以对本发明作进一步的改进。总之,按本发明的原理,本申请欲包括任何变更、用途或对本发明的改进,包括脱离了本申请中已公开范围,而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围,可以进行一些基本特征的应用。
序列表
<110> 宁夏伊品生物科技股份有限公司
<120> ppc突变体及其在制备L-缬氨酸中的应用
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 2760
<212> DNA
<213> Artificial Sequence
<400> 1
atgactgatt ttttacgcga tgacatcagg ttcctcggtc aaatcctcgg tgaggtaatt 60
gcggaacaag aaggccagga ggtttatgaa ctggtcgaac aagcgcgcct gacttctttt 120
gatatcgcca agggcaacgc cgaaatggat agcctggttc aggttttcga cggcattact 180
ccagccaagg caacaccgat tgctcgcgca ttttcccact tcgctctgct ggctaacctg 240
gcggaagacc tctacgatga agagcttcgt gaacaggctc tcgatgcagg cgacacccct 300
ccggacagca ctcttgatgc cacctggctg aaactcaatg agggcaatgt tggcgcagaa 360
gctgtggccg atgtgctgcg caatgctgag gtggcgccgg ttctgactgc gcacccaact 420
gagactcgcc gccgcactgt ttttgatgcg caaaagtgga tcaccaccca catgcgtgaa 480
cgccacgctt tgcagtctgc ggagcctacc gctcgtacgc aaagcaagtt ggatgagatc 540
gagaagaaca tccgcggtcg catcaccatt ttgtggcaga ccgcgttgat tcgtgtggcc 600
cgcccacgta tcgaggacga gatcgaagta gggctgcgct actacaagct gagccttttg 660
gaagagattc cacgtatcaa ccgtgatgtg gctgttgagc ttcgtgagcg tttcggcgag 720
gatgttcctt tgaagcccgt ggtcaagcca ggttcctgga ttggtggaga ccacgacggt 780
aacccttatg tcaccgcgga aacagttgag tattccactc accgcgctgc ggaaaccgtg 840
ctcaagtact atgcacgcca gctgcattcc ctcgagcatg agctcagcct gtcggaccgc 900
atgaataagg tcaccccgca gctgcttgcg ctggcagatg ccgggcacaa cgacgtgcca 960
agccgcgtgg atgagcctta tcgacgcgcc gtccatggcg ttcgcggacg tatcctcgcg 1020
acgacggccg agctgatcgg cgaggacgcc gttgagggcg tgtggttcaa ggtctttact 1080
ccatacgcat ctccggaaga attcttaaac gatgcgttga ccattgatca ttctctgcgt 1140
gaatccaatg acgttctcat tgccgatgat cgtttgtctg tgctgatttc tgccatcgag 1200
agctttggat tcaaccttta cgcactggat ctgcgccaaa actccgaaag ctacgaggac 1260
gtcctcaccg agcttttcga acgcgcccaa gtcaccgcaa actaccgcga gctgtctgaa 1320
gcagagaagc ttgaggtgct gctgaaggaa ctgcgcagcc ctcgtccgct gatcccgcac 1380
ggttcagatg aatacagcga ggtcaccgac cgcgagctcg gcatcttccg caccgcgtcg 1440
gaggctgtta agaaattcgg gccacggatg gtgcctcact gcatcatctc catggcatca 1500
tcggtcaccg atgtgctcga gccgatggta ttgctcaagg aattcggcct cattgcagcc 1560
aacggcgaca acccacgcgg caccgtcgat gtcatcccac tgttcgaaac catcgaagat 1620
ctccaggccg gcgccggaat cctcgacgaa ctgtggaaaa ttgatcttta ccgcaactac 1680
ctcctgcagc gcgacaacgt ccaggaagtc atgctcggtt actccgattc caacaaggat 1740
ggcggatatt tctccgcaaa ctgggcgctt tacgacgcgg aactgcagct cgtcgaacta 1800
tgccgatcag ccggggtcaa gcttcgcctg ttccacggcc gtggtggcac cgtcggccgc 1860
ggtggcggac cttcctacga cgcgattctt gcccagccca ggggggctgt ccaaggttcc 1920
gtgcgcatca ccgagcaggg cgagatcatc tccgctaagt acggcaaccc cgaaaccgcg 1980
cgccgaaacc tcgaagctct ggtctcagca acgcttgagg catcgcttct cgacgtctcc 2040
gaactcaccg atcaccaacg cgcgtacgac atcatgagtg agatctctga gctcagcttg 2100
aagaagtacg cctccttggt gcacgaggat caaggcttca tcgattactt cacccagtcc 2160
acgccgctgc aggagattgg atccctcaac atcggatcca ggccttcctc acgcaagcag 2220
acctcctcgg tggaagattt gcgagcaatc ccgtgggtgc tcagttggtc ccagtctcgt 2280
gtcatgctgc cgggctggtt tggtgtcggc accgcacttg agcaatggat tggcgaaggg 2340
gagcaggcca cccagcgcat tgccgagcta caaacactca acgagtcctg gccatttttc 2400
acctcagtgt tggataacat ggctcaggtg atgtccaagg cagagctgcg tttggcaaag 2460
ctctacgcag acctgatccc agatagggaa gtagctgagc gcgtttatgc cgtcatccgc 2520
gaggaatact tcctgaccaa gaagatgttc tgcgtaatca ccggttctga tgatctgctt 2580
gatgacaacc cgcttctcgc acgatccgtc cagcgccgat acccctacct gcttccactc 2640
aacgtgatcc aggtagagat gatgcgacgc taccgaaaag gcgaccaaag cgagcaagta 2700
tcccgcaaca tccagctgac catgaacggt ctttccactg cactgcgcaa ctctggctag 2760
<210> 2
<211> 919
<212> PRT
<213> Artificial Sequence
<400> 2
Met Thr Asp Phe Leu Arg Asp Asp Ile Arg Phe Leu Gly Gln Ile Leu
1 5 10 15
Gly Glu Val Ile Ala Glu Gln Glu Gly Gln Glu Val Tyr Glu Leu Val
20 25 30
Glu Gln Ala Arg Leu Thr Ser Phe Asp Ile Ala Lys Gly Asn Ala Glu
35 40 45
Met Asp Ser Leu Val Gln Val Phe Asp Gly Ile Thr Pro Ala Lys Ala
50 55 60
Thr Pro Ile Ala Arg Ala Phe Ser His Phe Ala Leu Leu Ala Asn Leu
65 70 75 80
Ala Glu Asp Leu Tyr Asp Glu Glu Leu Arg Glu Gln Ala Leu Asp Ala
85 90 95
Gly Asp Thr Pro Pro Asp Ser Thr Leu Asp Ala Thr Trp Leu Lys Leu
100 105 110
Asn Glu Gly Asn Val Gly Ala Glu Ala Val Ala Asp Val Leu Arg Asn
115 120 125
Ala Glu Val Ala Pro Val Leu Thr Ala His Pro Thr Glu Thr Arg Arg
130 135 140
Arg Thr Val Phe Asp Ala Gln Lys Trp Ile Thr Thr His Met Arg Glu
145 150 155 160
Arg His Ala Leu Gln Ser Ala Glu Pro Thr Ala Arg Thr Gln Ser Lys
165 170 175
Leu Asp Glu Ile Glu Lys Asn Ile Arg Gly Arg Ile Thr Ile Leu Trp
180 185 190
Gln Thr Ala Leu Ile Arg Val Ala Arg Pro Arg Ile Glu Asp Glu Ile
195 200 205
Glu Val Gly Leu Arg Tyr Tyr Lys Leu Ser Leu Leu Glu Glu Ile Pro
210 215 220
Arg Ile Asn Arg Asp Val Ala Val Glu Leu Arg Glu Arg Phe Gly Glu
225 230 235 240
Asp Val Pro Leu Lys Pro Val Val Lys Pro Gly Ser Trp Ile Gly Gly
245 250 255
Asp His Asp Gly Asn Pro Tyr Val Thr Ala Glu Thr Val Glu Tyr Ser
260 265 270
Thr His Arg Ala Ala Glu Thr Val Leu Lys Tyr Tyr Ala Arg Gln Leu
275 280 285
His Ser Leu Glu His Glu Leu Ser Leu Ser Asp Arg Met Asn Lys Val
290 295 300
Thr Pro Gln Leu Leu Ala Leu Ala Asp Ala Gly His Asn Asp Val Pro
305 310 315 320
Ser Arg Val Asp Glu Pro Tyr Arg Arg Ala Val His Gly Val Arg Gly
325 330 335
Arg Ile Leu Ala Thr Thr Ala Glu Leu Ile Gly Glu Asp Ala Val Glu
340 345 350
Gly Val Trp Phe Lys Val Phe Thr Pro Tyr Ala Ser Pro Glu Glu Phe
355 360 365
Leu Asn Asp Ala Leu Thr Ile Asp His Ser Leu Arg Glu Ser Asn Asp
370 375 380
Val Leu Ile Ala Asp Asp Arg Leu Ser Val Leu Ile Ser Ala Ile Glu
385 390 395 400
Ser Phe Gly Phe Asn Leu Tyr Ala Leu Asp Leu Arg Gln Asn Ser Glu
405 410 415
Ser Tyr Glu Asp Val Leu Thr Glu Leu Phe Glu Arg Ala Gln Val Thr
420 425 430
Ala Asn Tyr Arg Glu Leu Ser Glu Ala Glu Lys Leu Glu Val Leu Leu
435 440 445
Lys Glu Leu Arg Ser Pro Arg Pro Leu Ile Pro His Gly Ser Asp Glu
450 455 460
Tyr Ser Glu Val Thr Asp Arg Glu Leu Gly Ile Phe Arg Thr Ala Ser
465 470 475 480
Glu Ala Val Lys Lys Phe Gly Pro Arg Met Val Pro His Cys Ile Ile
485 490 495
Ser Met Ala Ser Ser Val Thr Asp Val Leu Glu Pro Met Val Leu Leu
500 505 510
Lys Glu Phe Gly Leu Ile Ala Ala Asn Gly Asp Asn Pro Arg Gly Thr
515 520 525
Val Asp Val Ile Pro Leu Phe Glu Thr Ile Glu Asp Leu Gln Ala Gly
530 535 540
Ala Gly Ile Leu Asp Glu Leu Trp Lys Ile Asp Leu Tyr Arg Asn Tyr
545 550 555 560
Leu Leu Gln Arg Asp Asn Val Gln Glu Val Met Leu Gly Tyr Ser Asp
565 570 575
Ser Asn Lys Asp Gly Gly Tyr Phe Ser Ala Asn Trp Ala Leu Tyr Asp
580 585 590
Ala Glu Leu Gln Leu Val Glu Leu Cys Arg Ser Ala Gly Val Lys Leu
595 600 605
Arg Leu Phe His Gly Arg Gly Gly Thr Val Gly Arg Gly Gly Gly Pro
610 615 620
Ser Tyr Asp Ala Ile Leu Ala Gln Pro Arg Gly Ala Val Gln Gly Ser
625 630 635 640
Val Arg Ile Thr Glu Gln Gly Glu Ile Ile Ser Ala Lys Tyr Gly Asn
645 650 655
Pro Glu Thr Ala Arg Arg Asn Leu Glu Ala Leu Val Ser Ala Thr Leu
660 665 670
Glu Ala Ser Leu Leu Asp Val Ser Glu Leu Thr Asp His Gln Arg Ala
675 680 685
Tyr Asp Ile Met Ser Glu Ile Ser Glu Leu Ser Leu Lys Lys Tyr Ala
690 695 700
Ser Leu Val His Glu Asp Gln Gly Phe Ile Asp Tyr Phe Thr Gln Ser
705 710 715 720
Thr Pro Leu Gln Glu Ile Gly Ser Leu Asn Ile Gly Ser Arg Pro Ser
725 730 735
Ser Arg Lys Gln Thr Ser Ser Val Glu Asp Leu Arg Ala Ile Pro Trp
740 745 750
Val Leu Ser Trp Ser Gln Ser Arg Val Met Leu Pro Gly Trp Phe Gly
755 760 765
Val Gly Thr Ala Leu Glu Gln Trp Ile Gly Glu Gly Glu Gln Ala Thr
770 775 780
Gln Arg Ile Ala Glu Leu Gln Thr Leu Asn Glu Ser Trp Pro Phe Phe
785 790 795 800
Thr Ser Val Leu Asp Asn Met Ala Gln Val Met Ser Lys Ala Glu Leu
805 810 815
Arg Leu Ala Lys Leu Tyr Ala Asp Leu Ile Pro Asp Arg Glu Val Ala
820 825 830
Glu Arg Val Tyr Ala Val Ile Arg Glu Glu Tyr Phe Leu Thr Lys Lys
835 840 845
Met Phe Cys Val Ile Thr Gly Ser Asp Asp Leu Leu Asp Asp Asn Pro
850 855 860
Leu Leu Ala Arg Ser Val Gln Arg Arg Tyr Pro Tyr Leu Leu Pro Leu
865 870 875 880
Asn Val Ile Gln Val Glu Met Met Arg Arg Tyr Arg Lys Gly Asp Gln
885 890 895
Ser Glu Gln Val Ser Arg Asn Ile Gln Leu Thr Met Asn Gly Leu Ser
900 905 910
Thr Ala Leu Arg Asn Ser Gly
915
<210> 3
<211> 2760
<212> DNA
<213> Artificial Sequence
<400> 3
atgactgatt ttttacgcga tgacatcagg ttcctcggtc aaatcctcgg tgaggtaatt 60
gcggaacaag aaggccagga ggtttatgaa ctggtcgaac aagcgcgcct gacttctttt 120
gatatcgcca agggcaacgc cgaaatggat agcctggttc aggttttcga cggcattact 180
ccagccaagg caacaccgat tgctcgcgca ttttcccact tcgctctgct ggctaacctg 240
gcggaagacc tctacgatga agagcttcgt gaacaggctc tcgatgcagg cgacacccct 300
ccggacagca ctcttgatgc cacctggctg aaactcaatg agggcaatgt tggcgcagaa 360
gctgtggccg atgtgctgcg caatgctgag gtggcgccgg ttctgactgc gcacccaact 420
gagactcgcc gccgcactgt ttttgatgcg caaaagtgga tcaccaccca catgcgtgaa 480
cgccacgctt tgcagtctgc ggagcctacc gctcgtacgc aaagcaagtt ggatgagatc 540
gagaagaaca tccgccgtcg catcaccatt ttgtggcaga ccgcgttgat tcgtgtggcc 600
cgcccacgta tcgaggacga gatcgaagta gggctgcgct actacaagct gagccttttg 660
gaagagattc cacgtatcaa ccgtgatgtg gctgttgagc ttcgtgagcg tttcggcgag 720
gatgttcctt tgaagcccgt ggtcaagcca ggttcctgga ttggtggaga ccacgacggt 780
aacccttatg tcaccgcgga aacagttgag tattccactc accgcgctgc ggaaaccgtg 840
ctcaagtact atgcacgcca gctgcattcc ctcgagcatg agctcagcct gtcggaccgc 900
atgaataagg tcaccccgca gctgcttgcg ctggcagatg ccgggcacaa cgacgtgcca 960
agccgcgtgg atgagcctta tcgacgcgcc gtccatggcg ttcgcggacg tatcctcgcg 1020
acgacggccg agctgatcgg cgaggacgcc gttgagggcg tgtggttcaa ggtctttact 1080
ccatacgcat ctccggaaga attcttaaac gatgcgttga ccattgatca ttctctgcgt 1140
gaatccaatg acgttctcat tgccgatgat cgtttgtctg tgctgatttc tgccatcgag 1200
agctttggat tcaaccttta cgcactggat ctgcgccaaa actccgaaag ctacgaggac 1260
gtcctcaccg agcttttcga acgcgcccaa gtcaccgcaa actaccgcga gctgtctgaa 1320
gcagagaagc ttgaggtgct gctgaaggaa ctgcgcagcc ctcgtccgct gatcccgcac 1380
ggttcagatg aatacagcga ggtcaccgac cgcgagctcg gcatcttccg caccgcgtcg 1440
gaggctgtta agaaattcgg gccacggatg gtgcctcact gcatcatctc catggcatca 1500
tcggtcaccg atgtgctcga gccgatggta ttgctcaagg aattcggcct cattgcagcc 1560
aacggcgaca acccacgcgg caccgtcgat gtcatcccac tgttcgaaac catcgaagat 1620
ctccaggccg gcgccggaat cctcgacgaa ctgtggaaaa ttgatcttta ccgcaactac 1680
ctcctgcagc gcgacaacgt ccaggaagtc atgctcggtt actccgattc caacaaggat 1740
ggcggatatt tctccgcaaa ctgggcgctt tacgacgcgg aactgcagct cgtcgaacta 1800
tgccgatcag ccggggtcaa gcttcgcctg ttccacggcc gtggtggcac cgtcggccgc 1860
ggtggcggac cttcctacga cgcgattctt gcccagccca ggggggctgt ccaaggttcc 1920
gtgcgcatca ccgagcaggg cgagatcatc tccgctaagt acggcaaccc cgaaaccgcg 1980
cgccgaaacc tcgaagctct ggtctcagca acgcttgagg catcgcttct cgacgtctcc 2040
gaactcaccg atcaccaacg cgcgtacgac atcatgagtg agatctctga gctcagcttg 2100
aagaagtacg cctccttggt gcacgaggat caaggcttca tcgattactt cacccagtcc 2160
acgccgctgc aggagattgg atccctcaac atcggatcca ggccttcctc acgcaagcag 2220
acctcctcgg tggaagattt gcgagcaatc ccgtgggtgc tcagttggtc ccagtctcgt 2280
gtcatgctgc cgggctggtt tggtgtcggc accgcacttg agcaatggat tggcgaaggg 2340
gagcaggcca cccagcgcat tgccgagcta caaacactca acgagtcctg gccatttttc 2400
acctcagtgt tggataacat ggctcaggtg atgtccaagg cagagctgcg tttggcaaag 2460
ctctacgcag acctgatccc agatagggaa gtagctgagc gcgtttatgc cgtcatccgc 2520
gaggaatact tcctgaccaa gaagatgttc tgcgtaatca ccggttctga tgatctgctt 2580
gatgacaacc cgcttctcgc acgatccgtc cagcgccgat acccctacct gcttccactc 2640
aacgtgatcc aggtagagat gatgcgacgc taccgaaaag gcgaccaaag cgagcaagta 2700
tcccgcaaca tccagctgac catgaacggt ctttccactg cactgcgcaa ctctggctag 2760
<210> 4
<211> 919
<212> PRT
<213> Artificial Sequence
<400> 4
Met Thr Asp Phe Leu Arg Asp Asp Ile Arg Phe Leu Gly Gln Ile Leu
1 5 10 15
Gly Glu Val Ile Ala Glu Gln Glu Gly Gln Glu Val Tyr Glu Leu Val
20 25 30
Glu Gln Ala Arg Leu Thr Ser Phe Asp Ile Ala Lys Gly Asn Ala Glu
35 40 45
Met Asp Ser Leu Val Gln Val Phe Asp Gly Ile Thr Pro Ala Lys Ala
50 55 60
Thr Pro Ile Ala Arg Ala Phe Ser His Phe Ala Leu Leu Ala Asn Leu
65 70 75 80
Ala Glu Asp Leu Tyr Asp Glu Glu Leu Arg Glu Gln Ala Leu Asp Ala
85 90 95
Gly Asp Thr Pro Pro Asp Ser Thr Leu Asp Ala Thr Trp Leu Lys Leu
100 105 110
Asn Glu Gly Asn Val Gly Ala Glu Ala Val Ala Asp Val Leu Arg Asn
115 120 125
Ala Glu Val Ala Pro Val Leu Thr Ala His Pro Thr Glu Thr Arg Arg
130 135 140
Arg Thr Val Phe Asp Ala Gln Lys Trp Ile Thr Thr His Met Arg Glu
145 150 155 160
Arg His Ala Leu Gln Ser Ala Glu Pro Thr Ala Arg Thr Gln Ser Lys
165 170 175
Leu Asp Glu Ile Glu Lys Asn Ile Arg Arg Arg Ile Thr Ile Leu Trp
180 185 190
Gln Thr Ala Leu Ile Arg Val Ala Arg Pro Arg Ile Glu Asp Glu Ile
195 200 205
Glu Val Gly Leu Arg Tyr Tyr Lys Leu Ser Leu Leu Glu Glu Ile Pro
210 215 220
Arg Ile Asn Arg Asp Val Ala Val Glu Leu Arg Glu Arg Phe Gly Glu
225 230 235 240
Asp Val Pro Leu Lys Pro Val Val Lys Pro Gly Ser Trp Ile Gly Gly
245 250 255
Asp His Asp Gly Asn Pro Tyr Val Thr Ala Glu Thr Val Glu Tyr Ser
260 265 270
Thr His Arg Ala Ala Glu Thr Val Leu Lys Tyr Tyr Ala Arg Gln Leu
275 280 285
His Ser Leu Glu His Glu Leu Ser Leu Ser Asp Arg Met Asn Lys Val
290 295 300
Thr Pro Gln Leu Leu Ala Leu Ala Asp Ala Gly His Asn Asp Val Pro
305 310 315 320
Ser Arg Val Asp Glu Pro Tyr Arg Arg Ala Val His Gly Val Arg Gly
325 330 335
Arg Ile Leu Ala Thr Thr Ala Glu Leu Ile Gly Glu Asp Ala Val Glu
340 345 350
Gly Val Trp Phe Lys Val Phe Thr Pro Tyr Ala Ser Pro Glu Glu Phe
355 360 365
Leu Asn Asp Ala Leu Thr Ile Asp His Ser Leu Arg Glu Ser Asn Asp
370 375 380
Val Leu Ile Ala Asp Asp Arg Leu Ser Val Leu Ile Ser Ala Ile Glu
385 390 395 400
Ser Phe Gly Phe Asn Leu Tyr Ala Leu Asp Leu Arg Gln Asn Ser Glu
405 410 415
Ser Tyr Glu Asp Val Leu Thr Glu Leu Phe Glu Arg Ala Gln Val Thr
420 425 430
Ala Asn Tyr Arg Glu Leu Ser Glu Ala Glu Lys Leu Glu Val Leu Leu
435 440 445
Lys Glu Leu Arg Ser Pro Arg Pro Leu Ile Pro His Gly Ser Asp Glu
450 455 460
Tyr Ser Glu Val Thr Asp Arg Glu Leu Gly Ile Phe Arg Thr Ala Ser
465 470 475 480
Glu Ala Val Lys Lys Phe Gly Pro Arg Met Val Pro His Cys Ile Ile
485 490 495
Ser Met Ala Ser Ser Val Thr Asp Val Leu Glu Pro Met Val Leu Leu
500 505 510
Lys Glu Phe Gly Leu Ile Ala Ala Asn Gly Asp Asn Pro Arg Gly Thr
515 520 525
Val Asp Val Ile Pro Leu Phe Glu Thr Ile Glu Asp Leu Gln Ala Gly
530 535 540
Ala Gly Ile Leu Asp Glu Leu Trp Lys Ile Asp Leu Tyr Arg Asn Tyr
545 550 555 560
Leu Leu Gln Arg Asp Asn Val Gln Glu Val Met Leu Gly Tyr Ser Asp
565 570 575
Ser Asn Lys Asp Gly Gly Tyr Phe Ser Ala Asn Trp Ala Leu Tyr Asp
580 585 590
Ala Glu Leu Gln Leu Val Glu Leu Cys Arg Ser Ala Gly Val Lys Leu
595 600 605
Arg Leu Phe His Gly Arg Gly Gly Thr Val Gly Arg Gly Gly Gly Pro
610 615 620
Ser Tyr Asp Ala Ile Leu Ala Gln Pro Arg Gly Ala Val Gln Gly Ser
625 630 635 640
Val Arg Ile Thr Glu Gln Gly Glu Ile Ile Ser Ala Lys Tyr Gly Asn
645 650 655
Pro Glu Thr Ala Arg Arg Asn Leu Glu Ala Leu Val Ser Ala Thr Leu
660 665 670
Glu Ala Ser Leu Leu Asp Val Ser Glu Leu Thr Asp His Gln Arg Ala
675 680 685
Tyr Asp Ile Met Ser Glu Ile Ser Glu Leu Ser Leu Lys Lys Tyr Ala
690 695 700
Ser Leu Val His Glu Asp Gln Gly Phe Ile Asp Tyr Phe Thr Gln Ser
705 710 715 720
Thr Pro Leu Gln Glu Ile Gly Ser Leu Asn Ile Gly Ser Arg Pro Ser
725 730 735
Ser Arg Lys Gln Thr Ser Ser Val Glu Asp Leu Arg Ala Ile Pro Trp
740 745 750
Val Leu Ser Trp Ser Gln Ser Arg Val Met Leu Pro Gly Trp Phe Gly
755 760 765
Val Gly Thr Ala Leu Glu Gln Trp Ile Gly Glu Gly Glu Gln Ala Thr
770 775 780
Gln Arg Ile Ala Glu Leu Gln Thr Leu Asn Glu Ser Trp Pro Phe Phe
785 790 795 800
Thr Ser Val Leu Asp Asn Met Ala Gln Val Met Ser Lys Ala Glu Leu
805 810 815
Arg Leu Ala Lys Leu Tyr Ala Asp Leu Ile Pro Asp Arg Glu Val Ala
820 825 830
Glu Arg Val Tyr Ala Val Ile Arg Glu Glu Tyr Phe Leu Thr Lys Lys
835 840 845
Met Phe Cys Val Ile Thr Gly Ser Asp Asp Leu Leu Asp Asp Asn Pro
850 855 860
Leu Leu Ala Arg Ser Val Gln Arg Arg Tyr Pro Tyr Leu Leu Pro Leu
865 870 875 880
Asn Val Ile Gln Val Glu Met Met Arg Arg Tyr Arg Lys Gly Asp Gln
885 890 895
Ser Glu Gln Val Ser Arg Asn Ile Gln Leu Thr Met Asn Gly Leu Ser
900 905 910
Thr Ala Leu Arg Asn Ser Gly
915
<210> 5
<211> 1294
<212> DNA
<213> Artificial Sequence
<400> 5
cagtgccaag cttgcatgcc tgcaggtcga ctctaggaga atgatcaatg gtcaacgcat 60
cgtttaagaa ttcttccgga gatgcgtatg gagtaaagac cttgaaccac acgccctcaa 120
cggcgtcctc gccgatcagc tcggccgtcg tcgcgaggat acgtccgcga acgccatgga 180
cggcgcgtcg ataaggctca tccacgcggc ttggcacgtc gttgtgcccg gcatctgcca 240
gcgcaagcag ctgcggggtg accttattca tgcggtccga caggctgagc tcatgctcga 300
gggaatgcag ctggcgtgca tagtacttga gcacggtttc cgcagcgcgg tgagtggaat 360
actcaactgt ttccgcggtg acataagggt taccgtcgtg gtctccacca atccaggaac 420
ctggcttgac cacgggcttc aaaggaacat cctcgccgaa acgctcacga agctcaacag 480
ccacatcacg gttgatacgt ggaatctctt ccaaaaggct cagcttgtag tagcgcagcc 540
ctacttcgat ctcgtcctcg atacgtgggc gggccacacg aatcaacgcg gtctgccaca 600
aaatggtgat gcgacggcgg atgttcttct cgatctcatc caacttgctt tgcgtacgag 660
cggtaggctc cgcagactgc aaagcgtggc gttcacgcat gtgggtggtg atccactttt 720
gcgcatcaaa aacagtgcgg cggcgagtct cagttgggtg cgcagtcaga accggcgcca 780
cctcagcatt gcgcagcaca tcggccacag cttctgcgcc aacattgccc tcattgagtt 840
tcagccaggt ggcatcaaga gtgctgtccg gaggggtgtc gcctgcatcg agagcctgtt 900
cacgaagctc ttcatcgtag aggtcttccg ccaggttagc cagcagagcg aagtgggaaa 960
atgcgcgagc aatcggtgtt gccttggctg gagtaatgcc gtcgaaaacc tgaaccaggc 1020
tatccatttc ggcgttgccc ttggcgatat caaaagaagt caggcgcgct tgttcgacca 1080
gttcataaac ctcctggcct tcttgttccg caattacctc accgaggatt tgaccgagga 1140
acctgatgtc atcgcgtaaa aaatcagtca taactacttt aaacactctt tcacattgag 1200
ggtgttggcg tgcatgagat aaaaaatcac ccagcaaaac aggtgtttag ctgggcgggt 1260
accgagctcg aattcgtaat catggtcata gctg 1294
<210> 6
<211> 795
<212> DNA
<213> Artificial Sequence
<400> 6
cagtgccaag cttgcatgcc tgcaggtcga ctctaggtag tgccgtgcgt accccattag 60
aaagtgaaaa ttcactgatt ctagccagtc acgctgggaa tcattacatg ggccttcttc 120
gatcattcca tgatcgacaa gaaaagcctc acgttcatca ggttgtaaat aggggacagt 180
agacattaat tacacctaaa aagaaaaggg cccccatgag gcgcatcgtt gagaggcgtt 240
gggggtgctg ttggcttcta cgatatatct aattttgcct gatgtgtcag tagctcgaac 300
gtcactttca cttgtcgtct gaagtttcga tgtttctgca ccataaacgg tgtttatgaa 360
ttatcccccc ctctaccccc cgggggtgag gttttcgctg agaaggctgg cttcaaacgg 420
gggctggaca cgtacgcgga gatggcgacg cgttctgtca cgaatcgtgc gttgcgtgct 480
ggccattccg ccacccaagc cagatccagg tcatgagggc taccaggcca cacagaagca 540
gcgctaccta gaacgccaga tcagggcgtc gaaacggatg gaagctgcag ccatcgaccc 600
tagagacatt gacaccgcaa aacagcgcat acgggcatac caggcaaaac tacgcgacca 660
catcaaacag cacgacctgc caaggcgcag acaccgagaa cagattaaaa tgcgctaaag 720
aagttaacat catgctgcca ccgcccaagc gggaaacatt gcgattgggg ttgggctagc 780
cagagttgcg cagtg 795
<210> 7
<211> 2985
<212> DNA
<213> Artificial Sequence
<400> 7
acattgcgat tggggttggg ctagccagag ttgcgcagtg cagtggaaag accgttcatg 60
gtcagctgga tgttgcggga tacttgctcg ctttggtcgc cttttcggta gcgtcgcatc 120
atctctacct ggatcacgtt gagtggaagc aggtaggggt atcggcgctg gacggatcgt 180
gcgagaagcg ggttgtcatc aagcagatca tcagaaccgg tgattacgca gaacatcttc 240
ttggtcagga agtattcctc gcggatgacg gcataaacgc gctcagctac ttccctatct 300
gggatcaggt ctgcgtagag ctttgccaaa cgcagctctg ccttggacat cacctgagcc 360
atgttatcca acactgaggt gaaaaatggc caggactcgt tgagtgtttg tagctcggca 420
atgcgctggg tggcctgctc cccttcgcca atccattgct caagtgcggt gccgacacca 480
aaccagcccg gcagcatgac acgagactgg gaccaactga gcacccacgg gattgctcgc 540
aaatcttcca ccgaggaggt ctgcttgcgt gaggaaggcc tggatccgat gttgagggat 600
ccaatctcct gcagcggcgt ggactgggtg aagtaatcga tgaagccttg atcctcgtgc 660
accaaggagg cgtacttctt caagctgagc tcagagatct cactcatgat gtcgtacgcg 720
cgttggtgat cggtgagttc ggagacgtcg agaagcgatg cctcaagcgt tgctgagacc 780
agagcttcga ggtttcggcg cgcggtttcg gggttgccgt acttagcgga gatgatctcg 840
ccctgctcgg tgatgcgcac ggaaccttgg acagcccccc tgggctgggc aagaatcgcg 900
tcgtaggaag gtccgccacc gcggccgacg gtgccaccac ggccgtggaa caggcgaagc 960
ttgaccccgg ctgatcggca tagttcgacg agctgcagtt ccgcgtcgta aagcgcccag 1020
tttgcggaga aatatccgcc atccttgttg gaatcggagt aaccgagcat gacttcctgg 1080
acgttgtcgc gctgcaggag gtagttgcgg taaagatcaa ttttccacag ttcgtcgagg 1140
attccggcgc cggcctggag atcttcgatg gtttcgaaca gtgggatgac atcgacggtg 1200
ccgcgtgggt tgtcgccgtt ggctgcaatg aggccgaatt ccttgagcaa taccatcggc 1260
tcgagcacat cggtgaccga tgatgccatg gagatgatgc agtgaggcac catccgtggc 1320
ccgaatttct taacagcctc cgacgcggtg cggaagatgc cgagctcgcg gtcggtgacc 1380
tcgctgtatt catctgaacc gtgcgggatc agcggacgag ggctgcgcag ttccttcagc 1440
agcacctcaa gcttctctgc ttcagacagc tcgcggtagt ttgcggtgac ttgggcgcgt 1500
tcgaaaagct cggtgaggac gtcctcgtag ctttcggagt tttggcgcag atccagtgcg 1560
taaaggttga atccaaagct ctcgatggca gaaatcagca cagacaaacg atcatcggca 1620
atgagaacgt cattggattc acgcagagaa tgatcaatgg tcaacgcatc gtttaagaat 1680
tcttccggag atgcgtatgg agtaaagacc ttgaaccaca cgccctcaac ggcgtcctcg 1740
ccgatcagct cggccgtcgt cgcgaggata cgtccgcgaa cgccatggac ggcgcgtcga 1800
taaggctcat ccacgcggct tggcacgtcg ttgtgcccgg catctgccag cgcaagcagc 1860
tgcggggtga ccttattcat gcggtccgac aggctgagct catgctcgag ggaatgcagc 1920
tggcgtgcat agtacttgag cacggtttcc gcagcgcggt gagtggaata ctcaactgtt 1980
tccgcggtga cataagggtt accgtcgtgg tctccaccaa tccaggaacc tggcttgacc 2040
acgggcttca aaggaacatc ctcgccgaaa cgctcacgaa gctcaacagc cacatcacgg 2100
ttgatacgtg gaatctcttc caaaaggctc agcttgtagt agcgcagccc tacttcgatc 2160
tcgtcctcga tacgtgggcg ggccacacga atcaacgcgg tctgccacaa aatggtgatg 2220
cgaccgcgga tgttcttctc gatctcatcc aacttgcttt gcgtacgagc ggtaggctcc 2280
gcagactgca aagcgtggcg ttcacgcatg tgggtggtga tccacttttg cgcatcaaaa 2340
acagtgcggc ggcgagtctc agttgggtgc gcagtcagaa ccggcgccac ctcagcattg 2400
cgcagcacat cggccacagc ttctgcgcca acattgccct cattgagttt cagccaggtg 2460
gcatcaagag tgctgtccgg aggggtgtcg cctgcatcga gagcctgttc acgaagctct 2520
tcatcgtaga ggtcttccgc caggttagcc agcagagcga agtgggaaaa tgcgcgagca 2580
atcggtgttg ccttggctgg agtaatgccg tcgaaaacct gaaccaggct atccatttcg 2640
gcgttgccct tggcgatatc aaaagaagtc aggcgcgctt gttcgaccag ttcataaacc 2700
tcctggcctt cttgttccgc aattacctca ccgaggattt gaccgaggaa cctgatgtca 2760
tcgcgtaaaa aatcagtcat aactacttta aacactcttt cacattgagg gtgttggcgt 2820
gcatgagata aaaaatcacc cagcaaaaca ggtgtttagc tgggcgattg atttcgatgg 2880
ctttcatggg ctcatcaaaa ccgcacttct acagtgcttg tgctttaagg ctgtgcttta 2940
aggctgtgct ttaaagctct gtacttcaaa aaccccaacc cgcac 2985
<210> 8
<211> 2985
<212> DNA
<213> Artificial Sequence
<400> 8
acattgcgat tggggttggg ctagccagag ttgcgcagtg cagtggaaag accgttcatg 60
gtcagctgga tgttgcggga tacttgctcg ctttggtcgc cttttcggta gcgtcgcatc 120
atctctacct ggatcacgtt gagtggaagc aggtaggggt atcggcgctg gacggatcgt 180
gcgagaagcg ggttgtcatc aagcagatca tcagaaccgg tgattacgca gaacatcttc 240
ttggtcagga agtattcctc gcggatgacg gcataaacgc gctcagctac ttccctatct 300
gggatcaggt ctgcgtagag ctttgccaaa cgcagctctg ccttggacat cacctgagcc 360
atgttatcca acactgaggt gaaaaatggc caggactcgt tgagtgtttg tagctcggca 420
atgcgctggg tggcctgctc cccttcgcca atccattgct caagtgcggt gccgacacca 480
aaccagcccg gcagcatgac acgagactgg gaccaactga gcacccacgg gattgctcgc 540
aaatcttcca ccgaggaggt ctgcttgcgt gaggaaggcc tggatccgat gttgagggat 600
ccaatctcct gcagcggcgt ggactgggtg aagtaatcga tgaagccttg atcctcgtgc 660
accaaggagg cgtacttctt caagctgagc tcagagatct cactcatgat gtcgtacgcg 720
cgttggtgat cggtgagttc ggagacgtcg agaagcgatg cctcaagcgt tgctgagacc 780
agagcttcga ggtttcggcg cgcggtttcg gggttgccgt acttagcgga gatgatctcg 840
ccctgctcgg tgatgcgcac ggaaccttgg acagcccccc tgggctgggc aagaatcgcg 900
tcgtaggaag gtccgccacc gcggccgacg gtgccaccac ggccgtggaa caggcgaagc 960
ttgaccccgg ctgatcggca tagttcgacg agctgcagtt ccgcgtcgta aagcgcccag 1020
tttgcggaga aatatccgcc atccttgttg gaatcggagt aaccgagcat gacttcctgg 1080
acgttgtcgc gctgcaggag gtagttgcgg taaagatcaa ttttccacag ttcgtcgagg 1140
attccggcgc cggcctggag atcttcgatg gtttcgaaca gtgggatgac atcgacggtg 1200
ccgcgtgggt tgtcgccgtt ggctgcaatg aggccgaatt ccttgagcaa taccatcggc 1260
tcgagcacat cggtgaccga tgatgccatg gagatgatgc agtgaggcac catccgtggc 1320
ccgaatttct taacagcctc cgacgcggtg cggaagatgc cgagctcgcg gtcggtgacc 1380
tcgctgtatt catctgaacc gtgcgggatc agcggacgag ggctgcgcag ttccttcagc 1440
agcacctcaa gcttctctgc ttcagacagc tcgcggtagt ttgcggtgac ttgggcgcgt 1500
tcgaaaagct cggtgaggac gtcctcgtag ctttcggagt tttggcgcag atccagtgcg 1560
taaaggttga atccaaagct ctcgatggca gaaatcagca cagacaaacg atcatcggca 1620
atgagaacgt cattggattc acgcagagaa tgatcaatgg tcaacgcatc gtttaagaat 1680
tcttccggag atgcgtatgg agtaaagacc ttgaaccaca cgccctcaac ggcgtcctcg 1740
ccgatcagct cggccgtcgt cgcgaggata cgtccgcgaa cgccatggac ggcgcgtcga 1800
taaggctcat ccacgcggct tggcacgtcg ttgtgcccgg catctgccag cgcaagcagc 1860
tgcggggtga ccttattcat gcggtccgac aggctgagct catgctcgag ggaatgcagc 1920
tggcgtgcat agtacttgag cacggtttcc gcagcgcggt gagtggaata ctcaactgtt 1980
tccgcggtga cataagggtt accgtcgtgg tctccaccaa tccaggaacc tggcttgacc 2040
acgggcttca aaggaacatc ctcgccgaaa cgctcacgaa gctcaacagc cacatcacgg 2100
ttgatacgtg gaatctcttc caaaaggctc agcttgtagt agcgcagccc tacttcgatc 2160
tcgtcctcga tacgtgggcg ggccacacga atcaacgcgg tctgccacaa aatggtgatg 2220
cgacggcgga tgttcttctc gatctcatcc aacttgcttt gcgtacgagc ggtaggctcc 2280
gcagactgca aagcgtggcg ttcacgcatg tgggtggtga tccacttttg cgcatcaaaa 2340
acagtgcggc ggcgagtctc agttgggtgc gcagtcagaa ccggcgccac ctcagcattg 2400
cgcagcacat cggccacagc ttctgcgcca acattgccct cattgagttt cagccaggtg 2460
gcatcaagag tgctgtccgg aggggtgtcg cctgcatcga gagcctgttc acgaagctct 2520
tcatcgtaga ggtcttccgc caggttagcc agcagagcga agtgggaaaa tgcgcgagca 2580
atcggtgttg ccttggctgg agtaatgccg tcgaaaacct gaaccaggct atccatttcg 2640
gcgttgccct tggcgatatc aaaagaagtc aggcgcgctt gttcgaccag ttcataaacc 2700
tcctggcctt cttgttccgc aattacctca ccgaggattt gaccgaggaa cctgatgtca 2760
tcgcgtaaaa aatcagtcat aactacttta aacactcttt cacattgagg gtgttggcgt 2820
gcatgagata aaaaatcacc cagcaaaaca ggtgtttagc tgggcgattg atttcgatgg 2880
ctttcatggg ctcatcaaaa ccgcacttct acagtgcttg tgctttaagg ctgtgcttta 2940
aggctgtgct ttaaagctct gtacttcaaa aaccccaacc cgcac 2985
<210> 9
<211> 769
<212> DNA
<213> Artificial Sequence
<400> 9
gtgctttaaa gctctgtact tcaaaaaccc caacccgcac atttttagat ttctattttg 60
tgtacatagg gttcggaaca aagcttaaac catccccaat tgaaatgtcg ttacacaccc 120
acatgtttga agtggagcaa accgaaaacc agttttcccc aacggcagcc gccccccacg 180
ttgaaccttc gaaatagtag gcaacaccat caagcggatc ttcatcaagc gaaatagtga 240
ttgactcttc accgttccgc ttacaaactg cgttagtgtc gctattttcc acccacttgt 300
cacactcgta cccgttttca tttagccatt tttcggcatg tcctattttc tcgaaccggg 360
caggagcgtc agggcttccg cagcccgcta gtagtagtcc ggctgcaatg atgcttaatg 420
tttttttcat gaattaaaca tagtactttg ctggtaaaaa tattggagaa ccccactggc 480
ctacatggtc agtgggggca tttttgcgtt tcacccctca aaaatcatca ccacacttgc 540
gggatttccc cctgatttcc cccactccca caccattccc agtggacagt gtggacgtat 600
tggacacatt aaacacattg cgaccaggta aaacgtcatg accaggtatc gtcaatgttc 660
ttgatgaatt tccgcaccgc aggattatca ttcgaggtgg aataaatagc ctgcagctcc 720
gctaaaccaa cgggtaccga gctcgaattc gtaatcatgg tcatagctg 769
<210> 10
<211> 1722
<212> DNA
<213> Artificial Sequence
<400> 10
cagtgccaag cttgcatgcc tgcaggtcga ctctagggct taccaacgca aaagctgcga 60
gaatccaggc tagatctgag gggtccatgt taaaggcctt tcagctaagt gatgctgtcg 120
agcggagtat tttctattcc gctatagata aacctagtcc agtttctacc tatcattcta 180
tagatttctg tttttataac gaaggtcacg tttttagccc gcacttacac cacgtctccc 240
ctgctgaaaa atcccccatg gcagcgtttt aacctgcggt tttatagact ccacgttccg 300
tcgggaaaca gttccccttt aaagatgtcc ttagggaaaa ttggaatgta tgtgaattag 360
gcatcaagat tttccaattt tgaccaccac tttgccgaaa tcttggtgtc gaaagtacac 420
accgagcaac agtggggaga cagcaggtcg acaaaaccca aaaaaagcgg ctcccctatg 480
aaaagggaac cgctcttttt aagctcgtgt cttatgaata agcctggatg aggttgagtg 540
cgacaatgca cacaatccag ataacggcaa ccaaaatggt gacgcgatcc aggttctttt 600
caacaacagt ggagcccgaa agattggact gcacaccgcc accgaagagg ctggagagtc 660
cgccgccttt gcccttgtgc agcaagacga aaaccgtcat gagcaggctg gcgacgacga 720
ggatgatttg aagcgtcaat gccatgtgga aaatgttcct ttgccgtttg agaacccagg 780
tgaaccttat tcccacctga tttcgaataa ctttagacag tatacacgag tactacctac 840
ccagcaggaa actactttaa acactctttc acattgaggg tgttggcgtg catgagataa 900
aaaatcaccc agcaaaacag gtgtttagct gggcgattga tttcgatggc tttcatgggc 960
tcatcaaaac cgcacttcta cagtgcttgt gctttaaggc tgtgctttaa ggctgtgctt 1020
taaagctctg tactttaagc aacgctcgca gcgttggcag ccagcttggc gaatgcttca 1080
ccgtcgaggg aagcgccacc gacaagtccg ccgtcgacgt caggctgacc gacgatctca 1140
gcgacggttt ctgccttaac agaaccaccg tagagaatac gcaggccctc agcgacctcg 1200
tcgcctgcaa gctccacgat cagaccgcgg atagccttgc acacttcctg agcgtcagct 1260
gcggaagcaa ccttaccggt gccgatagcc cacactggct catacgcgat aacggtgttg 1320
gccagttcag cagcgtccag gccagcaagg gacttgcggg tctgctcgac gacatactca 1380
acgtgggtgc cagcttcacg gatttccagt ggctcaccaa cgcagacgat cgggctgatg 1440
ccgttggaca gagctgcctt tgccttcgca gcaaccaact catcagactc gttgtggtac 1500
tcgcggcgct cggagtgtcc aacgacaacc caagagcagt tcaactttgc cagcatgctt 1560
gcagaaactt caccggtgta cgcaccggac tcgtgcgggg agacgtcctg agcaccgaaa 1620
gtgacctcaa gcttgtcgcc ctcaacgaga gtctggacgg agcggatgtc agtgaaagga 1680
acgggggtac cgagctcgaa ttcgtaatca tggtcatagc tg 1722
Claims (10)
1.一种ppc突变体,是将ppc蛋白质第186位氨基酸残基由甘氨酸突变为其他氨基酸残基得到的蛋白质;
所述ppc蛋白质为如下A1)-A3)中的任一种:
A1)SEQ ID No.2所示的氨基酸序列组成的蛋白质;
A2)将A1)所示的氨基酸序列经过除第186位氨基酸残基以外的一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与细菌产缬氨酸相关的蛋白质;
A3)来源于细菌且与A1)或A2)具有95%以上同一性且与细菌产缬氨酸相关的蛋白质。
2.根据权利要求1所述的ppc突变体,其特征在于:所述ppc突变体是将ppc蛋白质第186位氨基酸残基由甘氨酸突变为精氨酸得到的蛋白质。
3.与权利要求1所述ppc突变体相关的生物材料,所述生物材料为如下B1)至B4)中的任一种:
B1)编码权利要求1或2所述ppc突变体的核酸分子;
B2)含有B1)所述核酸分子的表达盒;
B3)含有B1)所述核酸分子的重组载体、或含有B2)所述表达盒的重组载体;
B4)含有B1)所述核酸分子的重组微生物、或含有B2)所述表达盒的重组微生物、或含有B3)所述重组载体的重组微生物。
4.根据权利要求3所述的生物材料,其特征在于:所述核酸分子为如下C1)或C2)中的任一种:
C1)核苷酸序列为SEQ ID No.3的DNA分子;
C2)将SEQ ID No.3所示的核苷酸序列经过修饰和/或一个或几个核苷酸的取代和/或缺失和/或添加得到的与C1)所示的DNA分子具有90%以上的同一性,且具有相同功能的DNA分子。
5.ppc蛋白质或与ppc蛋白质相关的生物材料或权利要求1所述的ppc突变体或权利要求2所述的生物材料在如下X1)至X4)中任一种中的应用:
X1)调控细菌缬氨酸产量;
X2)构建产缬氨酸工程菌;
X3)制备缬氨酸;
所述ppc蛋白质为权利要求1中所述ppc蛋白质;
与ppc蛋白质相关的生物材料为如下D1)至D4)中的任一种:
D1)编码所述ppc蛋白质的核酸分子;
D2)含有D1)所述核酸分子的表达盒;
D3)含有D1)所述核酸分子的重组载体、或含有D2)所述表达盒的重组载体;
D4)含有D1)所述核酸分子的重组微生物、或含有D2)所述表达盒的重组微生物、或含有D3)所述重组载体的重组微生物。
6.提高ppc蛋白质或ppc突变体含量和/或活性的物质或提高ppc基因或ppc突变体基因表达量的物质在如下Y1)至Y4)中任一种中的应用:
Y1)提高细菌缬氨酸产量;
Y2)构建产缬氨酸工程菌;
Y3)制备缬氨酸;
所述ppc蛋白质为权利要求1中所述ppc蛋白质;
所述ppc突变体为权利要求1所述ppc突变体;
所述ppc基因为编码权利要求1中所述ppc蛋白质的基因;
所述ppc突变体基因为编码权利要求1所述ppc突变体的基因。
7.一种提高细菌缬氨酸产量的方法,为如下M1)或M2):
所述M1)包括如下步骤:将细菌基因组中的ppc基因替换为ppc突变体基因,实现细菌缬氨酸产量的提高;
所述M2)包括如下步骤:提高细菌中ppc蛋白质或ppc突变体含量和/或活性,或提高细菌中ppc基因或ppc突变体基因表达量,实现细菌缬氨酸产量的提高;
所述ppc蛋白质为权利要求1中所述ppc蛋白质;
所述ppc突变体为权利要求1所述ppc突变体;
所述ppc基因为编码权利要求1中所述ppc蛋白质的基因;
所述ppc突变体基因为编码权利要求1所述ppc突变体的基因。
8.一种产缬氨酸工程菌的构建方法,为如下N1)或N2):
所述N1)包括如下步骤:将细菌基因组中的ppc基因替换为ppc突变体基因,得到所述产缬氨酸工程菌;
所述N2)包括如下步骤:提高细菌中ppc蛋白质或ppc突变体含量和/或活性,或提高细菌中ppc基因或ppc突变体基因表达量,得到所述产缬氨酸工程菌;
所述ppc蛋白质为权利要求1中所述ppc蛋白质;
所述ppc突变体为权利要求1所述ppc突变体;
所述ppc基因为编码权利要求1中所述ppc蛋白质的基因;
所述ppc突变体基因为编码权利要求1所述ppc突变体的基因。
9.按照权利要求8所述方法构建得到的产缬氨酸工程菌在制备缬氨酸中的应用。
10.一种制备缬氨酸的方法,包括如下步骤:发酵培养按照权利要求8所述方法构建得到的产缬氨酸工程菌,得到所述缬氨酸。
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1187539A (zh) * | 1996-12-05 | 1998-07-15 | 味之素株式会社 | 用于产生l-赖氨酸的方法 |
CN113667682A (zh) * | 2021-09-10 | 2021-11-19 | 宁夏伊品生物科技股份有限公司 | Yh66-rs11190基因突变体及其在制备l-缬氨酸中的应用 |
CN113683667A (zh) * | 2021-08-25 | 2021-11-23 | 宁夏伊品生物科技股份有限公司 | Yh66-rs10865基因改造得到的工程菌及其在制备缬氨酸中的应用 |
-
2021
- 2021-12-31 CN CN202111676686.2A patent/CN114292315A/zh active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1187539A (zh) * | 1996-12-05 | 1998-07-15 | 味之素株式会社 | 用于产生l-赖氨酸的方法 |
CN113683667A (zh) * | 2021-08-25 | 2021-11-23 | 宁夏伊品生物科技股份有限公司 | Yh66-rs10865基因改造得到的工程菌及其在制备缬氨酸中的应用 |
CN113667682A (zh) * | 2021-09-10 | 2021-11-19 | 宁夏伊品生物科技股份有限公司 | Yh66-rs11190基因突变体及其在制备l-缬氨酸中的应用 |
Non-Patent Citations (6)
Title |
---|
仇爱梅;窦文芳;李会;许正宏;: "磷酸烯醇式丙酮酸羧化酶基因的敲除对于谷氨酸棒杆菌V1生理代谢的影响", 微生物学通报, no. 09, 20 September 2012 (2012-09-20), pages 1215 - 1224 * |
仇爱梅等: "磷酸烯醇式丙酮酸羧化酶基因的敲除对于谷氨酸棒杆菌V1生理代谢的影响", 《微生物学通报》, vol. 39, no. 09, pages 1215 - 1224 * |
无: "MULTISPECIES: phosphoenolpyruvate carboxylase [Corynebacterium]", 《GENBANK DATABASE》, 18 January 2020 (2020-01-18), pages 040072736 * |
无: "MULTISPECIES: phosphoenolpyruvate carboxylase [Corynebacterium]", 《GENBANK DATABASE》, pages 040072736 * |
无: "phosphoenolpyruvate carboxylase [Corynebacterium glutamicum]", 《GENBANK DATABASE》, 18 January 2020 (2020-01-18), pages 003862256 * |
无: "phosphoenolpyruvate carboxylase [Corynebacterium glutamicum]", 《GENBANK DATABASE》, pages 003862256 * |
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