CN112280723A - 联产1,3-丙二醇和1,3-丁二醇的重组菌及其应用 - Google Patents
联产1,3-丙二醇和1,3-丁二醇的重组菌及其应用 Download PDFInfo
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Abstract
本发明提供一种联产1,3‑丙二醇和1,3‑丁二醇的重组菌及其应用。所述重组菌是将phaA、phaB、bld和yqhD基因通过质粒导入克雷伯氏菌(Klebsiella)中或通过基因工程手段整合到克雷伯氏菌染色体上而成。利用本发明提供的克雷伯氏菌工程菌,同时实现两种高附加值的二元醇(1,3‑丙二醇和1,3‑丁二醇)的生产,且两种二元醇相对于底物甘油的得率超过0.6g/g,远远大于单独生产1,3‑丙二醇或1,3‑丁二醇过程的收率。目标产物分离过程更加简化,从而极大提高整个生产过程的经济效益。
Description
技术领域
本发明属于生物化工技术领域,具体地说,涉及一种联产1,3-丙二醇和1,3-丁二醇的重组菌及其应用。
背景技术
1,3-丙二醇是一种重要的二元醇,主要用作单体与对苯二甲酸聚合生产新型的聚酯材料聚对苯二甲酸丙二醇脂(PTT)。1,3-丙二醇可以通过微生物发酵的方法,利用天然的微生物如克雷伯氏肺炎杆菌、丁酸梭菌等将甘油转化成1,3-丙二醇。由于克雷伯氏肺炎杆菌具有甘油代谢速率快、1,3-丙二醇产量高,能够耐受高浓度甘油和1,3-丙二醇,且发酵过程能在有氧条件下进行等特点,目前已被广泛应用于1,3-丙二醇的工业生产。
目前利用克雷伯氏肺炎杆菌生产1,3-丙二醇主要存在的问题是发酵过程产生大量的副产物,如2,3-丁二醇,乳酸,丁二酸,乙醇,乙酸等,不但极大降低了1,3-丙二醇的得率同时使得后提取过程变的十分的复杂,极大增加了发酵及后提取过程的成本。因此,提高1,3-丙二醇的得率同时降低副产物的产量,对于降低1,3-丙二醇生产成本具有极其重要的意义。
1,3-丁二醇是另外一种具有重要工业应用价值的二元醇,其可以被用作化妆品的溶剂,同时可以用作单体来合成聚酯、聚氨酯及生物增塑剂。同时光学纯的1,3-丁二醇可以用作医药中间体来合成许多药物。但化学法合成的1,3-丁二醇都是外消旋体,无法直接用作医药中间体。因此,利用生物法生产光学纯1,3-丁二醇具有重要应用价值。
目前还没有任何报道能够直接利用微生物同时合成1,3-丙二醇和1,3-丁二醇。
发明内容
本发明的目的是提供一种联产1,3-丙二醇和1,3-丁二醇的重组菌及其应用。
本发明构思如下:通过在克雷伯氏菌(如克雷伯氏肺炎杆菌)中引入1,3-丁二醇的生物合成途径,实现了在同一发酵过程同时生产两种具有重要工业价值的二元醇,使得两种二元醇相对于底物甘油的得率超过0.6g/g,远远大于单独生产1,3-丙二醇或1,3-丁二醇过程的收率。同时由于1,3-丁二醇与2,3-丁二醇竞争代谢底物且二者的分离十分困难,本发明通过阻断2,3-丁二醇的合成途径,完全消除了2,3-丁二醇的合成。本发明进一步通过敲除ldhA基因,adhE基因,mdh基因,消除了乳酸、乙醇、丁二酸的合成,使得重组克雷伯氏肺炎杆菌主要合成1,3-丙二醇和1,3-丁二醇两种产物,极大简化了后续提取的过程。
为了实现本发明目的,第一方面,本发明提供一种联产1,3-丙二醇和1,3-丁二醇的重组菌,所述重组菌是将phaA、phaB、bld和yqhD基因通过质粒导入克雷伯氏菌(Klebsiella)中或通过基因工程手段整合到克雷伯氏菌染色体上而成。
其中,所述phaA基因为乙酰CoA酰基转移酶基因,来源于Cupriavidus necator,其为编码如下蛋白质(a)或(b)的基因:
(a)由SEQ ID NO:3所示的氨基酸序列组成的蛋白质;
(b)SEQ ID NO:3所示序列经取代、缺失或添加一个或几个氨基酸且具有同等功能的由(a)衍生的蛋白质。
所述phaB基因为3-氧酰基-(酰基载体蛋白)还原酶基因,来源于Cupriavidusnecator,其为编码如下蛋白质(c)或(d)的基因:
(c)由SEQ ID NO:4所示的氨基酸序列组成的蛋白质;
(d)SEQ ID NO:4所示序列经取代、缺失或添加一个或几个氨基酸且具有同等功能的由(c)衍生的蛋白质。
所述bld基因来源于Clostridium saccharoperbutylacetonicum,其编码蛋白的氨基酸序列如SEQ ID NO:1或2所示。
所述yqhD基因为醇脱氢酶基因,来源于大肠杆菌(Escherichia coli),其为编码如下蛋白质(e)或(f)的基因:
(e)由SEQ ID NO:6所示的氨基酸序列组成的蛋白质;
(f)SEQ ID NO:6所示序列经取代、缺失或添加一个或几个氨基酸且具有同等功能的由(e)衍生的蛋白质。
优选地,所述克雷伯氏菌为克雷伯肺炎杆菌(Klebsiella pneumoniae),更优选保藏号为CGMCC NO.7824的克雷伯肺炎杆菌ACR30。公众可以通过中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)获得菌株ACR30。
所述重组菌可按如下方法构建得到:phaA、phaB基因经密码子优化后,与bld、yqhD基因一起构建到表达载体上,用重组载体转化克雷伯氏菌,筛选阳性转化子。
优选地,密码子优化的phaA-phaB操纵子的序列如SEQ ID NO:5所示。
更优选地,所述重组菌的构建如下:将bld-yqhD-phaAB串联基因表达盒构建到ptrc99a质粒上,用重组质粒转化克雷伯氏菌(如ACR30),筛选阳性转化子。
其中,bld-yqhD-phaAB串联基因表达盒的序列如SEQ ID NO:9和10所示。
第二方面,本发明提供一种联产1,3-丙二醇和1,3-丁二醇的重组菌,所述联产1,3-丙二醇和1,3-丁二醇的重组菌是以上述重组菌作为出发菌株,利用基因工程手段对出发菌株进行改造,得到的细胞内NADPH供给增强的工程菌。
优选地,通过增强出发菌株中与NADPH生物合成途径相关的基因,来提高细胞内NADPH的供给。
更优选地,所述与NADPH生物合成途径相关的基因为pntAB基因,核苷酸序列如下:
i)SEQ ID NO:7所示的核苷酸序列;
ii)SEQ ID NO:7所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列;
iii)在严格条件下与SEQ ID NO:7所示序列杂交且表达相同功能蛋白质的核苷酸序列,所述严格条件为在含0.1%SDS的0.1×SSPE或含0.1%SDS的0.1×SSC溶液中,在65℃下杂交,并用该溶液洗膜;或
iv)与i)、ii)或iii)的核苷酸序列具有90%以上同源性且表达相同功能蛋白质的核苷酸序列。
本发明中,所述增强的途径选自以下1)~6),或任选的组合:
1)通过导入具有所述基因的质粒而增强;
2)通过增加染色体上所述基因的拷贝数而增强;
3)通过改变染色体上所述基因的启动子序列而增强;
4)通过将强启动子与所述基因可操作地连接而增强;
5)通过导入增强子而增强;
6)通过使用具有编码高活性的相应酶或蛋白质的基因或等位基因而增强。
第三方面,本发明提供一种联产1,3-丙二醇和1,3-丁二醇的重组克雷伯氏菌,所述重组克雷伯氏菌是以上述重组菌作为出发菌株,通过弱化出发菌株中与2,3-丁二醇合成途径相关的基因,获得的基因弱化菌株。
所述与2,3-丁二醇合成途径相关的基因为budRABC基因,核苷酸序列如下:
i)SEQ ID NO:8所示的核苷酸序列;
ii)SEQ ID NO:8所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列;
iii)在严格条件下与SEQ ID NO:8所示序列杂交且表达相同功能蛋白质的核苷酸序列,所述严格条件为在含0.1%SDS的0.1×SSPE或含0.1%SDS的0.1×SSC溶液中,在65℃下杂交,并用该溶液洗膜;或
iv)与i)、ii)或iii)的核苷酸序列具有90%以上同源性且表达相同功能蛋白质的核苷酸序列。
第四方面,本发明提供一种高产1,3-丙二醇和1,3-丁二醇的工程菌,所述工程菌是以上述重组菌,或者上述重组克雷伯氏菌作为原始菌株,通过弱化原始菌株中ldhA、adhE、mdh基因中的至少一种基因,获得的基因弱化菌株;所述弱化包括敲除或降低基因的表达。
ldhA、adhE、mdh基因编码蛋白的氨基酸序列分别如SEQ ID NO:11-13所示。
优选地,弱化原始菌株中ldhA、adhE和mdh基因。
本发明中,所述弱化包括敲除或降低相应基因的表达。
进一步地,所述弱化是指利用同源重组的方法敲除相应基因。
第五方面,本发明提供上述重组菌,或上述重组克雷伯氏菌,或上述工程菌,在发酵联产1,3-丙二醇和1,3-丁二醇中的应用。
第六方面,本发明提供一种联产1,3-丙二醇和1,3-丁二醇的方法,包括在发酵培养基中对上述重组菌,或上述重组克雷伯氏菌,或上述工程菌进行培养以生产1,3-丙二醇和1,3-丁二醇。
可选地,所述发酵培养基为:甘油20g/L,K2HPO4 6g/L,KH2PO4 3g/L,NaCl 0.5g/L,NH4Cl 1g/L,MgSO4 0.5g/L,CaCl2 15mg/L,酵母粉2g/L,氨苄青霉素100mg/L,以水配制。
培养条件为:37℃,200rpm,控制溶氧低于10%的饱和溶氧值。
利用本发明提供的克雷伯氏菌工程菌,同时实现两种高附加值的二元醇(1,3-丙二醇和1,3-丁二醇)的生产,且两种二元醇相对于底物甘油的得率超过0.6g/g,远远大于单独生产1,3-丙二醇或1,3-丁二醇过程的收率。目标产物分离过程更加简化,从而极大提高整个生产过程的经济效益。
附图说明
图1为本发明所用质粒ptrc99a-bld-yqhD-phaAB的质粒图谱。
图2为本发明所用质粒ptrc99a-bld(L273T)-yqhD-phaAB的质粒图谱。
图3为本发明所用质粒ptrc99a-bld(L273T)-yqhD-phaAB-pntAB的质粒图谱。
具体实施方式
本发明具体的实施方式包括:(1)在克雷伯氏肺炎杆菌中引入1,3-丁二醇合成途径;(2)敲除2,3-丁二醇合成途径;(3)敲除其他副产物合成途径。
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,实施例均按照常规实验条件,如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning:a Laboratory Manual,2001),或按照制造厂商说明书建议的条件。
实施例1在克雷伯氏肺炎杆菌中构建1,3-丁二醇的生物合成途径
克雷伯氏肺炎杆菌本身不能直接合成1,3-丁二醇,本发明通过在克雷伯氏肺炎杆菌中引入外源的1,3-丁二醇合成途径,实现1,3-丙二醇与1,3-丁二醇的联产。
首先构建包含来源于Cupriavidus necator的phaA、phaB基因,来源于Clostridium saccharoperbutylacetonicum的醛脱氢酶基因bld基因(编码蛋白的氨基酸序列见SEQ ID NO:1),来源于大肠杆菌的yqhD基因(SEQ ID NO:4)的质粒ptrc99a-phaAB-bld-yqhD。以phaA(SEQ ID NO:3),phaB(SEQ ID NO:4)的氨基酸序列为基础,人工合成了密码子优化的phaA-phaB操纵子(SEQ ID NO:5)。将ptrc99a(从美国Addgene购买)用EcoRI/XbaI进行双酶切,利用Gibson组装试剂盒将人工合成的phaA-phaB操纵子、bld基因、yqhD基因***到ptrc99a质粒骨架上的EcoRI/XbaI酶切位点之间,获得的质粒命名为ptrc99a-phaAB-bld-yqhD(质粒图谱如图1所示)。
进一步地,本发明发现醛脱氢酶是影响1,3-丁二醇合成的关键制约因素。为此,本发明进行了大量的蛋白质工程改造,最终发现以ptrc99a-phaAB-bld-yqhD为模板,利用Biorad的基因突变试剂盒将bld基因的273位氨基酸由亮氨酸突变为苏氨酸(SEQ ID NO:2),获得质粒ptrc99a-phaAB-bld(L273T)-yqhD(质粒图谱如图2所示)。
进一步地,本发明发现NADPH供给对于克雷伯氏肺炎杆菌生产1,3-丁二醇的产量具有重要的影响,为此将ptrc99a-phaAB-bld(L273T)-yqhD用SbfI进行双酶切,利用Gibson组装试剂盒将大肠杆菌的pntAB基因(SEQ ID NO:7)***到ptrc99a-phaAB-bld(L273T)-yqhD质粒骨架上,获得的质粒命名为ptrc99a-phaAB-bld(L273T)-yqhD-pntAB(质粒图谱如图3所示)。
将上述三个质粒分别转化到克雷伯氏肺炎杆菌ACR30(CN 201310688092.2)中,获得的重组菌命名为ACR30/ptrc99a-bld-yqhD-phaAB,ACR30/ptrc99a-bld(L273T)-yqhD-phaAB,ACR30/ptrc99a-bld(L273T)-yqhD-phaAB-pntAB。将这三株菌与对照菌ACR30在250mL的摇瓶进行培养,培养基为:甘油20g/L,K2HPO4 6g/L,KH2PO4 3g/L,NaCl 0.5g/L,NH4Cl 1g/L,MgSO4 0.5g/L,CaCl2 15mg/L,酵母粉2g/L,氨苄青霉素100mg/L,培养温度为37℃,转速200rpm,待菌液OD600=0.6时,加入0.01mM的IPTG进行诱导,发酵48h时取样,利用高效液相色谱检测四株菌产物的分布情况。结果如表1所示,引入三个质粒后,三个重组菌株都可以同时生产1,3-丙二醇和1,3-丁二醇,其中ACR30/ptrc99a-bld(L273T)-yqhD-phaAB-pntAB的1,3-丙二醇和1,3-丁二醇的产量分别达到7.2g/L和2.8g/L,而其他副产物的产量大大减少。而对照菌株ACR30不能合成1,3-丁二醇,其主要产物为1,3-丙二醇和2,3-丁二醇,产量分别为6.8g/L和2.4g/L,同时还生产大量有机酸。
表1引入13-丁二醇合成途径对克雷伯氏肺炎杆菌发酵的影响
实施例2敲除2,3-丁二醇合成途径提高1,3-丙二醇和1,3-丁二醇的产量
上述改造实现了1,3-丙二醇和1,3-丁二醇的联产,但是同时还是积累了较多的副产物,尤其是2,3-丁二醇与1,3-丁二醇沸点接近,难以分离。为此通过敲除克雷伯氏肺炎杆菌中的2,3-丁二醇合成途径budRABC基因(SEQ ID NO:8),完全消除2,3-丁二醇的合成。
以克雷伯氏肺炎杆菌ACR30的基因组为模板以bud-up-F(atcgtatgtcttggatttaa)和bud-up-R(taataaaaaaagctctgacatggct cgaagcagctccagcctacagcgccatttt)为引物进行PCR,获得budRABC基因(SEQ ID NO:8)上游约1.0kb的DNA片段并进行PCR产物纯化。以克雷伯氏肺炎杆菌ACR30的基因组为模板以bud-down-F(gtcgacggatccccggaatattccctgtctcttgatcttcgcggcgccagcg)和bud-down-R(aaagcctcgcggtactgttt)为引物进行PCR,获得budRABC基因(SEQ ID NO:8)下游约1.0kb的DNA片段并进行PCR产物纯化。以质粒pIJ773(Journal of Industrial Microbiology 39(8):1219-26)为模板,以Apr-F(aaaatggcgctgtaggctggagctgcttcg)和Apr-R(agagacagggaatattccggggatccgtcgac)为引物进行PCR,获得安普霉素抗性基因约1.4kb的DNA片段并进行PCR产物纯化。以上面获得的三段PCR产物进行重叠PCR,获得约3.4kb的DNA片段并进行PCR产物纯化。
将包含有red重组***的质粒pDK-red(Journal of Industrial Microbiology39(8):1219-26)电转入克雷伯氏肺炎杆菌ACR30中,获得的菌株命名为ACR30/pDK-red。将上述获得的3.4kb重叠PCR产物电转到克雷伯氏肺炎杆菌ACR30/pDK-red,在带有50mg/L安普霉素的LB平板上筛选带有抗性的菌株。进一步利用Flp酶消除安普霉素抗性,将正确的菌株命名为ACR30:ΔbudRABC。将质粒ptrc99a-bld(L273T)-yqhD-phaAB-pntAB电转入ACR30:ΔbudRABC,获得的菌株命名为ACR30:ΔbudRABC/ptrc99a-bld(L273T)-yqhD-phaAB-pntAB。将该菌株与对照菌株ACR30/ptrc99a-bld(L273T)-yqhD-phaAB-pntAB进行发酵,发酵条件与实施例1相同,48h检测发酵产物的浓度。结果如表2所示,通过阻断2,3-丁二醇的合成途径,ACR30:ΔbudRABC/ptrc99a-phaAB-bld(L273T)-yqhD-pntAB完全不产2,3-丁二醇,同时1,3-丙二醇和1,3-丁二醇的产量提高到了7.3g/L和3.2g/L。
表2敲除2,3-丁二醇合成途径对克雷伯氏肺炎杆菌发酵的影响
实施例3阻断副产物合成进一步提高1,3-丙二醇和1,3-丁二醇产量
上述改造实现了1,3-丙二醇和1,3-丁二醇的联产,同时完全消除难以分离的2,3-丁二醇的合成,但仍然产生较多的副产物。为此通过敲除克雷伯氏肺炎杆菌中的乳酸合成基因ldhA、乙醇合成基因adhE,丁二酸合成基因mdh,极大降低副产物的合成,提高了1,3-丙二醇和1,3-丁二醇的产量,简化了后提取工艺。
利用red重组***,采用与实施例2相同的方法,以克雷伯氏肺炎杆菌ACR30的基因组为模板,分别以引物ldhA-up-F(tcacccgggcgatttcacagtc)、ldhA-up-R(aagacttttctccagtgattatacc)和ldhA-down-F(cgcctttcccttttgtgctcct)、ldhA-down-R(tggttcggcaattggcatgtgc)扩增ldhA上下游约1000bp片段,以引物adhE-up-F(aattaccaggagcgggggat)、adhE-up-R(ccaacagcgatagcatcgcg)和adhE-down-F(aatgctctcctgataatgtta)、adhE-down-R(cgatgctctcccggaccgccg)扩增adhE上下游约1000bp片段,以引物mdh-up-F(gtcatccccggcgatactgc)、mdh-up-R(cctaaactccttattatggttaatttagg)和mdh-down-F(tctcgccctaacagaagagc)、mdh-down-R(gaagcttaattttccctaatttaggc)扩增mdh上下游约1000bp片段,并分别与实施例2中的安普霉素抗性基因融合,将融合片段电转入ACR30:ΔbudRABC中用于敲除ldhA基因,adhE基因,mdh基因,获得重组菌分别命名为ACR30:ΔbudRABCΔldhA,ACR30:ΔbudRABCΔadhE,ACR30:ΔbudRABCΔmdh。进一步利用red重组***同时敲除ACR30:ΔbudRABC中的ldhA基因,adhE基因以及mdh基因,获得的重组菌株命名为ACR30:ΔbudRABCΔldhAΔadhEΔmdh。将质粒ptrc99a-bld(L273T)-yqhD-phaAB-pntAB分别电转入上述四株菌株,获得重组菌分别命名为ACR30:ΔbudRABCΔldhA/ptrc99a-bld(L273T)-yqhD-phaAB-pntAB,ACR30:ΔbudRABCΔadhE/ptrc99a-bld(L273T)-yqhD-phaAB-pntAB,ACR30:ΔbudRABCΔmdh/ptrc99a-bld(L273T)-yqhD-phaAB-pntAB,ACR30:ΔbudRABCΔldhAΔadhEΔmdh/ptrc99a-bld(L273T)-yqhD-phaAB-pntAB将上述四株菌与对照菌株ACR30:ΔbudRABC/ptrc99a-bld(L273T)-yqhD-phaAB-pntAB进行发酵,发酵条件与实施例1相同,48h检测发酵产物的浓度。结果如表3所示,通过阻断副产物的合成,1,3-丙二醇和1,3-丁二醇的产量都获得了提高。ACR30:ΔbudRABCΔldhAΔadhEΔmdh/ptrc99a-bld(L273T)-yqhD-phaAB-pntAB的1,3-丙二醇和1,3-丁二醇的产量提高到了7.5g/L和4.8g/L,两种二元醇的转化率超过0.6g/g甘油,其他副产物的产量都大大降低,极大简化了分离工艺,具有重要的经济价值。
表3阻断副产物合成途径对克雷伯氏肺炎杆菌发酵的影响
实施例4利用产酸克雷伯氏菌联产1,3-丙二醇和1,3-丁二醇
将实施例1中构建的质粒ptrc99a-bld-yqhD-phaAB、ptrc99a-bld(L273T)-yqhD-phaAB、ptrc99a-bld(L273T)-yqhD-phaAB-pntAB电转入产酸克雷伯氏菌M5al中(购自中国工业微生物菌种保藏中心),获得的重组菌命名为M5al/ptrc99a-bld-yqhD-phaAB、M5al/ptrc99a-bld(L273T)-yqhD-phaAB、M5al/ptrc99a-bld(L273T)-yqhD-phaAB-pntAB。将该菌株与对照菌M5al进行发酵,发酵条件与实施例1相同,48h检测发酵产物的浓度。结果见表4,说明在产酸克雷伯氏菌中引入本发明构建的1,3-丁二醇合成途径,同样实现了1,3-丙二醇和1,3-丁二醇的联产,并降低副产物乳酸、2,3-丁二醇的合成。
表4引入1,3-丁二醇合成途径对产酸克雷伯氏菌发酵的影响
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之做一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
序列表
<110> 清华大学 广东清大智兴生物技术有限公司
<120> 联产1,3-丙二醇和1,3-丁二醇的重组菌及其应用
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cgtggctggg gccgcatcgt caacatctcg tcggtgaacg ggcagaaggg ccagttcggc 1680
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gaagtggcga ccaagggcgt gaccgtcaac acggtctctc cgggctatat cgccaccgac 1800
atggtcaagg cgatccgcca ggacgtgctc gacaagatcg tcgcgacgat cccggtcaag 1860
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<210> 6
<211> 387
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Met Asn Asn Phe Asn Leu His Thr Pro Thr Arg Ile Leu Phe Gly Lys
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Gly Ala Ile Ala Gly Leu Arg Glu Gln Ile Pro His Asp Ala Arg Val
20 25 30
Leu Ile Thr Tyr Gly Gly Gly Ser Val Lys Lys Thr Gly Val Leu Asp
35 40 45
Gln Val Leu Asp Ala Leu Lys Gly Met Asp Val Leu Glu Phe Gly Gly
50 55 60
Ile Glu Pro Asn Pro Ala Tyr Glu Thr Leu Met Asn Ala Val Lys Leu
65 70 75 80
Val Arg Glu Gln Lys Val Thr Phe Leu Leu Ala Val Gly Gly Gly Ser
85 90 95
Val Leu Asp Gly Thr Lys Phe Ile Ala Ala Ala Ala Asn Tyr Pro Glu
100 105 110
Asn Ile Asp Pro Trp His Ile Leu Gln Thr Gly Gly Lys Glu Ile Lys
115 120 125
Ser Ala Ile Pro Met Gly Cys Val Leu Thr Leu Pro Ala Thr Gly Ser
130 135 140
Glu Ser Asn Ala Gly Ala Val Ile Ser Arg Lys Thr Thr Gly Asp Lys
145 150 155 160
Gln Ala Phe His Ser Ala His Val Gln Pro Val Phe Ala Val Leu Asp
165 170 175
Pro Val Tyr Thr Tyr Thr Leu Pro Pro Arg Gln Val Ala Asn Gly Val
180 185 190
Val Asp Ala Phe Val His Thr Val Glu Gln Tyr Val Thr Lys Pro Val
195 200 205
Asp Ala Lys Ile Gln Asp Arg Phe Ala Glu Gly Ile Leu Leu Thr Leu
210 215 220
Ile Glu Asp Gly Pro Lys Ala Leu Lys Glu Pro Glu Asn Tyr Asp Val
225 230 235 240
Arg Ala Asn Val Met Trp Ala Ala Thr Gln Ala Leu Asn Gly Leu Ile
245 250 255
Gly Ala Gly Val Pro Gln Asp Trp Ala Thr His Met Leu Gly His Glu
260 265 270
Leu Thr Ala Met His Gly Leu Asp His Ala Gln Thr Leu Ala Ile Val
275 280 285
Leu Pro Ala Leu Trp Asn Glu Lys Arg Asp Thr Lys Arg Ala Lys Leu
290 295 300
Leu Gln Tyr Ala Glu Arg Val Trp Asn Ile Thr Glu Gly Ser Asp Asp
305 310 315 320
Glu Arg Ile Asp Ala Ala Ile Ala Ala Thr Arg Asn Phe Phe Glu Gln
325 330 335
Leu Gly Val Pro Thr His Leu Ser Asp Tyr Gly Leu Asp Gly Ser Ser
340 345 350
Ile Pro Ala Leu Leu Lys Lys Leu Glu Glu His Gly Met Thr Gln Leu
355 360 365
Gly Glu Asn His Asp Ile Thr Leu Asp Val Ser Arg Arg Ile Tyr Glu
370 375 380
Ala Ala Arg
385
<210> 7
<211> 2948
<212> DNA
<213> 克雷伯肺炎杆菌(Klebsiella pneumoniae)
<400> 7
gatggaaggg aatatcatgc gaattggcat accaagagaa cggttaacca atgaaacccg 60
tgttgcagca acgccaaaaa cagtggaaca gctgctgaaa ctgggtttta ccgtcgcggt 120
agagagcggc gcgggtcaac tggcaagttt tgacgataaa gcgtttgtgc aagcgggcgc 180
tgaaattgta gaagggaata gcgtctggca gtcagagatc attctgaagg tcaatgcgcc 240
gttagatgat gaaattgcgt tactgaatcc tgggacaacg ctggtgagtt ttatctggcc 300
tgcgcagaat ccggaattaa tgcaaaaact tgcggaacgt aacgtgaccg tgatggcgat 360
ggactctgtg ccgcgtatct cacgcgcaca atcgctggac gcactaagct cgatggcgaa 420
catcgccggt tatcgcgcca ttgttgaagc ggcacatgaa tttgggcgct tctttaccgg 480
gcaaattact gcggccggga aagtgccacc ggcaaaagtg atggtgattg gtgcgggtgt 540
tgcaggtctg gccgccattg gcgcagcaaa cagtctcggc gcgattgtgc gtgcattcga 600
cacccgcccg gaagtgaaag aacaagttca aagtatgggc gcggaattcc tcgagctgga 660
ttttaaagag gaagctggca gcggcgatgg ctatgccaaa gtgatgtcgg acgcgttcat 720
caaagcggaa atggaactct ttgccgccca ggcaaaagag gtcgatatca ttgtcaccac 780
cgcgcttatt ccaggcaaac cagcgccgaa gctaattacc cgtgaaatgg ttgactccat 840
gaaggcgggc agtgtgattg tcgacctggc agcccaaaac ggcggcaact gtgaatacac 900
cgtgccgggt gaaatcttca ctacggaaaa tggtgtcaaa gtgattggtt ataccgatct 960
tccgggccgt ctgccgacgc aatcctcaca gctttacggc acaaacctcg ttaatctgct 1020
gaaactgttg tgcaaagaga aagacggcaa tatcactgtt gattttgatg atgtggtgat 1080
tcgcggcgtg accgtgatcc gtgcgggcga aattacctgg ccggcaccgc cgattcaggt 1140
atcagctcag ccgcaggcgg cacaaaaagc ggcaccggaa gtgaaaactg aggaaaaatg 1200
tacctgctca ccgtggcgta aatacgcgtt gatggcgctg gcaatcattc tttttggctg 1260
gatggcaagc gttgcgccga aagaattcct tgggcacttc accgttttcg cgctggcctg 1320
cgttgtcggt tattacgtgg tgtggaatgt atcgcacgcg ctgcatacac cgttgatgtc 1380
ggtcaccaac gcgatttcag ggattattgt tgtcggagca ctgttgcaga ttggccaggg 1440
cggctgggtt agcttcctta gttttatcgc ggtgcttata gccagcatta atattttcgg 1500
tggcttcacc gtgactcagc gcatgctgaa aatgttccgc aaaaattaag gggtaacata 1560
tgtctggagg attagttaca gctgcataca ttgttgccgc gatcctgttt atcttcagtc 1620
tggccggtct ttcgaaacat gaaacgtctc gccagggtaa caacttcggt atcgccggga 1680
tggcgattgc gttaatcgca accatttttg gaccggatac gggtaatgtt ggctggatct 1740
tgctggcgat ggtcattggt ggggcaattg gtatccgtct ggcgaagaaa gttgaaatga 1800
ccgaaatgcc agaactggtg gcgatcctgc atagcttcgt gggtctggcg gcagtgctgg 1860
ttggctttaa cagctatctg catcatgacg cgggaatggc accgattctg gtcaatattc 1920
acctgacgga agtgttcctc ggtatcttca tcggggcggt aacgttcacg ggttcggtgg 1980
tggcgttcgg caaactgtgt ggcaagattt cgtctaaacc attgatgctg ccaaaccgtc 2040
acaaaatgaa cctggcggct ctggtcgttt ccttcctgct gctgattgta tttgttcgca 2100
cggacagcgt cggcctgcaa gtgctggcat tgctgataat gaccgcaatt gcgctggtat 2160
tcggctggca tttagtcgcc tccatcggtg gtgcagatat gccagtggtg gtgtcgatgc 2220
tgaactcgta ctccggctgg gcggctgcgg ctgcgggctt tatgctcagc aacgacctgc 2280
tgattgtgac cggtgcgctg gtcggttctt cgggggctat cctttcttac attatgtgta 2340
aggcgatgaa ccgttccttt atcagcgtta ttgcgggtgg tttcggcacc gacggctctt 2400
ctactggcga tgatcaggaa gtgggtgagc accgcgaaat caccgcagaa gagacagcgg 2460
aactgctgaa aaactcccat tcagtgatca ttactccggg gtacggcatg gcagtcgcgc 2520
aggcgcaata tcctgtcgct gaaattactg agaaattgcg cgctcgtggt attaatgtgc 2580
gtttcggtat ccacccggtc gcggggcgtt tgcctggaca tatgaacgta ttgctggctg 2640
aagcaaaagt accgtatgac atcgtgctgg aaatggacga gatcaatgat gactttgctg 2700
ataccgatac cgtactggtg attggtgcta acgatacggt taacccggcg gcgcaggatg 2760
atccgaagag tccgattgct ggtatgcctg tgctggaagt gtggaaagcg cagaacgtga 2820
ttgtctttaa acgttcgatg aacactggct atgctggtgt gcaaaacccg ctgttcttca 2880
aggaaaacac ccacatgctg tttggtgacg ccaaagccag cgtggatgca atcctgaaag 2940
ctctgtaa 2948
<210> 8
<211> 4242
<212> DNA
<213> 克雷伯肺炎杆菌(Klebsiella pneumoniae)
<400> 8
tcagaacatc gccagaaagc gtttcaccgt acgcgagcgc tcgaagcgcc gccaggcgat 60
ggcgatatcg gtcttcagcg gcgccccgct aagcgggtga tagctgacgt tcggctgctg 120
gatgcaggtc atcgactgcg gaaccagcgc gaagccgaag cctgcattga ccatgctcag 180
cgacgatgaa atttgcgacg actgccaggc gcgcgccata tcgatcccgg cgcgcaggca 240
actgttatac accagctcat acagcccggg ggccacctcc cgcgggaaga ggatcggcgc 300
cacgtcgcgc agctgctcca gggccagggt cggctgcgtc gccagcgggt tatcgcgcgg 360
cagcgcgata accatcggct cctcatcgat aatccgcaga ttaaaggctt tactgctctc 420
gcacggcagg cggacgaagg cgatatccag ctcggcctcg ctcagggcgg tcatcagatt 480
ggccatattg tcttccatct ggtgcagggt caccccgggg tggtcgagct gaaaacggtg 540
cagcagcgtg aagatttgcg gatggaaagc atcagaactg gtaatgccta gcgacaggct 600
gccgttcatc ccgcgcgcaa tgcccttggc cttctccagc gccgcatcgc tcatggcgag 660
gatctggcgg gcatcctcat agaaagactc tcccgcttcc gtcagctcca ccccgcgggt 720
taaacgccgg aacagcgggg ttcccacttc gcgctcaagc cgctgaattt gctgacttaa 780
cggaggctgt gaaataccca gctccttggc ggcctgggtg aagtgccgcg tcctggcgac 840
ggcgacaaaa tagcgaagat aacgaagttc catatcgaaa acgtctcaaa ccagcatggt 900
ttctatattg gaactgtgag ctgaatcggg tcaacattta tttaaccttt cttatatttg 960
ttgaacgagg aagtggtata tgaatcattc tgctgaatgc acctgcgaag agagtctatg 1020
cgaaaccctg cgggcgtttt ccgcgcagca tcccgagagc gtgctctatc agacatcgct 1080
catgagcgcc ctgctgagcg gggtttacga aggcagcacc accatcgcgg acctgctgaa 1140
acacggcgat ttcggcctcg gcacctttaa tgagctggac ggggagctga tcgccttcag 1200
cagtcaggtc tatcagctgc gcgccgacgg cagcgcgcgc aaagcccagc cggagcagaa 1260
aacgccgttc gcggtgatga cctggttcca gccgcagtac cggaaaacct ttgaccatcc 1320
ggtgagccgc cagcaactgc acgaggtgat tgaccagcaa atcccctctg acaacctgtt 1380
ctgcgccctg cgcatcgacg gccatttccg ccatgcccat acccgcaccg tgccgcgcca 1440
gacaccgccg taccgggcga tgaccgacgt cctcgacgat cagccggtgt tccgctttaa 1500
ccagcgcgaa ggggtgctgg tcggcttccg gaccccgcag catatgcagg ggatcaacgt 1560
cgccgggtat cacgagcatt ttattaccga tgaccgcaaa ggcggcggtc acctgctgga 1620
ttaccagctc gaccacgggg tgctgacctt cggcgaaatt cacaagctga tgatcgacct 1680
gcccgccgac agcgcgttcc tgcaggctaa tctgcatccc gataatctcg atgccgccat 1740
ccgttccgta gaaagttaag ggggtcacat ggacaaacag tatccggtac gccagtgggc 1800
gcacggcgcc gatctcgtcg tcagtcagct ggaagctcag ggggtacgcc aggtgttcgg 1860
catccccggc gctaaaatcg acaaggtctt cgattcactg ctggattcct ccattcgcat 1920
tattccggta cgccacgaag ccaacgccgc atttatggcc gccgccgtcg gacgtattac 1980
cggcaaagcg ggcgtggcgc tggtcacctc cggtccgggt tgttctaacc tgatcaccgg 2040
catggccacc gcgaacagcg aaggcgaccc ggtggtggcc ctgggcggcg cggtaaaacg 2100
cgccgataaa gccaaacagg tccatcagag tatggatacg gtggcgatgt tcagcccggt 2160
aaccaaatac gccgtcgagg tgacggcgcc ggatgcgctg gcggaagtgg tctccaacgc 2220
cttccgcgcc gccgagcagg gccggccggg cagcgcgttc gttagcctgc cgcaggatgt 2280
ggtcgatggc ccggtcagcg gcaaagtgct gccggccagc ggggccccgc agatgggcgc 2340
cgcgccggat gatgccatcg accaggtggc gaagcttatc gcccaggcga agaacccgat 2400
cttcctgctc ggcctgatgg ccagccagcc ggaaaacagc aaggcgctgc gccgtttgct 2460
ggagaccagc catattccag tcaccagcac ctatcaggcc gccggagcgg tgaatcagga 2520
taacttctct cgcttcgccg gccgggttgg gctgtttaac aaccaggccg gggaccgtct 2580
gctgcagctc gccgacctgg tgatctgcat cggctacagc ccggtggaat acgaaccggc 2640
gatgtggaac agcggcaacg cgacgctggt gcacatcgac gtgctgcccg cctatgaaga 2700
gcgcaactat accccggatg tcgagctggt gggcgatatc gccggcactc tcaacaagct 2760
ggcgcaaaat atcgatcatc ggctggtgct ctccccgcag gcggcggaga tcctccgcga 2820
ccgccagcac cagcgcgagc tgctggaccg ccgcggcgcg cagctcaacc agtttgccct 2880
gcatcccctg cgcatcgttc gcgccatgca ggacatcgtc aacagcgacg tcacgctgac 2940
cgtggacatg ggcagcttcc atatctggat tgcccgctac ctgtacagct tccgcgcccg 3000
ccaggtgatg atctccaacg gccagcagac catgggcgtc gccctgccct gggccatcgg 3060
cgcctggctg gtcaatcctg agcgcaaagt ggtctccgtc tccggcgacg gcggcttcct 3120
gcagtcgagc atggagctgg agaccgccgt ccgcctgaaa gccaacgtgc tgcacctgat 3180
ctgggtcgat aacggctaca acatggtcgc tatccaggaa gagaaaaaat atcagcgcct 3240
gtccggcgtc gagttcgggc cgatggattt taaagcctat gccgaatcct tcggcgcgaa 3300
agggtttgcc gtggaaagcg ccgaggcgct tgagccgacc ctgcgcgcgg cgatggacgt 3360
cgacggcccg gcggtagtgg ccatcccggt ggattatcgc gataacccgc tgctgatggg 3420
ccagctgcat ctgagtcaga ttctgtaagt catcacaata aggaaaggaa aatgaaaaaa 3480
gtcgcacttg ttaccggcgc cggccagggg attggtaaag ctatcgccct tcgtctggtg 3540
aaggatggat ttgccgtggc cattgccgat tataacgacg ccaccgccaa agcggtcgcc 3600
tccgaaatca accaggccgg cggccgcgcc atggcggtga aagtggatgt ctccgaccgc 3660
gaccaggtat ttgccgccgt cgaacaggcg cgcaaaacgc tgggcggctt cgacgtcatc 3720
gtcaataacg ccggcgtagc gccgtccacg ccgatcgagt ccattacccc ggagattgtc 3780
gacaaagtct acaatatcaa cgtcaaaggg gtgatctggg gcatccaggc ggcggttgag 3840
gcctttaaga aagagggtca cggcgggaaa atcatcaacg cctgttccca ggccggccac 3900
gtcggcaacc cggagctggc ggtgtatagc tccagtaaat tcgcggtacg cggcttaacc 3960
cagaccgccg ctcgcgacct cgcgccgctg ggcatcacgg tcaacggcta ctgcccgggg 4020
attgtcaaaa cgccaatgtg ggccgaaatt gaccgccagg tgtccgaagc cgccggtaaa 4080
ccgctgggct acggtaccgc cgagttcgcc aaacgcatca ccctcggtcg tctgtccgag 4140
ccggaagatg tcgccgcctg cgtctcctat cttgccagcc cggattctga ttatatgacc 4200
ggtcagtcat tgctgatcga cggcgggatg gtgtttaact aa 4242
<210> 9
<211> 4627
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
atgaacaaag atactttaat cccgacgacc aaagatttaa aacttaaaac gaatgtcgaa 60
aatatcaatt tgaaaaatta taaagataat tcctcttgct ttggggtctt cgaaaatgtg 120
gaaaatgcga ttaatagcgc agttcacgct caaaaaatcc tttcgttgca ttacaccaag 180
gaacagcgtg agaaaattat caccgaaatt cgtaaagctg cacttgagaa taaggaggtg 240
cttgctacta tgattttgga ggaaacacat atgggccgct acgaagacaa aatccttaaa 300
catgaactgg ttgcgaaata taccccggga accgaagacc ttacaaccac tgcgtggagt 360
ggtgacaacg gcttgactgt tgtagagatg tcgccctatg gggtaattgg ggcgattacc 420
ccgtctacca acccgacgga gaccgtgatc tgcaactcaa ttggaatgat tgctgcagga 480
aatgcagtcg tattcaacgg ccacccgggt gccaaaaagt gcgttgcatt cgcgattgaa 540
atgattaata aggcgattat ttcttgcggt ggtccggaaa atctggtcac cacgatcaag 600
aacccgacaa tggaaagtct ggatgcgatc atcaaacacc ctttgattaa gcttctttgc 660
gggacgggtg gaccgggtat ggttaagact ttgctgaact caggtaagaa agcgattggt 720
gctggcgcag gtaacccgcc tgtgatcgtc gacgatactg ccgacatcga gaaagcaggg 780
aagtccatta ttgaaggatg tagctttgac aacaatttgc cttgcatcgc agagaaagag 840
gtattcgttt ttgagaacgt cgcggacgat ctgattagta acatgctgaa gaacaacgca 900
gtcattatta acgaggatca ggtgtccaaa ctgatcgact tggtgttgca aaaaaataat 960
gaaactcagg agtacttcat caacaagaaa tgggttggaa aggatgccaa gttattcagt 1020
gatgaaattg atgttgaatc gccctcgaac attaagtgta ttgtctgtga ggtcaacgct 1080
aaccatccat ttgttatgac cgaattaatg atgcctattc tgcccatcgt tcgcgtgaag 1140
gacattgatg aagctgtaaa gtataccaaa atcgcggaac aaaaccgtaa gcacagcgca 1200
tacatctaca gtaaaaacat tgacaacctt aaccgttttg agcgcgaaat tgatacgacg 1260
attttcgtta agaacgcaaa aagtttcgct ggggtagggt atgaggcaga aggatttaca 1320
acttttacaa ttgcgggatc tacgggagaa gggattacct ccgcacgcaa tttcacgcgc 1380
caacgtcgtt gtgtgttggc ggggtgaaac tttaagaagg agatatacat gaacaacttt 1440
aatctgcaca ccccaacccg cattctgttt ggtaaaggcg caatcgctgg tttacgcgaa 1500
caaattcctc acgatgctcg cgtattgatt acctacggcg gcggcagcgt gaaaaaaacc 1560
ggcgttctcg atcaagttct ggatgccctg aaaggcatgg acgtgctgga atttggcggt 1620
attgagccaa acccggctta tgaaacgctg atgaacgccg tgaaactggt tcgcgaacag 1680
aaagtgactt tcctgctggc ggttggcggc ggttctgtac tggacggcac caaatttatc 1740
gccgcagcgg ctaactatcc ggaaaatatc gatccgtggc acattctgca aacgggcggt 1800
aaagagatta aaagcgccat cccgatgggc tgtgtgctga cgctgccagc aaccggttca 1860
gaatccaacg caggcgcggt gatctcccgt aaaaccacag gcgacaagca ggcgttccat 1920
tctgcccatg ttcagccggt atttgccgtg ctcgatccgg tttataccta caccctgccg 1980
ccgcgtcagg tggctaacgg cgtagtggac gcctttgtac acaccgtgga acagtatgtt 2040
accaaaccgg ttgatgccaa aattcaggac cgtttcgcag aaggcatttt gctgacgcta 2100
atcgaagatg gtccgaaagc cctgaaagag ccagaaaact acgatgtgcg cgccaacgtc 2160
atgtgggcgg cgactcaggc gctgaacggt ttgattggcg ctggcgtacc gcaggactgg 2220
gcaacgcata tgctgggcca cgaactgact gcgatgcacg gtctggatca cgcgcaaaca 2280
ctggctatcg tcctgcctgc actgtggaat gaaaaacgcg ataccaagcg cgctaagctg 2340
ctgcaatatg ctgaacgcgt ctggaacatc actgaaggtt ccgatgatga gcgtattgac 2400
gccgcgattg ccgcaacccg caatttcttt gagcaattag gcgtgccgac ccacctctcc 2460
gactacggtc tggacggcag ctccatcccg gctttgctga aaaaactgga agagcacggc 2520
atgacccaac tgggcgaaaa tcatgacatt acgttggatg tcagccgccg tatatacgaa 2580
gccgcccgct aattgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat 2640
aacaatttca cccattgaaa ggactacaca atgactgacg ttgtcatcgt atccgccgcc 2700
cgcaccgcgg tcggcaagtt tggcggctcg ctggccaaga tcccggcacc ggaactgggt 2760
gccgtggtca tcaaggccgc gctggagcgc gccggcgtca agccggagca ggtgagcgaa 2820
gtcatcatgg gccaggtgct gaccgccggt tcgggccaga accccgcacg ccaggccgcg 2880
atcaaggccg gcctgccggc gatggtgccg gccatgacca tcaacaaggt gtgcggctcg 2940
ggcctgaagg ccgtgatgct ggccgccaac gcgatcatgg cgggcgacgc cgagatcgtg 3000
gtggccggcg gccaggaaaa catgagcgcc gccccgcacg tgctgccggg ctcgcgcgat 3060
ggtttccgca tgggcgatgc caagctggtc gacaccatga tcgtcgacgg cctgtgggac 3120
gtgtacaacc agtaccacat gggcatcacc gccgagaacg tggccaagga atacggcatc 3180
acacgcgagg cgcaggatga gttcgccgtc ggctcgcaga acaaggccga agccgcgcag 3240
aaggccggca agtttgacga agagatcgtc ccggtgctga tcccgcagcg caagggcgac 3300
ccggtggcct tcaagaccga cgagttcgtg cgccagggcg ccacgctgga cagcatgtcc 3360
ggcctcaagc ccgccttcga caaggccggc acggtgaccg cggccaacgc ctcgggcctg 3420
aacgacggcg ccgccgcggt ggtggtgatg tcggcggcca aggccaagga actgggcctg 3480
accccgctgg ccacgatcaa gagctatgcc aacgccggtg tcgatcccaa ggtgatgggc 3540
atgggcccgg tgccggcctc caagcgcgcc ctgtcgcgcg ccgagtggac cccgcaagac 3600
ctggacctga tggagatcaa cgaggccttt gccgcgcagg cgctggcggt gcaccagcag 3660
atgggctggg acacctccaa ggtcaatgtg aacggcggcg ccatcgccat cggccacccg 3720
atcggcgcgt cgggctgccg tatcctggtg acgctgctgc acgagatgaa gcgccgtgac 3780
gcgaagaagg gcctggcctc gctgtgcatc ggcggcggca tgggcgtggc gctggcagtc 3840
gagcgcaaat aacggcgacg ataacgaagc caatcaagga gtggacatga ctcagcgcat 3900
tgcgtatgtg accggcggca tgggtggtat cggaaccgcc atttgccagc ggctggccaa 3960
ggatggcttt cgtgtggtgg ccggttgcgg ccccaactcg ccgcgccgcg aaaagtggct 4020
ggagcagcag aaggccctgg gcttcgattt cattgcctcg gaaggcaatg tggctgactg 4080
ggactcgacc aagaccgcat tcgacaaggt caagtccgag gtcggcgagg ttgatgtgct 4140
gatcaacaac gccggtatca cccgcgacgt ggtgttccgc aagatgaccc gcgccgactg 4200
ggatgcggtg atcgacacca acctgacctc gctgttcaac gtcaccaagc aggtgatcga 4260
cggcatggcc gaccgtggct ggggccgcat cgtcaacatc tcgtcggtga acgggcagaa 4320
gggccagttc ggccagacca actactccac cgccaaggcc ggcctgcatg gcttcaccat 4380
ggcactggcg caggaagtgg cgaccaaggg cgtgaccgtc aacacggtct ctccgggcta 4440
tatcgccacc gacatggtca aggcgatccg ccaggacgtg ctcgacaaga tcgtcgcgac 4500
gatcccggtc aagcgcctgg gcctgccgga agagatcgcc tcgatctgcg cctggttgtc 4560
gtcggaggag tccggtttct cgaccggcgc cgacttctcg ctcaacggcg gcctgcatat 4620
gggctga 4627
<210> 10
<211> 4627
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
atgatcaagg acaccctggt ttcgattaca aaagacttaa agttgaaaac aaacgtagaa 60
aacgccaacc ttaaaaacta taaagatgac tcgtcttgct tcggggtgtt tgagaacgtg 120
gaaaatgcga tttcgaacgc agtccacgcc cagaagattt tatcgcttca ctacactaag 180
gagcagcgcg aaaagattat cacggagatt cgtaaagctg cacttgagaa caaagagatt 240
ttggcaacca tgatcttgga ggagactcat atggggcgtt atgaagataa aattttaaag 300
catgaacttg tcgctaaata cactccaggg actgaagact taacgaccac ggcatggagc 360
ggagataatg ggcttactgt tgttgaaatg agcccctacg gggtgattgg ggcaatcact 420
cccagcacaa atcctaccga gactgtgatt tgtaattcta ttggcatgat tgcggctggc 480
aatacggtgg tcttcaatgg acatccgggg gccaagaagt gtgttgcatt tgctgttgag 540
atgatcaaca aagccattat ttcatgtggt gggcccgaga atttggttac aaccattaag 600
aatccaacta tggactctct ggacgctatt attaaacacc cgtcgattaa acttttatgc 660
ggaaccggag gaccgggaat ggtgaaaacc cttctgaatt ccgggaaaaa ggcgatcggt 720
gcgggtgccg gcaacccacc tgtcattgtt gatgacacag cagacattga aaaggcgggc 780
aagagcatca tcgagggctg tagttttgat aacaacacgc cctgcattgc tgaaaaggaa 840
gttttcgtct tcgagaatgt tgcggacgac cttatttcca atatgcttaa gaacaacgcg 900
gtaatcatta acgaagacca agtatcaaag ttaatcgatt tggtcctgca gaagaacaat 960
gagactcaag agtatagcat taataaaaaa tgggttggga aggacgcaaa attgtttctt 1020
gatgagatcg atgtggaatc cccttcctct gtcaaatgca tcatttgcga agtatctgcg 1080
cgtcacccat tcgtcatgac agagctgatg atgcccattc tgcctattgt acgcgttaaa 1140
gatatcgacg aagctattga atatgcgaaa atcgccgagc agaaccgtaa gcactcagcg 1200
tatatttatt ccaaaaatat cgataactta aatcgttttg agcgcgaaat cgatacaact 1260
atcttcgtca agaacgccaa aagctttgct ggcgtgggct atgaagctga agggtttacc 1320
acgttcacca tcgcagggag caccggcgaa gggattacaa gtgcgcgcaa tttcactcgt 1380
caacgtcgct gcgtgttagc cggttaaaac tttaagaagg agatatacat gaacaacttt 1440
aatctgcaca ccccaacccg cattctgttt ggtaaaggcg caatcgctgg tttacgcgaa 1500
caaattcctc acgatgctcg cgtattgatt acctacggcg gcggcagcgt gaaaaaaacc 1560
ggcgttctcg atcaagttct ggatgccctg aaaggcatgg acgtgctgga atttggcggt 1620
attgagccaa acccggctta tgaaacgctg atgaacgccg tgaaactggt tcgcgaacag 1680
aaagtgactt tcctgctggc ggttggcggc ggttctgtac tggacggcac caaatttatc 1740
gccgcagcgg ctaactatcc ggaaaatatc gatccgtggc acattctgca aacgggcggt 1800
aaagagatta aaagcgccat cccgatgggc tgtgtgctga cgctgccagc aaccggttca 1860
gaatccaacg caggcgcggt gatctcccgt aaaaccacag gcgacaagca ggcgttccat 1920
tctgcccatg ttcagccggt atttgccgtg ctcgatccgg tttataccta caccctgccg 1980
ccgcgtcagg tggctaacgg cgtagtggac gcctttgtac acaccgtgga acagtatgtt 2040
accaaaccgg ttgatgccaa aattcaggac cgtttcgcag aaggcatttt gctgacgcta 2100
atcgaagatg gtccgaaagc cctgaaagag ccagaaaact acgatgtgcg cgccaacgtc 2160
atgtgggcgg cgactcaggc gctgaacggt ttgattggcg ctggcgtacc gcaggactgg 2220
gcaacgcata tgctgggcca cgaactgact gcgatgcacg gtctggatca cgcgcaaaca 2280
ctggctatcg tcctgcctgc actgtggaat gaaaaacgcg ataccaagcg cgctaagctg 2340
ctgcaatatg ctgaacgcgt ctggaacatc actgaaggtt ccgatgatga gcgtattgac 2400
gccgcgattg ccgcaacccg caatttcttt gagcaattag gcgtgccgac ccacctctcc 2460
gactacggtc tggacggcag ctccatcccg gctttgctga aaaaactgga agagcacggc 2520
atgacccaac tgggcgaaaa tcatgacatt acgttggatg tcagccgccg tatatacgaa 2580
gccgcccgct aattgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat 2640
aacaatttca cccattgaaa ggactacaca atgactgacg ttgtcatcgt atccgccgcc 2700
cgcaccgcgg tcggcaagtt tggcggctcg ctggccaaga tcccggcacc ggaactgggt 2760
gccgtggtca tcaaggccgc gctggagcgc gccggcgtca agccggagca ggtgagcgaa 2820
gtcatcatgg gccaggtgct gaccgccggt tcgggccaga accccgcacg ccaggccgcg 2880
atcaaggccg gcctgccggc gatggtgccg gccatgacca tcaacaaggt gtgcggctcg 2940
ggcctgaagg ccgtgatgct ggccgccaac gcgatcatgg cgggcgacgc cgagatcgtg 3000
gtggccggcg gccaggaaaa catgagcgcc gccccgcacg tgctgccggg ctcgcgcgat 3060
ggtttccgca tgggcgatgc caagctggtc gacaccatga tcgtcgacgg cctgtgggac 3120
gtgtacaacc agtaccacat gggcatcacc gccgagaacg tggccaagga atacggcatc 3180
acacgcgagg cgcaggatga gttcgccgtc ggctcgcaga acaaggccga agccgcgcag 3240
aaggccggca agtttgacga agagatcgtc ccggtgctga tcccgcagcg caagggcgac 3300
ccggtggcct tcaagaccga cgagttcgtg cgccagggcg ccacgctgga cagcatgtcc 3360
ggcctcaagc ccgccttcga caaggccggc acggtgaccg cggccaacgc ctcgggcctg 3420
aacgacggcg ccgccgcggt ggtggtgatg tcggcggcca aggccaagga actgggcctg 3480
accccgctgg ccacgatcaa gagctatgcc aacgccggtg tcgatcccaa ggtgatgggc 3540
atgggcccgg tgccggcctc caagcgcgcc ctgtcgcgcg ccgagtggac cccgcaagac 3600
ctggacctga tggagatcaa cgaggccttt gccgcgcagg cgctggcggt gcaccagcag 3660
atgggctggg acacctccaa ggtcaatgtg aacggcggcg ccatcgccat cggccacccg 3720
atcggcgcgt cgggctgccg tatcctggtg acgctgctgc acgagatgaa gcgccgtgac 3780
gcgaagaagg gcctggcctc gctgtgcatc ggcggcggca tgggcgtggc gctggcagtc 3840
gagcgcaaat aacggcgacg ataacgaagc caatcaagga gtggacatga ctcagcgcat 3900
tgcgtatgtg accggcggca tgggtggtat cggaaccgcc atttgccagc ggctggccaa 3960
ggatggcttt cgtgtggtgg ccggttgcgg ccccaactcg ccgcgccgcg aaaagtggct 4020
ggagcagcag aaggccctgg gcttcgattt cattgcctcg gaaggcaatg tggctgactg 4080
ggactcgacc aagaccgcat tcgacaaggt caagtccgag gtcggcgagg ttgatgtgct 4140
gatcaacaac gccggtatca cccgcgacgt ggtgttccgc aagatgaccc gcgccgactg 4200
ggatgcggtg atcgacacca acctgacctc gctgttcaac gtcaccaagc aggtgatcga 4260
cggcatggcc gaccgtggct ggggccgcat cgtcaacatc tcgtcggtga acgggcagaa 4320
gggccagttc ggccagacca actactccac cgccaaggcc ggcctgcatg gcttcaccat 4380
ggcactggcg caggaagtgg cgaccaaggg cgtgaccgtc aacacggtct ctccgggcta 4440
tatcgccacc gacatggtca aggcgatccg ccaggacgtg ctcgacaaga tcgtcgcgac 4500
gatcccggtc aagcgcctgg gcctgccgga agagatcgcc tcgatctgcg cctggttgtc 4560
gtcggaggag tccggtttct cgaccggcgc cgacttctcg ctcaacggcg gcctgcatat 4620
gggctga 4627
<210> 11
<211> 329
<212> PRT
<213> 克雷伯肺炎杆菌(Klebsiella pneumoniae)
<400> 11
Met Lys Ile Ala Val Tyr Ser Thr Lys Gln Tyr Asp Lys Lys Tyr Leu
1 5 10 15
Gln His Val Asn Asp Ala Tyr Gly Phe Glu Leu Glu Phe Phe Asp Phe
20 25 30
Leu Leu Thr Ala Lys Thr Ala Lys Thr Ala Asn Gly Cys Glu Ala Val
35 40 45
Cys Ile Phe Val Asn Asp Asp Gly Ser Arg Pro Val Leu Glu Glu Leu
50 55 60
Lys Ala His Gly Val Lys Tyr Ile Ala Leu Arg Cys Ala Gly Phe Asn
65 70 75 80
Asn Val Asp Leu Glu Ala Ala Lys Glu Leu Gly Leu Arg Val Val Arg
85 90 95
Val Pro Ala Tyr Ser Pro Glu Ala Val Ala Glu His Ala Ile Gly Met
100 105 110
Met Met Ser Leu Asn Arg Arg Ile His Arg Ala Tyr Gln Arg Thr Arg
115 120 125
Asp Ala Asn Phe Ser Leu Glu Gly Leu Thr Gly Phe Thr Met Tyr Gly
130 135 140
Lys Thr Ala Gly Val Ile Gly Thr Gly Lys Ile Gly Val Ala Met Leu
145 150 155 160
Arg Ile Leu Lys Gly Phe Gly Met Arg Leu Leu Ala Phe Asp Pro Tyr
165 170 175
Pro Ser Ala Ala Ala Leu Glu Leu Gly Val Glu Tyr Val Asp Leu Ala
180 185 190
Thr Leu Tyr Lys Glu Ser Asp Val Ile Ser Leu His Cys Pro Leu Thr
195 200 205
Asp Glu Asn Tyr His Leu Leu Asn Arg Glu Ala Phe Asp Gln Met Lys
210 215 220
Asp Gly Val Met Val Ile Asn Thr Ser Arg Gly Ala Leu Ile Asp Ser
225 230 235 240
Gln Ala Ala Ile Asp Ala Leu Lys His Gln Lys Ile Gly Ala Leu Gly
245 250 255
Leu Asp Val Tyr Glu Asn Glu Arg Asp Leu Phe Phe Glu Asp Lys Ser
260 265 270
Asn Asp Val Ile Gln Asp Asp Val Phe Arg Arg Leu Ser Ala Cys His
275 280 285
Asn Val Leu Phe Thr Gly His Gln Ala Phe Leu Thr Ala Glu Ala Leu
290 295 300
Ile Ser Ile Ser Glu Thr Thr Leu Gly Asn Leu Gln Gln Val Ala Asn
305 310 315 320
Gly Glu Thr Cys Pro Asn Ala Ile Val
325
<210> 12
<211> 891
<212> PRT
<213> 克雷伯肺炎杆菌(Klebsiella pneumoniae)
<400> 12
Met Ala Val Thr Asn Ile Ala Glu Leu Asn Ala Leu Val Glu Arg Val
1 5 10 15
Lys Lys Ala Gln Arg Glu Tyr Ala Ser Phe Thr Gln Glu Gln Val Asp
20 25 30
Lys Ile Phe Arg Ala Ala Ala Leu Ala Ala Ala Asp Ala Arg Ile Pro
35 40 45
Leu Ala Lys Met Ala Val Ala Glu Ser Gly Met Gly Ile Val Glu Asp
50 55 60
Lys Val Ile Lys Asn His Phe Ala Ser Glu Tyr Ile Tyr Asn Ala Tyr
65 70 75 80
Lys Asp Glu Lys Thr Cys Gly Val Leu Ser Glu Asp Asp Thr Phe Gly
85 90 95
Thr Ile Thr Ile Ala Glu Pro Ile Gly Ile Ile Cys Gly Ile Val Pro
100 105 110
Thr Thr Asn Pro Thr Ser Thr Ala Ile Phe Lys Ser Leu Ile Ser Leu
115 120 125
Lys Thr Arg Asn Ala Ile Ile Phe Ser Pro His Pro Arg Ala Lys Glu
130 135 140
Ala Thr Asn Lys Ala Ala Asp Ile Val Leu Gln Ala Ala Ile Ala Ala
145 150 155 160
Gly Ala Pro Lys Asp Leu Ile Gly Trp Ile Asp Gln Pro Ser Val Glu
165 170 175
Leu Ser Asn Ala Leu Met His His Pro Asp Ile Asn Leu Ile Leu Ala
180 185 190
Thr Gly Gly Pro Gly Met Val Lys Ala Ala Tyr Ser Ser Gly Lys Pro
195 200 205
Ala Ile Gly Val Gly Ala Gly Asn Thr Pro Val Val Ile Asp Glu Thr
210 215 220
Ala Asp Ile Lys Arg Ala Val Ala Ser Val Leu Met Ser Lys Thr Phe
225 230 235 240
Asp Asn Gly Val Ile Cys Ala Ser Glu Gln Ser Val Val Val Val Asp
245 250 255
Ser Val Tyr Asp Ala Val Arg Glu Arg Phe Ala Ser His Gly Gly Tyr
260 265 270
Leu Leu Gln Gly Lys Glu Leu Lys Ala Val Gln Asp Ile Ile Leu Lys
275 280 285
Asn Gly Ala Leu Asn Ala Ala Ile Val Gly Gln Pro Ala Ala Lys Ile
290 295 300
Ala Glu Leu Ala Gly Phe Thr Val Pro Ala Thr Thr Lys Ile Leu Ile
305 310 315 320
Gly Glu Val Thr Asn Val Asp Glu Ser Glu Pro Phe Ala His Glu Lys
325 330 335
Leu Ser Pro Thr Leu Ala Met Tyr Arg Ala Lys Asp Phe Glu Asp Ala
340 345 350
Val Ala Lys Ala Glu Lys Leu Val Ala Met Gly Gly Ile Gly His Thr
355 360 365
Ser Cys Leu Tyr Thr Asp Gln Asp Asn Gln Pro Ala Arg Val Ala Tyr
370 375 380
Phe Gly Gln Met Met Lys Thr Ala Arg Ile Leu Ile Asn Thr Pro Ala
385 390 395 400
Ser Gln Gly Gly Ile Gly Asp Leu Tyr Asn Phe Lys Leu Ala Pro Ser
405 410 415
Leu Thr Leu Gly Cys Gly Ser Trp Gly Gly Asn Ser Ile Ser Glu Asn
420 425 430
Val Gly Pro Lys His Leu Ile Asn Lys Lys Thr Val Ala Lys Arg Ala
435 440 445
Glu Asn Met Leu Trp His Lys Leu Pro Lys Ser Ile Tyr Phe Arg Arg
450 455 460
Gly Ser Leu Pro Ile Ala Leu Asp Glu Val Ile Thr Asp Gly His Lys
465 470 475 480
Arg Ala Leu Ile Val Thr Asp Arg Phe Leu Phe Asn Asn Gly Tyr Ala
485 490 495
Asp Gln Ile Thr Ser Val Leu Lys Ala Ala Gly Val Glu Thr Glu Val
500 505 510
Phe Phe Glu Val Glu Ala Asp Pro Thr Leu Thr Ile Val Arg Lys Gly
515 520 525
Ala Asp Leu Ala Asn Ser Phe Lys Pro Asp Val Ile Ile Ala Leu Gly
530 535 540
Gly Gly Ser Pro Met Asp Ala Ala Lys Ile Met Trp Val Met Tyr Glu
545 550 555 560
His Pro Glu Thr His Phe Glu Glu Leu Ala Leu Arg Phe Met Asp Ile
565 570 575
Arg Lys Arg Ile Tyr Lys Phe Pro Lys Met Gly Val Lys Ala Lys Met
580 585 590
Val Ala Ile Thr Thr Thr Ser Gly Thr Gly Ser Glu Val Thr Pro Phe
595 600 605
Ala Val Val Thr Asp Asp Ala Thr Gly Gln Lys Tyr Pro Leu Ala Asp
610 615 620
Tyr Ala Leu Thr Pro Asp Met Ala Ile Val Asp Ala Asn Leu Val Met
625 630 635 640
Asp Met Pro Lys Ser Leu Cys Ala Phe Gly Gly Leu Asp Ala Val Thr
645 650 655
His Ala Leu Glu Ala Tyr Val Ser Val Leu Ala Ser Glu Phe Ser Asp
660 665 670
Gly Gln Ala Leu Gln Ala Leu Lys Leu Leu Lys Glu Tyr Leu Pro Ala
675 680 685
Ser Tyr His Glu Gly Ser Lys Asn Pro Val Ala Arg Glu Arg Val His
690 695 700
Ser Ala Ala Thr Ile Ala Gly Ile Ala Phe Ala Asn Ala Phe Leu Gly
705 710 715 720
Val Cys His Ser Met Ala His Lys Leu Gly Ser Gln Phe His Ile Pro
725 730 735
His Gly Leu Ala Asn Ala Leu Leu Ile Cys Asn Val Ile Arg Tyr Asn
740 745 750
Ala Asn Asp Asn Pro Thr Lys Gln Thr Ala Phe Ser Gln Tyr Asp Arg
755 760 765
Pro Gln Ala Arg Arg Arg Tyr Ala Glu Ile Ala Asp His Leu Gly Leu
770 775 780
Ser Ala Pro Gly Asp Arg Thr Ala Ala Lys Ile Glu Lys Leu Leu Ala
785 790 795 800
Trp Leu Glu Ser Ile Lys Ala Glu Leu Gly Ile Pro Lys Ser Ile Arg
805 810 815
Glu Ala Gly Val Gln Glu Ala Asp Phe Leu Ala His Val Asp Lys Leu
820 825 830
Ser Glu Asp Ala Phe Asp Asp Gln Cys Thr Gly Ala Asn Pro Arg Tyr
835 840 845
Pro Leu Ile Ser Glu Leu Lys Gln Ile Leu Leu Asp Thr Tyr Tyr Gly
850 855 860
Arg Glu Phe Val Glu Gly Glu Ala Gly Ala Lys Ala Glu Val Ala Pro
865 870 875 880
Val Lys Ala Glu Lys Lys Ala Lys Lys Ser Ala
885 890
<210> 13
<211> 312
<212> PRT
<213> 克雷伯肺炎杆菌(Klebsiella pneumoniae)
<400> 13
Met Lys Val Ala Val Leu Gly Ala Ala Gly Gly Ile Gly Gln Ala Leu
1 5 10 15
Ala Leu Leu Leu Lys Thr Gln Leu Pro Ser Gly Ser Glu Leu Ser Leu
20 25 30
Tyr Asp Ile Ala Pro Val Thr Pro Gly Val Ala Val Asp Leu Ser His
35 40 45
Ile Pro Thr Asp Val Lys Ile Lys Gly Phe Ser Gly Glu Asp Ala Thr
50 55 60
Pro Ala Leu Glu Gly Ala Asp Val Val Leu Ile Ser Ala Gly Val Ala
65 70 75 80
Arg Lys Pro Gly Met Asp Arg Ser Asp Leu Phe Asn Val Asn Ala Gly
85 90 95
Ile Val Lys Asn Leu Val Gln Gln Ile Ala Lys Thr Cys Pro Gln Ala
100 105 110
Cys Ile Gly Ile Ile Thr Asn Pro Val Asn Thr Thr Val Ala Ile Ala
115 120 125
Ala Glu Val Leu Lys Lys Ala Gly Val Tyr Asp Lys Asn Lys Leu Phe
130 135 140
Gly Val Thr Thr Leu Asp Ile Ile Arg Ser Asn Thr Phe Val Ala Glu
145 150 155 160
Leu Lys Gly Lys Ser Ala Thr Glu Val Glu Val Pro Val Ile Gly Gly
165 170 175
His Ser Gly Val Thr Ile Leu Pro Leu Leu Ser Gln Ile Pro Gly Val
180 185 190
Ser Phe Ser Asp Gln Glu Ile Ala Asp Leu Thr Lys Arg Ile Gln Asn
195 200 205
Ala Gly Thr Glu Val Val Glu Ala Lys Ala Gly Gly Gly Ser Ala Thr
210 215 220
Leu Ser Met Gly Gln Ala Ala Ala Arg Phe Gly Leu Ser Leu Val Arg
225 230 235 240
Ala Met Gln Gly Glu Lys Gly Val Val Glu Cys Ala Tyr Val Glu Gly
245 250 255
Asp Gly His Tyr Ala Arg Phe Phe Ser Gln Pro Leu Leu Leu Gly Lys
260 265 270
Asn Gly Val Glu Glu Arg Gln Ser Ile Gly Lys Leu Ser Ala Phe Glu
275 280 285
Gln Gln Ala Leu Glu Gly Met Leu Asp Thr Leu Lys Lys Asp Ile Ala
290 295 300
Leu Gly Glu Asp Phe Val Asn Lys
305 310
Claims (10)
1.联产1,3-丙二醇和1,3-丁二醇的重组菌,其特征在于,所述重组菌是将phaA、phaB、bld和yqhD基因通过质粒导入克雷伯氏菌(Klebsiella)中或通过基因工程手段整合到克雷伯氏菌染色体上而成;
其中,所述phaA基因来源于Cupriavidus necator,其为编码如下蛋白质(a)或(b)的基因:
(a)由SEQ ID NO:3所示的氨基酸序列组成的蛋白质;
(b)SEQ ID NO:3所示序列经取代、缺失或添加一个或几个氨基酸且具有同等功能的由(a)衍生的蛋白质;
所述phaB基因来源于Cupriavidus necator,其为编码如下蛋白质(c)或(d)的基因:
(c)由SEQ ID NO:4所示的氨基酸序列组成的蛋白质;
(d)SEQ ID NO:4所示序列经取代、缺失或添加一个或几个氨基酸且具有同等功能的由(c)衍生的蛋白质;
所述bld基因来源于Clostridium saccharoperbutylacetonicum,其编码蛋白的氨基酸序列如SEQ ID NO:1或2所示;
所述yqhD基因来源于大肠杆菌(Escherichia coli),其为编码如下蛋白质(e)或(f)的基因:
(e)由SEQ ID NO:6所示的氨基酸序列组成的蛋白质;
(f)SEQ ID NO:6所示序列经取代、缺失或添加一个或几个氨基酸且具有同等功能的由(e)衍生的蛋白质;
优选地,所述克雷伯氏菌为克雷伯肺炎杆菌(Klebsiella pneumoniae),更优选保藏号为CGMCC NO.7824的克雷伯肺炎杆菌ACR30。
2.根据权利要求1所述的重组菌,其特征在于,所述重组菌的构建如下:phaA、phaB基因经密码子优化后,与bld、yqhD基因一起构建到表达载体上,用重组载体转化克雷伯氏菌,筛选阳性转化子;
优选地,密码子优化的phaA-phaB操纵子的序列如SEQ ID NO:5所示。
3.根据权利要求2所述的重组菌,其特征在于,所述重组菌的构建如下:将bld-yqhD-phaAB串联基因表达盒构建到ptrc99a质粒上,用重组质粒转化克雷伯氏菌,筛选阳性转化子;
其中,bld-yqhD-phaAB串联基因表达盒的序列如SEQ ID NO:9和10所示。
4.联产1,3-丙二醇和1,3-丁二醇的重组菌,其特征在于,所述联产1,3-丙二醇和1,3-丁二醇的重组菌是以权利要求2或3所述的重组菌作为出发菌株,利用基因工程手段对出发菌株进行改造,得到的细胞内NADPH供给增强的工程菌;
优选地,通过增强出发菌株中与NADPH生物合成途径相关的基因,来提高细胞内NADPH的供给;
更优选地,所述与NADPH生物合成途径相关的基因为pntAB基因,核苷酸序列如下:
i)SEQ ID NO:7所示的核苷酸序列;
ii)SEQ ID NO:7所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列;
iii)在严格条件下与SEQ ID NO:7所示序列杂交且表达相同功能蛋白质的核苷酸序列,所述严格条件为在含0.1%SDS的0.1×SSPE或含0.1%SDS的0.1×SSC溶液中,在65℃下杂交,并用该溶液洗膜;或
iv)与i)、ii)或iii)的核苷酸序列具有90%以上同源性且表达相同功能蛋白质的核苷酸序列。
5.联产1,3-丙二醇和1,3-丁二醇的重组克雷伯氏菌,其特征在于,所述重组克雷伯氏菌是以权利要求2-4任一项所述的重组菌作为出发菌株,通过弱化出发菌株中与2,3-丁二醇合成途径相关的基因,获得的基因弱化菌株;所述弱化包括敲除或降低基因的表达。
6.根据权利要求5所述的重组克雷伯氏菌,其特征在于,所述与2,3-丁二醇合成途径相关的基因为budRABC基因,核苷酸序列如下:
i)SEQ ID NO:8所示的核苷酸序列;
ii)SEQ ID NO:8所示的核苷酸序列经取代、缺失和/或增加一个或多个核苷酸且表达相同功能蛋白质的核苷酸序列;
iii)在严格条件下与SEQ ID NO:8所示序列杂交且表达相同功能蛋白质的核苷酸序列,所述严格条件为在含0.1%SDS的0.1×SSPE或含0.1%SDS的0.1×SSC溶液中,在65℃下杂交,并用该溶液洗膜;或
iv)与i)、ii)或iii)的核苷酸序列具有90%以上同源性且表达相同功能蛋白质的核苷酸序列。
7.高产1,3-丙二醇和1,3-丁二醇的工程菌,其特征在于,所述工程菌是以权利要求2-4任一项所述的重组菌,或者权利要求5或6所述的重组克雷伯氏菌作为原始菌株,通过弱化原始菌株中ldhA、adhE、mdh基因中的至少一种基因,获得的基因弱化菌株;所述弱化包括敲除或降低基因的表达;
ldhA、adhE、mdh基因编码蛋白的氨基酸序列分别如SEQ ID NO:11-13所示;
优选地,弱化原始菌株中ldhA、adhE和mdh基因。
8.根据权利要求5或6所述的重组克雷伯氏菌,或者权利要求7或8所述的工程菌,其特征在于,所述弱化是指利用同源重组的方法敲除相应基因。
9.权利要求1-4任一项所述的重组菌,或权利要求5或6所述的重组克雷伯氏菌,或权利要求7所述的工程菌,或者权利要求8所述的重组克雷伯氏菌或工程菌在发酵联产1,3-丙二醇和1,3-丁二醇中的应用。
10.联产1,3-丙二醇和1,3-丁二醇的方法,其特征在于,包括在发酵培养基中对权利要求1-4任一项所述的重组菌,或权利要求5或6所述的重组克雷伯氏菌,或权利要求7所述的工程菌,或者权利要求8所述的重组克雷伯氏菌或工程菌进行培养以生产1,3-丙二醇和1,3-丁二醇。
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