CN109929887A - 改进的发酵生产l-赖氨酸的方法 - Google Patents
改进的发酵生产l-赖氨酸的方法 Download PDFInfo
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- CN109929887A CN109929887A CN201711350942.2A CN201711350942A CN109929887A CN 109929887 A CN109929887 A CN 109929887A CN 201711350942 A CN201711350942 A CN 201711350942A CN 109929887 A CN109929887 A CN 109929887A
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- leu
- ala
- thr
- gly
- val
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Abstract
本发明提供了发酵生产L‑赖氨酸的方法,其包括改造棒杆菌属细菌染色体上编码NCBI参考序列NP_602070.1的基因;和,用改造而得到的细菌发酵生产l‑赖氨酸。另外,本发明还提供了由该方法衍生的方法和应用,以及可以用在这些方法和应用中的细菌等。
Description
技术领域
本发明属于氨基酸发酵领域,具体而言,本发明涉及发酵生产L-赖氨酸的方法和应用,和可以用在这些方法和应用中的细菌等。
背景技术
通过产L-赖氨酸的细菌(如,埃希氏菌属的大肠杆菌和棒杆菌属的杆状细菌)发酵来生产L-赖氨酸已经得到了产业化应用。这些细菌,可以是从自然界分离的细菌,也可以是通过诱变或基因工程改造获得的细菌,或者两者兼而有之。
产L-赖氨酸的细菌包括棒杆菌属的细菌。例如,中国专利CN1017906B公开了生产L-赖氨酸的方法,包括使用含合成二氢二吡啶羧酸合成酶和/或琥珀酰四氢吡啶羧酸合成酶的重组DNA的棒状杆菌属细菌来发酵的步骤。
中国专利申请CN1187539A公开了生产L-赖氨酸的方法,包括使用含编码天冬氨酸激酶和编码二氨基庚二酸脱羧酶的重组DNA的棒杆菌属细菌来发酵的步骤。
中国专利申请CN1310234A公开了生产L-赖氨酸的方法,包括使用含α-酮戊二酸脱氢酶的基因的棒杆菌属细菌来发酵的步骤。
中国专利申请CN1890372A公开了生产L-赖氨酸的方法,包括使用果糖-1,6-二磷酸酶活性增加谷氨酸棒杆菌来发酵的步骤。
中国专利申请CN101065484A公开了具有产生L-氨基酸的能力的棒菌属细菌,其被修饰使得乙酰辅酶A水解酶活性减少。
中国专利申请CN101855357A公开了生产L-赖氨酸的方法,包括使用在编码果糖-PTS酶的ptsF基因上具有突变的谷氨酸棒杆菌来发酵的步骤。
中国专利申请CN104245921A公开了生产L-赖氨酸的方法,包括使用含编码木糖异构酶和木酮糖激酶的基因的棒杆菌属细菌来发酵的步骤。
上述生产L-赖氨酸的文献都是基于已知生物学功能的酶或基因。
在已经测序完成的谷氨酸棒杆菌ATCC 13032的全基因组(可参见NCBI参考序列:NC_003450.3)中,有约3057个基因。然而,在这些基因中,除了生物学功能明确的基因外,有约1196个基因编码生物学功能不清楚的“假设的蛋白质”。在这些“假设的蛋白质”中,有约236个基因编码的蛋白质被通过生物信息学方法注释以表明它们与某些蛋白质或酶相似,或包含某些结构域。然而,仍旧有约960个基因编码的蛋白质的生物学功能没有被启示生物学功能,也不清楚它们在发酵生产L-赖氨酸中的效用。
本发明人的中国专利申请第201610800601.X号开创性地对两个未知功能的基因进行了研究,发现对它们进行敲除,可以提高L-赖氨酸的发酵生产量。然而,此后并没有新的未知功能的基因被研究。
本发明人经过长期研究和实践,经历了众多的失败,凭借了一些运气,偶然发现在棒杆菌的染色体中对一个编码“假设的蛋白质”的基因的非敲除式的突变能够有助于提高L-赖氨酸的产量。该方法与现有改造的大量高产L-赖氨酸的细菌的染色体改造位点没有冲突,可以叠加提高的效果,从而在实践上可用于多种细菌发酵生产L-赖氨酸。
发明内容
本发明要解决的技术问题在于提供新的发酵生产L-赖氨酸的方法及其相关的方法,包括相对于未改造细菌提高L-赖氨酸的发酵生产量的方法,改造的细菌在发酵生产L-赖氨酸中的应用,改造的细菌在相对于未改造细菌提高L-赖氨酸的发酵生产量的应用,和/或,改造细菌的方法等。
具体而言,在第一方面,本发明提供了发酵生产L-赖氨酸的方法,其包括:
(1)改造棒杆菌属细菌染色体上编码NCBI参考序列NP_602070.1的基因;和,
(2)用步骤(1)改造而得到的细菌发酵生产L-赖氨酸。
在本文中,术语“改造”指的是是相应被改造的对象发生变化,从而达到一定的效果。改造位于染色体上的基因的手段包括但是不限于,诱变、定点突变、和/或同源重组,优选是后两者。改造位于染色体上的基因使得该基因的核苷酸序列被添加、缺失或替换一个或多个核苷酸,优选替换一个或多个核苷酸,更优选替换一个核苷酸。在本发明的具体实施方式中,进行V351L突变。
这些技术手段广泛记载于分子生物学和微生物学文献中,有许多甚至已经商品化了。在本发明的具体实施方式中,根据同源重组的原理,可以采用Addgene公司商品化的pKOV质粒系统来进行改造,也可以采用pK18mobsacB质粒系统来进行改造。因此,在本文中,改造优选是通过同源重组进行的改造,更优选是通过同源重组进行的基因突变。
在本文中,NCBI参考序列NP_602070.1(简称为NP_602070.1)是一种“假设的蛋白质”NCgl2780,其氨基酸序列如SEQ ID NO:3所示(也可获取自网站http://www.ncbi.nlm.nih.gov,NP_602070.1)。编码NP_602070.1的基因的(互补)核苷酸序列如SEQ ID NO:1所示(也可获取自网站http://www.ncbi.nlm.nih.gov)。尽管NP_602070.1的具体活性还未知,但是在本发明的具体实施方式中,其基因被突变后,赖氨酸的产量提高了。
相应地,本发明还提供了其他的应用或方法。例如,在第二方面,本发明提供了提高L-赖氨酸的发酵量的方法,其包括:
(1)改造棒杆菌属细菌染色体上编码NCBI参考序列NP_602070.1的基因;和,
(2)用步骤(1)改造而得到的细菌发酵生产L-赖氨酸。
又如,在第三方面,本发明提供了改造获得的细菌在发酵生产L-赖氨酸中的应用,其中,所述改造获得是改造棒杆菌属细菌染色体上编码NCBI参考序列NP_602070.1的基因。
还如,在第四方面,本发明提供了改造获得的细菌在提高L-赖氨酸的发酵量中的应用,其中,所述改造获得是改造棒杆菌属细菌染色体上编码NCBI参考序列NP_602070.1的基因。
在本文中,如无特别限定(如未以“改造获得”来限定),术语“细菌”或“棒杆菌属细菌”是未改造或改造前的细菌或棒杆菌属细菌,其染色体具有野生型的编码NCBI参考序列NP_602070.1的基因。
L-赖氨酸作为细菌的重要代谢产物,大多数棒杆菌属细菌或多或少都能够发酵产生一定量的L-赖氨酸。现有技术没有在赖氨酸生产/发酵中关注过编码NCBI参考序列NP_602070.1的基因,因此现有技术中的产L-赖氨酸的棒杆菌属细菌通常都带有野生型的编码NCBI参考序列NP_602070.1的基因基本上都可以采用本发明的方法进行改造,提高L-赖氨酸的发酵量。在本文中,棒杆菌属细菌包括谷氨酸棒杆菌或北京棒杆菌,优选是谷氨酸棒杆菌。
更本质地,在第五方面,本发明提供了改造细菌的方法,其包括改造棒杆菌属细菌的方法,其包括改造棒杆菌属细菌染色体上编码NCBI参考序列NP_602070.1的基因。
本发明第五方面的方法改造而获得的细菌能够用于发酵生产或产生l-赖氨酸。因此,在第六方面,本发明提供了本发明第五方面的方法改造而获得的细菌。本发明第六方面的细菌是棒杆菌属细菌,其染色体上编码NCBI参考序列NP_602070.1的基因座位的核苷酸序列不同于编码NCBI参考序列NP_602070.1的基因的核苷酸序列,优选其染色体上编码NCBI参考序列NP_601029.1和/或NP_599350.1的基因的核苷酸序列进行添加、缺失或替换一个或多个核苷酸,例如替换一个或多个核苷酸,更优选替换一个核苷酸,最优选进行V351L突变。
在第六方面,本发明提供了突变的蛋白质,其包含突变V351L,即第351位氨基酸残基由V突变为L。该突变的蛋白质的氨基酸序列如SEQ ID No:4所示。
在第七方面,本发明提供了编码本发明第六方面所述的蛋白质的基因。优选该基因的核苷酸序列如SEQ ID No:2所示。
在第八方面,本发明提供了包含本发明第七方面所述的基因的载体。
在第九方面,本发明提供了细胞,其包含本发明第七方面所述的基因,或用本发明的第八方面所述的载体转化或者转染而得。优选细胞是菌种,优选为细菌,更有选为棒杆菌属细菌,如谷氨酸棒杆菌或北京棒杆菌。
在第十方面,本发明提供了本发明第六方面所述的蛋白质和/或本发明第七方面所述的基因在棒杆菌属细菌发酵生产L-赖氨酸中的应用。本发明第六方面所述的蛋白质替换了野生型的NP_602070.1,能提高棒杆菌属细菌发酵生产L-赖氨酸的产量。其中,NCBI参考序列NP_601029.1的氨基酸序列如SEQ ID NO:3,其编码基因的核苷酸序列如SEQ ID NO:1所示;本发明第六方面所述的蛋白质的氨基酸序列如SEQ ID NO:4所示,其优选的编码基因的核苷酸序列如SEQ ID NO:2所示。
本发明的有益效果在于,开辟并且实践证明了新的提高L-赖氨酸的发酵量的方式,而且与现有改造的大量高产L-赖氨酸的棒杆菌属细菌的染色体改造位点没有冲突,从而在实践上可用于进一步提高L-赖氨酸的产量。
为了便于理解,以下将通过具体的实施例对本发明进行详细地描述。需要特别指出的是,这些描述仅仅是示例性的描述,并不构成对本发明范围的限制。依据本说明书的论述,本发明的许多变化、改变对所属领域技术人员来说都是显而易见的。
另外,本发明引用了公开文献,这些文献是为了更清楚地描述本发明,它们的全文内容均纳入本文进行参考,就好像它们的全文已经在本文中重复叙述过一样。
具体实施方式
以下通过实施例进一步说明本发明的内容。如未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段和市售的常用仪器、试剂,可参见《分子克隆实验指南(第3版)》(科学出版社)、《微生物学实验(第4版)》(高等教育出版社)以及相应仪器和试剂的厂商说明书等参考。
实施例1 包含突变的NCgl2780基因的转化载体pK18-NCgl2780V351L的构建
根据NCBI公布的谷氨酸棒杆菌ATCC13032的基因组序列,合成两对扩增NCgl2780基因(其核苷酸序列如SEQ ID NO:1所示,编码的蛋白质的氨基酸序列如SEQ ID NO:3所示)编码区片段的引物,以通过等位基因置换在菌株YPL-4的背景中的NCgl2780基因中引入点突变,获得编码如SEQ ID NO:4所示的蛋白质的突变基因(其核苷酸序列如SEQ ID NO:2所示)。引物设计如下(上海英俊公司合成):
P1: 5' CAAGCTT CGAAGCCAACACATCTACTC 3' (HindⅢ)
P2: 5' CGATACCGAA GGGGCGGCTG GGGCTGCGCA CTCCGGGTAT GGC 3'
P3: 5' GCCATACCCG GAGTGCGCAG CCCCAGCCGC CCCTTCGGTA TCG 3'
P4: 5' GGAATTCTCTAACCCGTCCTGAATCTC 3' (EcoRⅠ)
以谷氨酸棒杆菌ATCC13032为模板,分别以引物P1/P2及P3/P4,进行PCR扩增,获得两条含有NCgl2780基因部分编码区(ORF)分离的DNA片段,大小分别为768bp及964bp,再用引物P1/P4进行OVER PCR得到整个等位替换的片段1689bp,,此片段包含部分NCgl2780基因1689bp的DNA片段,片段导致野生型NCgl2780基因的第1051位的核酸改变,最终导致编码的蛋白第351位由缬氨酸到亮氨酸的氨基酸取代,且片段两端分别含有HindⅢ和EcoRⅠ酶切位点。PCR反应结束后,对扩增的产物进行电泳回收,采用柱式DNA凝胶回收试剂盒进行回收所需要的1730bp的DNA片段,并通过酶切回收连接,与穿梭质粒pk18mobsacB质粒相连接,获得等位替换质粒pK18-NCgl2780V351L。该质粒上含有卡那霉素抗性标记。
实施例2包含突变的NCgl2780V351L的菌株的构建
将等位替换电转化入赖氨酸生产菌株专利菌株YP97136中(其构建方法可参见WO2014121669A1;经测序确认该菌株染色体上保留有野生型的NCgl2780基因),对培养产生的单菌落分别通过如下引物(上海英俊公司合成)进行PCR鉴定:
P5:5' GGGTTATTACGGGCTGATG 3'
P6: 5' AGAAACAGGCAGCAGAATG 3'
上述PCR扩增产物通过高温变性、冰浴后进行sscp电泳,同时以质粒扩增片段位阳性对照,野生型扩增片段为阴性对照,水作为空白对照,片段大小与阴性对照片段大小不一致且与阳性对照片段大小一致的菌株为等位替换成功的菌株。再次通过PCR扩增片段,并连接到PMD19-T载体,并测序,通过序列比对,碱基序列发生突变的序列验证菌株的等位替换成功,并被命名为YPL-4-001。
实施例3 赖氨酸发酵实验
将实施例2构建的菌株和原始菌株在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1所示的培养基和表2所示的过程进行发酵实验。每个菌株重复三次,结果如表3所示。
表1 发酵培养基配方
表2 发酵过程
表3 赖氨酸发酵实验结果
结果如表3所示,在棒杆菌中对NCgl2780进行点突变,有助于L-赖氨酸产量的提高。
序列表
<110> 宁夏伊品生物科技股份有限公司
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atgacgttta gcccccagcg tccggagttt gagaccggta agcagccaga tccagaaact 60
gaacacgcag gtgacttctt tgaagaaacc tcaagcagtg ctcctcgcgc cgcatctaac 120
ggttcttctg gtccgaacta caccctcatt acaacgtttc tagcggccct tactgctggc 180
atctttgctt tctgggcagg ctggacccgc aaatggatca gcgacgacgg actgatcgtc 240
ctacgcaccg tccgaaacct cctggctgga aacgggccag tattcaacgc tggcgaacgc 300
gtcgaagcca acacatctac tctgtggcaa tactgcatct atctggttgc cttagtaact 360
gactatcgcc tcgaagatat tgctctgtgg cttgcgctgc tgttcaccac cgcagcgtcc 420
atcatcggtg tcctgggtac cgcgcatctc caccgcaaac gcattgccgt attgcttcct 480
gcaggcgtga tcggctactt cagcctttcc ccggcgcgag actttgccac ttccggattg 540
gagtggggcc tatctttgat gtggatttcc atccaatggc tgctgctggt gttgtgggcg 600
acttcgggca agacctcggg caagaaggct tcgggcgcaa aaacttcaaa tcctatcgtt 660
aatgccggtg caataaccta tgctttggcc ttttggtcag gcttgagctg gctggttcgc 720
ccagaactgg cgatgtatgg cggtttgact ggagtgttgc tgctgcttac tgcgccacga 780
tggcgggtag ttttggggat cctggtggcg gctttgcctc ttccagctgc gtaccaaatc 840
ttccgcatgg gttattacgg gctgatggtg ccgcacacgg ctgtagcgaa atcagcctca 900
gatgcggtgt gggggactgg ttgggagtat gttgaggatt tcacggggcc ttacaacctg 960
tggctcggtt tggccttgct gttggccgca ggcgcgttga cagtgtggaa aactgacaag 1020
cacttagcga taccgaaggg gcggctgggg gtgcgcactc cgggtatggc tatagcgttg 1080
ctggttatct gtgcgctcgt ccacttcctt tacgttatcc gtgttggtgg cgacttcatg 1140
catggacgca tgctgctcct tccacttttt gccattctgc tgcctgtttc tgtcattccg 1200
gtcaatgttg ttgatcgagg ttggcaggat ttggttgcgc tggttctcgt tttctctacg 1260
tgggtgtggt ccactgtggt ttttgtgcag gggcaccaat gggaaaatac cggccagcat 1320
gtggttgatg agcgtgattt ttggattgat ttcaccaacc gagatgaaga tcatcctccg 1380
ctttatgcag aggatttcct cactgttgat tccatgaatg attacgcaga ggttatgcgc 1440
gatcagacgt tggttaatcc aacaggccag caactcaata ttctggccag cagtgacccg 1500
accacttatt cgtggatcac cacacctcgc gtggaggggg ttgaagccgg tgatttggct 1560
aacctctcgc caactgtttt ccatgtgaac ctcggcatga cctccatgaa cgcaccgctc 1620
aacgtgcgtg tgacagacct gattggtttg gcaacgccac tggcagcccg ccagccacgc 1680
attgaaggtg gtcgaatcgg ccacgataaa ttgatggact tggaatggca ggtcgcggaa 1740
tccgccactc cgctggcgta cacaccgggt tggttggata ctcaaaagac ttatgaggcc 1800
cgccaggcgc tacgccaccc agaattggtt caccttttcc agacttaccg tgagccaatg 1860
tcctaccacc ggtttgtgga caatattaaa tacgcactca ctaccggaag aacactggaa 1920
atttcagata atcctgaaga tcttttgaaa gaatttaacc cgacccctgc agagattcag 1980
gacgggttag aaactattgc ttggcctggg gaaattaaac ttgatgaacc tcgcggagaa 2040
cctttatata gctctcagta a 2061
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atgacgttta gcccccagcg tccggagttt gagaccggta agcagccaga tccagaaact 60
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ggttcttctg gtccgaacta caccctcatt acaacgtttc tagcggccct tactgctggc 180
atctttgctt tctgggcagg ctggacccgc aaatggatca gcgacgacgg actgatcgtc 240
ctacgcaccg tccgaaacct cctggctgga aacgggccag tattcaacgc tggcgaacgc 300
gtcgaagcca acacatctac tctgtggcaa tactgcatct atctggttgc cttagtaact 360
gactatcgcc tcgaagatat tgctctgtgg cttgcgctgc tgttcaccac cgcagcgtcc 420
atcatcggtg tcctgggtac cgcgcatctc caccgcaaac gcattgccgt attgcttcct 480
gcaggcgtga tcggctactt cagcctttcc ccggcgcgag actttgccac ttccggattg 540
gagtggggcc tatctttgat gtggatttcc atccaatggc tgctgctggt gttgtgggcg 600
acttcgggca agacctcggg caagaaggct tcgggcgcaa aaacttcaaa tcctatcgtt 660
aatgccggtg caataaccta tgctttggcc ttttggtcag gcttgagctg gctggttcgc 720
ccagaactgg cgatgtatgg cggtttgact ggagtgttgc tgctgcttac tgcgccacga 780
tggcgggtag ttttggggat cctggtggcg gctttgcctc ttccagctgc gtaccaaatc 840
ttccgcatgg gttattacgg gctgatggtg ccgcacacgg ctgtagcgaa atcagcctca 900
gatgcggtgt gggggactgg ttgggagtat gttgaggatt tcacggggcc ttacaacctg 960
tggctcggtt tggccttgct gttggccgca ggcgcgttga cagtgtggaa aactgacaag 1020
cacttagcga taccgaaggg gcggctgggg ctgcgcactc cgggtatggc tatagcgttg 1080
ctggttatct gtgcgctcgt ccacttcctt tacgttatcc gtgttggtgg cgacttcatg 1140
catggacgca tgctgctcct tccacttttt gccattctgc tgcctgtttc tgtcattccg 1200
gtcaatgttg ttgatcgagg ttggcaggat ttggttgcgc tggttctcgt tttctctacg 1260
tgggtgtggt ccactgtggt ttttgtgcag gggcaccaat gggaaaatac cggccagcat 1320
gtggttgatg agcgtgattt ttggattgat ttcaccaacc gagatgaaga tcatcctccg 1380
ctttatgcag aggatttcct cactgttgat tccatgaatg attacgcaga ggttatgcgc 1440
gatcagacgt tggttaatcc aacaggccag caactcaata ttctggccag cagtgacccg 1500
accacttatt cgtggatcac cacacctcgc gtggaggggg ttgaagccgg tgatttggct 1560
aacctctcgc caactgtttt ccatgtgaac ctcggcatga cctccatgaa cgcaccgctc 1620
aacgtgcgtg tgacagacct gattggtttg gcaacgccac tggcagcccg ccagccacgc 1680
attgaaggtg gtcgaatcgg ccacgataaa ttgatggact tggaatggca ggtcgcggaa 1740
tccgccactc cgctggcgta cacaccgggt tggttggata ctcaaaagac ttatgaggcc 1800
cgccaggcgc tacgccaccc agaattggtt caccttttcc agacttaccg tgagccaatg 1860
tcctaccacc ggtttgtgga caatattaaa tacgcactca ctaccggaag aacactggaa 1920
atttcagata atcctgaaga tcttttgaaa gaatttaacc cgacccctgc agagattcag 1980
gacgggttag aaactattgc ttggcctggg gaaattaaac ttgatgaacc tcgcggagaa 2040
cctttatata gctctcagta a 2061
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Met Thr Phe Ser Pro Gln Arg Pro Glu Phe Glu Thr Gly Lys Gln Pro
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Ser Ala Pro Arg Ala Ala Ser Asn Gly Ser Ser Gly Pro Asn Tyr Thr
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Leu Ile Thr Thr Phe Leu Ala Ala Leu Thr Ala Gly Ile Phe Ala Phe
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Trp Ala Gly Trp Thr Arg Lys Trp Ile Ser Asp Asp Gly Leu Ile Val
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Leu Arg Thr Val Arg Asn Leu Leu Ala Gly Asn Gly Pro Val Phe Asn
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Ala Gly Glu Arg Val Glu Ala Asn Thr Ser Thr Leu Trp Gln Tyr Cys
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Leu Gly Thr Ala His Leu His Arg Lys Arg Ile Ala Val Leu Leu Pro
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Ala Gly Val Ile Gly Tyr Phe Ser Leu Ser Pro Ala Arg Asp Phe Ala
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Thr Ser Gly Leu Glu Trp Gly Leu Ser Leu Met Trp Ile Ser Ile Gln
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Pro Glu Leu Ala Met Tyr Gly Gly Leu Thr Gly Val Leu Leu Leu Leu
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Pro Leu Pro Ala Ala Tyr Gln Ile Phe Arg Met Gly Tyr Tyr Gly Leu
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Met Val Pro His Thr Ala Val Ala Lys Ser Ala Ser Asp Ala Val Trp
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Gly Thr Gly Trp Glu Tyr Val Glu Asp Phe Thr Gly Pro Tyr Asn Leu
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Trp Leu Gly Leu Ala Leu Leu Leu Ala Ala Gly Ala Leu Thr Val Trp
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Thr Pro Gly Met Ala Ile Ala Leu Leu Val Ile Cys Ala Leu Val His
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Phe Leu Tyr Val Ile Arg Val Gly Gly Asp Phe Met His Gly Arg Met
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Leu Leu Leu Pro Leu Phe Ala Ile Leu Leu Pro Val Ser Val Ile Pro
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Val Asn Val Val Asp Arg Gly Trp Gln Asp Leu Val Ala Leu Val Leu
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Val Phe Ser Thr Trp Val Trp Ser Thr Val Val Phe Val Gln Gly His
420 425 430
Gln Trp Glu Asn Thr Gly Gln His Val Val Asp Glu Arg Asp Phe Trp
435 440 445
Ile Asp Phe Thr Asn Arg Asp Glu Asp His Pro Pro Leu Tyr Ala Glu
450 455 460
Asp Phe Leu Thr Val Asp Ser Met Asn Asp Tyr Ala Glu Val Met Arg
465 470 475 480
Asp Gln Thr Leu Val Asn Pro Thr Gly Gln Gln Leu Asn Ile Leu Ala
485 490 495
Ser Ser Asp Pro Thr Thr Tyr Ser Trp Ile Thr Thr Pro Arg Val Glu
500 505 510
Gly Val Glu Ala Gly Asp Leu Ala Asn Leu Ser Pro Thr Val Phe His
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Val Asn Leu Gly Met Thr Ser Met Asn Ala Pro Leu Asn Val Arg Val
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Thr Asp Leu Ile Gly Leu Ala Thr Pro Leu Ala Ala Arg Gln Pro Arg
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Ile Glu Gly Gly Arg Ile Gly His Asp Lys Leu Met Asp Leu Glu Trp
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Gln Val Ala Glu Ser Ala Thr Pro Leu Ala Tyr Thr Pro Gly Trp Leu
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Asp Thr Gln Lys Thr Tyr Glu Ala Arg Gln Ala Leu Arg His Pro Glu
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Leu Val His Leu Phe Gln Thr Tyr Arg Glu Pro Met Ser Tyr His Arg
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Phe Val Asp Asn Ile Lys Tyr Ala Leu Thr Thr Gly Arg Thr Leu Glu
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Ile Ser Asp Asn Pro Glu Asp Leu Leu Lys Glu Phe Asn Pro Thr Pro
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Ala Glu Ile Gln Asp Gly Leu Glu Thr Ile Ala Trp Pro Gly Glu Ile
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<210> 4
<211> 686
<212> PRT
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Met Thr Phe Ser Pro Gln Arg Pro Glu Phe Glu Thr Gly Lys Gln Pro
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Asp Pro Glu Thr Glu His Ala Gly Asp Phe Phe Glu Glu Thr Ser Ser
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Ser Ala Pro Arg Ala Ala Ser Asn Gly Ser Ser Gly Pro Asn Tyr Thr
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Leu Ile Thr Thr Phe Leu Ala Ala Leu Thr Ala Gly Ile Phe Ala Phe
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Trp Ala Gly Trp Thr Arg Lys Trp Ile Ser Asp Asp Gly Leu Ile Val
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Leu Arg Thr Val Arg Asn Leu Leu Ala Gly Asn Gly Pro Val Phe Asn
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Ala Gly Glu Arg Val Glu Ala Asn Thr Ser Thr Leu Trp Gln Tyr Cys
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Ile Tyr Leu Val Ala Leu Val Thr Asp Tyr Arg Leu Glu Asp Ile Ala
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Leu Trp Leu Ala Leu Leu Phe Thr Thr Ala Ala Ser Ile Ile Gly Val
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Leu Gly Thr Ala His Leu His Arg Lys Arg Ile Ala Val Leu Leu Pro
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Ala Gly Val Ile Gly Tyr Phe Ser Leu Ser Pro Ala Arg Asp Phe Ala
165 170 175
Thr Ser Gly Leu Glu Trp Gly Leu Ser Leu Met Trp Ile Ser Ile Gln
180 185 190
Trp Leu Leu Leu Val Leu Trp Ala Thr Ser Gly Lys Thr Ser Gly Lys
195 200 205
Lys Ala Ser Gly Ala Lys Thr Ser Asn Pro Ile Val Asn Ala Gly Ala
210 215 220
Ile Thr Tyr Ala Leu Ala Phe Trp Ser Gly Leu Ser Trp Leu Val Arg
225 230 235 240
Pro Glu Leu Ala Met Tyr Gly Gly Leu Thr Gly Val Leu Leu Leu Leu
245 250 255
Thr Ala Pro Arg Trp Arg Val Val Leu Gly Ile Leu Val Ala Ala Leu
260 265 270
Pro Leu Pro Ala Ala Tyr Gln Ile Phe Arg Met Gly Tyr Tyr Gly Leu
275 280 285
Met Val Pro His Thr Ala Val Ala Lys Ser Ala Ser Asp Ala Val Trp
290 295 300
Gly Thr Gly Trp Glu Tyr Val Glu Asp Phe Thr Gly Pro Tyr Asn Leu
305 310 315 320
Trp Leu Gly Leu Ala Leu Leu Leu Ala Ala Gly Ala Leu Thr Val Trp
325 330 335
Lys Thr Asp Lys His Leu Ala Ile Pro Lys Gly Arg Leu Gly Leu Arg
340 345 350
Thr Pro Gly Met Ala Ile Ala Leu Leu Val Ile Cys Ala Leu Val His
355 360 365
Phe Leu Tyr Val Ile Arg Val Gly Gly Asp Phe Met His Gly Arg Met
370 375 380
Leu Leu Leu Pro Leu Phe Ala Ile Leu Leu Pro Val Ser Val Ile Pro
385 390 395 400
Val Asn Val Val Asp Arg Gly Trp Gln Asp Leu Val Ala Leu Val Leu
405 410 415
Val Phe Ser Thr Trp Val Trp Ser Thr Val Val Phe Val Gln Gly His
420 425 430
Gln Trp Glu Asn Thr Gly Gln His Val Val Asp Glu Arg Asp Phe Trp
435 440 445
Ile Asp Phe Thr Asn Arg Asp Glu Asp His Pro Pro Leu Tyr Ala Glu
450 455 460
Asp Phe Leu Thr Val Asp Ser Met Asn Asp Tyr Ala Glu Val Met Arg
465 470 475 480
Asp Gln Thr Leu Val Asn Pro Thr Gly Gln Gln Leu Asn Ile Leu Ala
485 490 495
Ser Ser Asp Pro Thr Thr Tyr Ser Trp Ile Thr Thr Pro Arg Val Glu
500 505 510
Gly Val Glu Ala Gly Asp Leu Ala Asn Leu Ser Pro Thr Val Phe His
515 520 525
Val Asn Leu Gly Met Thr Ser Met Asn Ala Pro Leu Asn Val Arg Val
530 535 540
Thr Asp Leu Ile Gly Leu Ala Thr Pro Leu Ala Ala Arg Gln Pro Arg
545 550 555 560
Ile Glu Gly Gly Arg Ile Gly His Asp Lys Leu Met Asp Leu Glu Trp
565 570 575
Gln Val Ala Glu Ser Ala Thr Pro Leu Ala Tyr Thr Pro Gly Trp Leu
580 585 590
Asp Thr Gln Lys Thr Tyr Glu Ala Arg Gln Ala Leu Arg His Pro Glu
595 600 605
Leu Val His Leu Phe Gln Thr Tyr Arg Glu Pro Met Ser Tyr His Arg
610 615 620
Phe Val Asp Asn Ile Lys Tyr Ala Leu Thr Thr Gly Arg Thr Leu Glu
625 630 635 640
Ile Ser Asp Asn Pro Glu Asp Leu Leu Lys Glu Phe Asn Pro Thr Pro
645 650 655
Ala Glu Ile Gln Asp Gly Leu Glu Thr Ile Ala Trp Pro Gly Glu Ile
660 665 670
Lys Leu Asp Glu Pro Arg Gly Glu Pro Leu Tyr Ser Ser Gln
675 680 685
Claims (10)
1.发酵生产L-赖氨酸的方法或者提高L-赖氨酸的发酵量的方法,其包括:
(1)改造棒杆菌属细菌染色体上编码NCBI参考序列NP_602070.1的基因;和,
(2)用步骤(1)改造而得到的细菌发酵生产L-赖氨酸。
2.改造获得的细菌在发酵生产L-赖氨酸或者提高L-赖氨酸的发酵量中的应用,其中,所述改造获得是改造棒杆菌属细菌染色体上编码NCBI参考序列NP_602070.1的基因。
3.改造棒杆菌属细菌的方法,其包括改造棒杆菌属细菌染色体上编码NCBI参考序列NP_602070.1的基因。
4.权利要求1-3之任一所述的方法或应用,其中,改造棒杆菌属细菌染色体上编码NCBI参考序列NP_602070.1的基因是对所述基因的核苷酸序列进行添加、缺失或替换一个或多个核苷酸,优选替换一个或多个核苷酸,更优选替换一个核苷酸,最优选进行V351L突变。
5.权利要求1-4之任一所述的方法或应用,其中,所述棒杆菌属细菌是谷氨酸棒杆菌。
6.权利要求3-5之任一所述的方法改造而得到的细菌。
7.蛋白质,其氨基酸序列如SEQ ID NO:4所示。
8.编码权利要求7所述的蛋白质的基因,优选其核苷酸序列如SEQ ID NO:2所示。
9.包含权利要求8所述的基因的载体或细胞,优选是棒杆菌属细菌细胞。
10.权利要求7所述的蛋白质和/或权利要求8所述的基因在棒杆菌属细菌发酵生产L-赖氨酸中的应用。
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US20020197605A1 (en) * | 1999-12-16 | 2002-12-26 | Satoshi Nakagawa | Novel Polynucleotides |
CN1582300A (zh) * | 2001-11-05 | 2005-02-16 | 巴斯福股份公司 | 编码新蛋白的基因 |
CN106367432A (zh) * | 2016-09-01 | 2017-02-01 | 宁夏伊品生物科技股份有限公司 | 发酵生产l‑赖氨酸的方法及改造的棒杆菌 |
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US20020197605A1 (en) * | 1999-12-16 | 2002-12-26 | Satoshi Nakagawa | Novel Polynucleotides |
CN1582300A (zh) * | 2001-11-05 | 2005-02-16 | 巴斯福股份公司 | 编码新蛋白的基因 |
CN106367432A (zh) * | 2016-09-01 | 2017-02-01 | 宁夏伊品生物科技股份有限公司 | 发酵生产l‑赖氨酸的方法及改造的棒杆菌 |
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