CN113249286B - 一种构建l-肌氨酸生产菌的方法 - Google Patents
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- CN113249286B CN113249286B CN202110571087.8A CN202110571087A CN113249286B CN 113249286 B CN113249286 B CN 113249286B CN 202110571087 A CN202110571087 A CN 202110571087A CN 113249286 B CN113249286 B CN 113249286B
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0044—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12N9/1025—Acyltransferases (2.3)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
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Abstract
本发明公开了一种构建L‑肌氨酸生产菌的方法,该基因工程菌能够以葡萄糖和甲胺为主要原料,通过发酵直接产生L‑肌氨酸,5L发酵罐发酵72小时,可产L‑肌氨酸18.3g/L,具有工业化应用前景。
Description
技术领域
本发明属于代谢工程和基因工程领域,具体地说,涉及一种构建L-肌氨酸生产菌的方法、构建得到的基因工程菌及其应用。
背景技术
L-肌氨酸是一种有机物,别名N-甲基甘氨酸,化学式为C3H7NO2,白色结晶,微有甜味,具潮解性,能溶于水,微溶于醇,不溶于醚,天然存在于海星和海胆中。主要应用于生化试剂、染料稳定剂、日用化学品、氨基酸型表面活性剂、保健药品疲劳恢复剂,是胆碱和肌酸降解的中间产物。肌氨酸作为导入神经胶质细胞的抑制剂,显示出作为抗精神病药物的潜力;肌氨酸酰化产生离子表面活性剂,用作洗发水、剃须泡沫和清洁剂的添加剂,广泛用日化行业。
L-肌氨酸主要通过化学法和发酵法制备:目前化学法主要有氯乙酸法和羟基乙腈法,但生成成本高、回收困难且污染严重。发酵法制备L-肌氨酸产物浓度仍较低,最高仅达到10g/L,难以满足工业化生成需求。2014年,Zahoor et al.(Journal of Biotechnology,V192,2014,P366-375.)报道了利用谷氨酸棒杆菌通过敲除内源苹果酸合成酶aceB、弱化异柠檬酸脱氢酶icd和过表达大肠杆菌来源的乙醛酸还原酶ycdW,在基本盐培养基中,可产5.3g/L乙醇酸。
来自假单胞菌的N-烷基氨基酸脱氢酶(DpkA)被证明为提供微生物发酵法生产N-烷基化氨基酸的新途径,利用乙醛酸经此途径制备肌氨酸成为了可能(BiotechnologyJournal,2020,15(7).)。2019年美国伯克利大学Mindt et al.(Bioresource Technology,2019,281.)报道了在谷氨酸棒杆菌敲除aceB、弱化icd、过表达Pseudomonas putidaKT2440 dpkA基因,在以木糖和乙酸钾为碳源的发酵培养中,肌氨酸产量达到8.7g/L左右;同年Mindt et al.(Frontiers in engineering and biotechnolgy,2019,26.)利用氨基酰基残基在底物结合位点突变的DpkAF117L,使得乙醛酸还原烷基胺化的比活性提高20%左右,并以单乙胺为基质,首次报道了N-乙基甘氨酸的发酵生产。
发明内容
为了探索发酵法生产L-肌氨酸的可行性,本发明以谷氨酸棒杆菌ATCC13032为出发菌株,通过过表达大肠杆菌来源的pntAB和恶臭假单胞菌KT2440(Pseudomonas putidaKT2440)来源的DpkAF117L,敲除glcB(NCgl2247)和alaT(NCgl2747),将异柠檬酸脱氢酶(icd,NCgl0634)基因起始密码子由ATG变为GTG,构建的基因工程菌株能够以葡萄糖和甲胺为原料,通过发酵产生L-肌氨酸。具体而言,本发明包括如下技术方案。
一种构建L-肌氨酸生产菌的方法,其包括以下步骤:
A.以谷氨酸棒杆菌ATCC13032为出发菌株,在基因组中整合NADPH-NAD+转氢酶(嘧啶核苷酸转氢酶A亚基(PNTA)和B亚基(PNTB))的编码基因pntAB(即pntA和pntB),得到过表达pntAB的菌株A;
B.在菌株A的基因组中整合恶臭假单胞菌KT2440(Pseudomonas putida KT2440)来源的亚胺还原酶突变体DpkAF117L编码基因,得到过表达DpkAF117L的菌株B;
C.敲除菌株B基因组中的基因glcB(NCgl2247)和alaT(NCgl2747),得到glcB(NCgl2247)和alaT(NCgl2747)缺失的菌株C;
D.将菌株C基因组中的异柠檬酸脱氢酶(icd,NCgl0634)基因起始密码子由ATG变为GTG,筛选阳性克隆,得到L-肌氨酸生产菌。
优选地,上述基因pntAB是大肠杆菌来源的pntAB。
上述DpkAF117L编码基因是GenBank:AAN69191.1。
在一种实施方式中,步骤A中基因pntAB的整合和步骤C中基因glcB(NCgl2247)的敲除可以包括下述步骤:将核苷酸序列为SEQ ID NO:1的质粒pK18mobsacB-glc-pntAB转入宿主细胞中;进行SacB蔗糖反筛。
上述步骤B中基因DpkAF117L的整合和步骤C中基因alaT(NCgl2747)的敲除可以包括下述步骤:将核苷酸序列为SEQ ID NO:3的质粒pK18mobsacB-alaT-dpkA转入宿主细胞中;进行SacB蔗糖反筛。
上述步骤D中异柠檬酸脱氢酶(icd,NCgl0634)基因起始密码子由ATG变为GTG的可以包括下述步骤:将核苷酸序列为SEQ ID NO:4的质粒pK18mobsacB-icd转入宿主细胞中;进行SacB蔗糖反筛。
上述步骤中的质粒转入可以是氯化钙转化法或电转化,优选电转化。
本发明的第二个目的在于提供一种L-肌氨酸生产菌,其通过上述的方法构建和筛选得到。
优选地,上述L-肌氨酸生产菌可以是谷氨酸棒杆菌(Corynebacteriumglutamicum),保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCCNo.22265。
本发明的第三个目的在于提供上述L-肌氨酸生产菌比如CGMCC No.22265用于生产L-肌氨酸的用途。
例如,可以通过上述L-肌氨酸生产菌比如CGMCC No.22265的发酵来生产L-肌氨酸。
在发酵时,需要将葡萄糖和甲胺作为主要原料。
发酵所用的培养基可以是任何适用于谷氨酸棒杆菌生长发酵的培养基。例如,发酵培养基组成如下:葡萄糖100g/L,硫酸镁100mg/L,酵母粉35g/L,硫酸铵20g/L,FeSO4·7H2O 11mg/L,柠檬酸钠10mg/L,H3PO4(85%)250μl/L,消泡剂204 500mg/L,泛酸钙60mg/L,维生素PP 18mg/L,thiamin HCl 15mg/L,生物素15mg/L,pH 7.0;发酵过程中流加甲胺。
发酵温度优选是30℃。
本发明构建的基因工程菌能够通过发酵直接产生L-肌氨酸,具有工业化开发利用价值。
本发明构建的L-肌氨酸高产基因工程菌的拉丁学名是Corynebacteriumglutamicum,中文名称是谷氨酸棒杆菌或谷氨酸棒状杆菌,已保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏日期是2021年5月6日,保藏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,保藏编号为CGMCC No.22265。
附图说明
图1为本发明构建的生物合成L-肌氨酸的代谢路线图。
图2是本发明构建的质粒pK18mobsacB-glc-pntAB的结构示意图。其核苷酸序列为SEQ ID NO:1。
图3是本发明构建的质粒pTrc99a-dpkAF117L的结构示意图。其核苷酸序列为SEQID NO:2。
图4是本发明构建的质粒pK18mobsacB-alaT-dpkA的结构示意图。其核苷酸序列为SEQ ID NO:3。
图5是本发明构建的质粒pK18mobsacB-icd的结构示意图。其核苷酸序列为SEQ IDNO:4。
具体实施方式
本发明对谷氨酸棒杆菌内在的代谢途径进行了调控,引入亚胺还原酶比如DpkAF117L,如图1所示,可以从葡萄糖出发并将甲胺作为原料,通过微生物发酵实现L-肌氨酸的生物合成。
这些过表达的基因大肠杆菌来源的pntAB和恶臭假单胞菌KT2440来源的DpkAF117L可以分别单独地克隆在一个质粒上,然后分别地、或者同时地转入同一个谷氨酸棒杆菌感受态细胞中;也可以将两个基因克隆在一个质粒上,然后分别地、或者同时地转入同一个谷氨酸棒杆菌感受态细胞中。
其中,可用的质粒载体包括pK18mobsacB等,但不限于此。
应理解,在构建本发明的基因工程菌的具体操作中,步骤A、步骤B、步骤C和步骤D的排序并非完全根据英文字母顺序由前到后地固定不变,它们可以交叉、颠倒地操作,只要每个步骤能实现各自的功能、完成宿主细胞基因型的定向改变即可。
在本文中,为了描述简便,有时会将某种酶比如异柠檬酸脱氢酶icd与其编码基因(DNA)名称混用,本领域技术人员应能理解它们在不同描述场合表示不同的物质。本领域技术人员根据语境和上下文容易理解它们的含义。例如,对于icd,用于描述异柠檬酸脱氢酶的功能或类别时,指的是蛋白质;在作为一种基因描述时,指的是编码该酶的基因。
以下结合具体实施例对本发明做进一步详细说明。应理解,以下实施例仅用于说明本发明而非用于限定本发明的范围。
本文中涉及到多种物质的添加量、含量及浓度,其中所述的百分含量,除特别说明外,皆指质量百分含量。
实施例
材料和方法
实施例中的全基因合成、引物合成及测序皆由苏州金唯智生物科技有限公司完成。
实施例中的分子生物学实验包括质粒构建、酶切、连接、感受态细胞制备、转化、培养基配制等等,主要参照《分子克隆实验指南》(第三版),J.萨姆布鲁克,D.W.拉塞尔(美)编著,黄培堂等译,科学出版社,北京,2002)进行。必要时可以通过简单试验确定具体实验条件。
PCR扩增实验根据质粒或DNA模板供应商提供的反应条件或试剂盒说明书进行。必要时可以通过简单试验予以调整。
LB液体培养基:10g/L胰蛋白胨,5g/L酵母提取物,10g/L氯化钠。
LB固体培养基:10g/L胰蛋白胨,5g/L酵母提取物,10g/L氯化钠,20g/L琼脂粉。
BHIS液体培养基:37g/L BHI,91g/L山梨醇。
BHIS固体培养基:37g/L BHI,91g/L山梨醇,20g/L琼脂粉。
BHIS-suc固体培养基:37g/LBHI,91g/L山梨醇,200g/L蔗糖,10g/L葡萄糖。
20X电转母液:80g/L甘氨酸,2%吐温80。
以下实施例中,当使用含卡那霉素的培养基时,所述卡那霉素在培养基中的终浓度为50μg/ml。
L-肌氨酸的HPLC检测方法:安捷伦1260高效液相色谱仪;色谱条件,色谱柱:Venusil HILIC,5μm,Ф4.6mm×250mm;流动相:20mM磷酸二氢钾:乙腈=30:70;流速:1mL/min;柱温:20℃;进样量:5uL;检测器波长:210nm;运行时间:15min;稀释液:超纯水;肌氨酸成品出峰时间:7.365min。
20mM磷酸二氢钾流动相的制备:用称取磷酸二氢钾(AR)2.7218g至1000mL棕色试剂瓶中,加入适量超纯水溶解定容到1000mL,摇匀,用无机微孔(0.45μm)滤膜抽滤3次。
实施例中所使用的出发菌株为谷氨酸棒杆菌ATCC13032(简称ATCC13032),由上海工业生物技术研发中心惠赠。
实施例中所使用的质粒pTrc99a-dpkAF117L委托苏州金唯智生物科技有限公司合成,质粒pK18mobsacB-glc-pntAB、pK18mobsacB-alaT-dpkA和pK18mobsacB-icd由浙江华睿生物技术有限公司构建惠赠,任何单位和个人都可以获得这些质粒用于验证本发明,但未经允许不得用作其他用途,包括开发利用、科学研究和教学。
实施例中使用的引物序列信息如表1所示。
表1、引物序列
注:表中引物名称后缀F代表正向引物,R代表反向引物。
实施例1:基因改造靶点质粒构建
1.1分别使用引物对GlcB-F1/GlcB-R1和GlcB-F2/GlcB-R2,以谷氨酸棒杆菌ATCC13032基因组为模板,PCR扩增扩增GlcB上游同源臂和下游同源臂片段,用KOD-neo DNA聚合酶进行扩增。
PCR扩增条件:95℃5min;94℃30s,58℃30s,68℃35s,30个循环;68℃10min。
琼脂糖凝胶回收1.0kb大小PCR片段一和片段二(Axygen DNA凝胶回收试剂盒AP-GX-50)。
使用引物对Trc-pntAB-F/Trc-pntAB-R,以大肠杆菌W3110基因组为模板,PCR扩增pntAB片段,扩增条件同上,68℃延伸时间为90s。使用引物对Trc-F/Trc-pntAB-R,以该PCR产物为模板进行PCR扩增,PCR产物用琼脂糖凝胶进行回收,片段三大小为3.0kb左右。
使用引物对GlcB-F1/GlcB-R2,以上述三个片段为模板进行OE-PCR,片段大小约为5.0kb,此片段纯化回收后与HindIII/EcoRI线性化pK18mobsacB质粒进行同源拼接(BasicSeamless Cloning and Assembly Kit,TransGene),然后转化DH5α感受态细胞(上海唯地生物),转化子抽质粒测序鉴定,获得质粒pK18sacB-glcB-pntAB(图1)。
1.2分别使用引物对AlaT-F1/AlaT-R1和AlaT-F2/AlaT-R2,以谷氨酸棒杆菌ATCC13032基因组为模板,PCR扩增alaT上游同源臂和下游同源臂片段,长度约为1.0kb,琼脂糖凝胶回收。
使用引物对Trc-dpkA-F/Trc-dpkA-R,以合成质粒pTrc99a-dpkAF114L(图2)为模板,PCR扩增dpkA基因片段,大小约为1kb;使用引物对Trc-Fdpk/Trc-dpkA-R,以此PCR产物为模板,PCR扩增PtrcdpkA片段,大小约1kb;使用引物对AlaT-F1/AlaT-R2,以alaT上游同源臂、下游同源臂和PtrcdpkA片段这三个片段为模板,进行OE-PCR,片段长度为3kb,此片段纯化回收后与HindIII/EcoRI线性化pK18mobsacB质粒进行同源重组,转化DH5α感受态细胞,转化子抽质粒测序鉴定,获得质粒pK18sacB-alaT-dpkA(图3)。
1.3分别使用引物对Icd-F1/Icd-R1和Icd-F2/Icd-R2,分别以谷氨酸棒杆菌ATCC13032基因组为模板,PCR扩增扩增icd突变碱基上游同源臂和下游同源臂片段,长度约为1.0kb,琼脂糖凝胶回收,回收片段与HindIII/EcoRI线性化pK18mobsacB质粒进行同源重组,转化DH5α感受态细胞,转化子抽质粒测序鉴定,获得质粒pK18mobsacB-icd(图4)。
实施例2:基因工程菌株构建
2.1制备谷氨酸棒杆菌ATCC13032感受态:谷氨酸棒杆菌ATCC13032甘油菌在BHIS平板上划线,30℃恒温培养箱中过夜培养48h,挑取单菌落到BHIS试管培养基中,30℃摇床220rpm过夜培养。取1ml菌液到50ml的BHIS液体培养基摇瓶中,同时加入2.5ml 20X电转母液,恒温摇床上30℃,220rpm培养4-6h至OD600值达到1.0左右。在超净工作台上,将菌液全部转移至50ml离心管中,4℃,4500×g离心,弃上清,用10%甘油洗涤菌体,使菌体重悬,4℃,4500×g离心,重复洗涤1遍,弃上清。最后加入250μl的10%甘油,使菌体悬浮,分装到1.5ml离心管中,每80μl制备为一支感受态细胞,感受态细胞可放置在-80℃冰箱中保存。
2.2菌株ATCC13032感受态细胞电转化:选取测序正确的pK18mobsacB-glc-pntAB质粒(图1),吸取3μl(500ng以上)到谷氨酸棒状杆菌ATCC13032感受态细胞中,混匀后转移至2μm电转杯中,在2.5kV的条件下进行电击,电击时间为5.3ms,电击后立即转入800μl 46℃预热的BHIS液体培养基中,并在46℃水浴锅中水浴6min,然后置于恒温摇床中30℃,220rpm培养1h,使菌体复苏。复苏之后取100μl菌体涂布在含卡那霉素的BHIS平板上,将平板倒置在30℃恒温培养箱中培养48小时。
2.3SacB蔗糖反筛:挑取含卡那霉素的BHIS平板上的转化子,接种到无抗性的BHIS试管培养基中,在恒温摇床中30℃,220rpm培养24h,使其发生双交换。将菌体稀释1000倍后涂布在含20%蔗糖的BHIS-suc平板上,倒置在30℃恒温培养箱中培养48小时。挑取BHIS-suc平板转化子,分别点板BHIS平板和含卡那霉素的BHIS平板,倒置在30℃恒温培养箱中培养24小时。使用引物glcB-V-F和glcB-V-R进行PCR扩增验证在BHIS平板生长而在含卡那霉素的BHIS平板不能生长的转化子,阳性转化子PCR扩增条带约为1.5Kb,阴性转化子PCR扩增无条带。挑取阳性转化子到4ml的BHIS试管培养基中,在恒温摇床上30℃,220rpm培养24小时,使用20%甘油保菌,得到了基因型为ATCC13032::pntAB,△glcB的菌株。
2.4将步骤2.3中得到的ATCC13032::pntAB,△glcB菌株制备电转感受态细胞,将pK18mobsacB-alaT-dpkA质粒转入感受态细胞(相关感受态制备、转化剂蔗糖反筛步骤同上),转化子使用引物alaT-V-F和alaT-V-R进行PCR扩增验证,阳性转化子PCR扩增条带约为2.0Kb,阴性转化子PCR扩增无条带。挑取阳性转化子到4ml的BHIS试管培养基中,在恒温摇床上30℃,220rpm培养24小时,使用20%甘油保菌,得到了基因型为ATCC13032::pntAB::dpkA△glcB△alaT的菌株。
2.5将步骤2.4中得到的ATCC13032::pntAB::dpkA△glcB△alaT的菌株制备电转感受态细胞,将pK18mobsacB-icd质粒转入感受态细胞(相关感受态制备、转化剂蔗糖反筛步骤同上),转化子使用引物icd-V-F和icd-V-R进行PCR扩增验证,转化子PCR扩增条带约为0.9kb,送测序鉴定。挑取阳性转化子到4ml的BHIS试管培养基中,在恒温摇床上30℃,220rpm培养24小时,使用20%甘油保菌,得到了基因型为ATCC13032::pntAB::dpkA△glcB△alaT,icd*菌株。
实施例3:工程菌的发酵罐发酵
将实施例2中得到的ATCC13032::pntAB::dpkA△glcB△alaT,icd*菌株BHI平板划线活化,挑单克隆接接BHI试管,30℃、220rpm振荡培养14h左右,按照5v/v%接种量转接种子摇瓶(50/500ml),30℃220rpm振荡培养7h。然后按照10v/v%接种量(200ml/2L)转接5L发酵罐,发酵温度30℃,溶氧维持30%,25%氨水维持pH 7.0,发酵时间为72h。发酵8h后按照加入0.1g/L/min流加甲胺,发酵24h后加入5g/L乙酸钾。
种子培养基:蛋白胨10g/L,酵母粉5g/L,牛肉膏5g/L,氯化钠2.5g/L,葡萄糖10g/L,pH 7.0。
发酵培养基见表2。
表2、发酵培养基组成
物料 | 含量/L |
葡萄糖 | 100g |
硫酸镁 | 100mg |
酵母粉 | 35g |
硫酸铵 | 20g |
FeSO4·7H2O | 11mg |
柠檬酸钠 | 10mg |
H3PO4(85%) | 250μl |
消泡剂204 | 500mg |
泛酸钙 | 60mg |
维生素PP | 18mg |
thiamin HCl | 15mg |
生物素 | 15mg |
L-肌氨酸HPLC检测:使用柱前衍生氨基酸分析法(参见安捷伦公司手册)来测定发酵液中L-肌氨酸含量。氨基酸与氯甲酸-9-芴甲酯(FMOC)反应生成FMOC-氨基酸,生成的氨基酸衍生物经反相高效液相色谱分离后用紫外或荧光检测,在一定的范围内其吸光值与氨基酸浓度成正比。发酵72h后HPLC检测,产物L-肌氨酸最高浓度达到18.3g/L。
上述实施例表明,采用本发明方法构建的菌株能够实现发酵液中L-肌氨酸的积累,具有工业化开发前景,已对其进行菌种保藏,保藏编号为CGMCC No.22265。
应理解,这些实施例仅用于举例说明目的,而不是对本发明的限制。本领域技术人员在阅读了本发明的构思之后,对其作出的各种改变或调整,均应落入本发明的保护范围内,这些等价形式同样属于本申请所附权利要求书限定的范围。
序列表
<110> 洛阳华荣生物技术有限公司
<120> 一种构建L-肌氨酸生产菌的方法
<130> SHPI2110101
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 10582
<212> DNA
<213> 人工序列()
<400> 1
gcaattccgg ttcgcttgct gtccataaaa ccgcccagtc tagctatcgc catgtaagcc 60
cactgcaagc tacctgcttt ctctttgcgc ttgcgttttc ccttgtccag atagcccagt 120
agctgacatt catccggggt cagcaccgtt tctgcggact ggctttctac gtgttccgct 180
tcctttagca gcccttgcgc cctgagtgct tgcggcagcg tgaagctagc ttatcgcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gctttccggg aagataaacg 420
caatgatcgc gcccagcaga atgaaagttg gaaacgcgta aataattatg gccgctgaac 480
ggccttcttc tgttttggac actagaaaac tccaagaaag ttaaaactga aatggtcgtg 540
ctagtggtgg gcacagaatt aaatcgctgg tgtgcgaact ttcatacctt aacgcagtag 600
tgatgcttaa ggcacaacgt gggggcaatt ccggcctata ctttggaagt atgactactt 660
ttattacctc cggtggcttg gaaatctccc ccgctggcgc tcatattgtt cacgccgaat 720
cacctgaagg tgagctgttg tttgttagct ccgcttccca atatggggag ggaaatgcaa 780
ttaggggtgg tgtgcccatc attgctccat ggtttggtgg actgcttggt ttggaccctg 840
cacatggttg ggcgaagcgt tccgcgtggg acgtgactga acatgacggc caaattcacg 900
ctgaatatgg ccgcgatggt ttactgctgg atattcgtgc gaacagcact aagaatggtt 960
ttgagatcac cctgcgcgct tacaacgaca ccgatgaggc acgcactgtg cagttggcct 1020
tccaccctta tttcaaggtg gatgatgtag aaaagatcga ggtccgtggc cttgatgggg 1080
tggacattct caatcgcctg aacaatgagg tggagaccca agatggtccc gttacttttg 1140
atggcgagtt cgatcgcatt gcgctaggga ctccggttgt gaggattttt gataccgatc 1200
gcatcatcac cattgagggc gatggtcatg attccactgt ggtgtggaat ccaggcgaaa 1260
gtcgcgcctc caccgtggcc gatattggcg aaggtgaatg gcgcgacttt gtgtgtgttg 1320
aaccggcgct tttgggtgct gaccaaaaag gagtgagggt ggctccgggg cagtcagtca 1380
ccgttgggat gcaggtaagc gtcgaaaagc gtgcttagtt gacaattaat catccggctc 1440
gtataatgtg tggaattgtg agcggataac aatttcacac aggaaacaga ccatgcgaat 1500
tggcatacca agagaacggt taaccaatga aacccgtgtt gcagcaacgc caaaaacagt 1560
ggaacagctg ctgaaactgg gttttaccgt cgcggtagag agcggcgcgg gtcaactggc 1620
aagttttgac gataaagcgt ttgtgcaagc gggcgctgaa attgtagaag ggaatagcgt 1680
ctggcagtca gagatcattc tgaaggtcaa tgcgccgtta gatgatgaaa ttgcgttact 1740
gaatcctggg acaacgctgg tgagttttat ctggcctgcg cagaatccgg aattaatgca 1800
aaaacttgcg gaacgtaacg tgaccgtgat ggcgatggac tctgtgccgc gtatctcacg 1860
cgcacaatcg ctggacgcac taagctcgat ggcgaacatc gccggttatc gcgccattgt 1920
tgaagcggca catgaatttg ggcgcttctt taccgggcaa attactgcgg ccgggaaagt 1980
gccaccggca aaagtgatgg tgattggtgc gggtgttgca ggtctggccg ccattggcgc 2040
agcaaacagt ctcggcgcga ttgtgcgtgc attcgacacc cgcccggaag tgaaagaaca 2100
agttcaaagt atgggcgcgg aattcctcga gctggatttt aaagaggaag ctggcagcgg 2160
cgatggctat gccaaagtga tgtcggacgc gttcatcaaa gcggaaatgg aactctttgc 2220
cgcccaggca aaagaggtcg atatcattgt caccaccgcg cttattccag gcaaaccagc 2280
gccgaagcta attacccgtg aaatggttga ctccatgaag gcgggcagtg tgattgtcga 2340
cctggcagcc caaaacggcg gcaactgtga atacaccgtg ccgggtgaaa tcttcactac 2400
ggaaaatggt gtcaaagtga ttggttatac cgatcttccg ggccgtctgc cgacgcaatc 2460
ctcacagctt tacggcacaa acctcgttaa tctgctgaaa ctgttgtgca aagagaaaga 2520
cggcaatatc actgttgatt ttgatgatgt ggtgattcgc ggcgtgaccg tgatccgtgc 2580
gggcgaaatt acctggccgg caccgccgat tcaggtatca gctcagccgc aggcggcaca 2640
aaaagcggca ccggaagtga aaactgagga aaaatgtacc tgctcaccgt ggcgtaaata 2700
cgcgttgatg gcgctggcaa tcattctttt tggctggatg gcaagcgttg cgccgaaaga 2760
attccttggg cacttcaccg ttttcgcgct ggcctgcgtt gtcggttatt acgtggtgtg 2820
gaatgtatcg cacgcgctgc atacaccgtt gatgtcggtc accaacgcga tttcagggat 2880
tattgttgtc ggagcactgt tgcagattgg ccagggcggc tgggttagct tccttagttt 2940
tatcgcggtg cttatagcca gcattaatat tttcggtggc ttcaccgtga ctcagcgcat 3000
gctgaaaatg ttccgcaaaa attaaggggt aacatatgtc tggaggatta gttacagctg 3060
catacattgt tgccgcgatc ctgtttatct tcagtctggc cggtctttcg aaacatgaaa 3120
cgtctcgcca gggtaacaac ttcggtatcg ccgggatggc gattgcgtta atcgcaacca 3180
tttttggacc ggatacgggt aatgttggct ggatcttgct ggcgatggtc attggtgggg 3240
caattggtat ccgtctggcg aagaaagttg aaatgaccga aatgccagaa ctggtggcga 3300
tcctgcatag cttcgtgggt ctggcggcag tgctggttgg ctttaacagc tatctgcatc 3360
atgacgcggg aatggcaccg attctggtca atattcacct gacggaagtg ttcctcggta 3420
tcttcatcgg ggcggtaacg ttcacgggtt cggtggtggc gttcggcaaa ctgtgtggca 3480
agatttcgtc taaaccattg atgctgccaa accgtcacaa aatgaacctg gcggctctgg 3540
tcgtttcctt cctgctgctg attgtatttg ttcgcacgga cagcgtcggc ctgcaagtgc 3600
tggcattgct gataatgacc gcaattgcgc tggtattcgg ctggcattta gtcgcctcca 3660
tcggtggtgc agatatgcca gtggtggtgt cgatgctgaa ctcgtactcc ggctgggcgg 3720
ctgcggctgc gggctttatg ctcagcaacg acctgctgat tgtgaccggt gcgctggtcg 3780
gttcttcggg ggctatcctt tcttacatta tgtgtaaggc gatgaaccgt tcctttatca 3840
gcgttattgc gggtggtttc ggcaccgacg gctcttctac tggcgatgat caggaagtgg 3900
gtgagcaccg cgaaatcacc gcagaagaga cagcggaact gctgaaaaac tcccattcag 3960
tgatcattac tccggggtac ggcatggcag tcgcgcaggc gcaatatcct gtcgctgaaa 4020
ttactgagaa attgcgcgct cgtggtatta atgtgcgttt cggtatccac ccggtcgcgg 4080
ggcgtttgcc tggacatatg aacgtattgc tggctgaagc aaaagtaccg tatgacatcg 4140
tgctggaaat ggacgagatc aatgatgact ttgctgatac cgataccgta ctggtgattg 4200
gtgctaacga tacggttaac ccggcggcgc aggatgatcc gaagagtccg attgctggta 4260
tgcctgtgct ggaagtgtgg aaagcgcaga acgtgattgt ctttaaacgt tcgatgaaca 4320
ctggctatgc tggtgtgcaa aacccgctgt tcttcaagga aaacacccac atgctgtttg 4380
gtgacgccaa agccagcgtg gatgcaatcc tgaaagctct gtaaccctga cggcctctgc 4440
tgaggccggt gccactcttc tttcgatttc aacccttatc gtgtttggcg atgtgatcag 4500
actaagtgat caccgtcacc agcaaaaggg gtttgcgaac tttactaagt cattaccccc 4560
gcctaacccc gacttttatc taggtcacac cttcgaaacc tacggaacgt tgcggtgcct 4620
gcattttccc atttcagagc atttgcccag tacatccgta ctagcaactc ccccgcccac 4680
tttttctgcg aagccagaac tttgcaaact tcacaacagg ggtgaccacc cccgcacaaa 4740
acttaaaaac ccaaaccgat tgacgcacca atgcccgatg gagcaatgtg tgaaccacgc 4800
caccacgcaa accgatgcac atcacgtcga aacagtgaca gtgcattagc tcatactttg 4860
tggtcggcac cgcccattgc gaatcagcac ttaaggaagt gactttgatg tcaaacgttg 4920
gaaagccacg taccgcacag gaaatccagc aggattggga caccaaccct cgttggaacg 4980
gcatcacccg cgactacacc gcagaccagg tagctgatct gcagggttcc gtcatcgagg 5040
agcacactct tgctcgccgc ggctcagaga tcctctggga cgcagtcacc caggaaggtg 5100
acggatacat caacgcgctt ggcgcactca ccggtaacca ggctgttcag caggttcgtg 5160
caggcctgaa ggctgtctac ctgtccggtt ggcaggtcgc aggtgacgcc aacctctccg 5220
gccacaccta ccctgaccag tccctctacc cagcgaactc cgttccaagc gtcgttcgtc 5280
gcatcaacaa cgcactgctg cgttccgatg gaattcgtaa tcatggtcat agctgtttcc 5340
tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg 5400
taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc 5460
cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg 5520
gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc 5580
ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 5640
agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 5700
ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 5760
caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 5820
gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 5880
cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta 5940
tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 6000
gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 6060
cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 6120
tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg 6180
tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 6240
caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 6300
aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 6360
cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 6420
ccttttgggg tgggcgaaga actccagcat gagatccccg cgctggagga tcatccagcc 6480
ctgatagaaa cagaagccac tggagcacct caaaaacacc atcatacact aaatcagtaa 6540
gttggcagca tcacccgacg cactttgcgc cgaataaata cctgtgacgg aagatcactt 6600
cgcagaataa ataaatcctg gtgtccctgt tgataccggg aagccctggg ccaacttttg 6660
gcgaaaatga gacgttgatc ggcacgtaag aggttccaac tttcaccata atgaaataag 6720
atcactaccg ggcgtatttt ttgagttatc gagattttca ggagctgata gaaacagaag 6780
ccactggagc acctcaaaaa caccatcata cactaaatca gtaagttggc agcatcaccc 6840
gacgcacttt gcgccgaata aatacctgtg acggaagatc acttcgcaga ataaataaat 6900
cctggtgtcc ctgttgatac cgggaagccc tgggccaact tttggcgaaa atgagacgtt 6960
gatcggcacg taagaggttc caactttcac cataatgaaa taagatcact accgggcgta 7020
ttttttgagt tatcgagatt ttcaggagct ctttggcatc gtctctcgcc tgtcccctca 7080
gttcagtaat ttcctgcatt tgcctgtttc cagtcggtag atattccaca aaacagcagg 7140
gaagcagcgc ttttccgctg cataaccctg cttcggggtc attatagcga ttttttcggt 7200
atatccatcc tttttcgcac gatatacagg attttgccaa agggttcgtg tagactttcc 7260
ttggtgtatc caacggcgtc agcggggcag gataggtgaa gtaggcccac ccgcgagcgg 7320
gtgttccttc ttcactgtcc cttattcgca cctggcggtg ctcaacggga atcctgctct 7380
gcgaggctgg ccggctaccg ccggcgtaac agatgagggc aagcggatgg ctgatgaaac 7440
caagccaacc aggaagggca gcccacctat caaggtgtac tgccttccag acgaacgaag 7500
agcgattgag gaaaaggcgg cggcggccgg catgagcctg tcggcctacc tgctggccgt 7560
cggccagggc tacaaaatca cgggcgtcgt ggactatgag cacgtccgcg agggcgtccc 7620
ggaaaacgat tccgaagccc aacctttcat agaaggcggc ggtggaatcg aaatctcgtg 7680
atggcaggtt gggcgtcgct tggtcggtca tttcgctcgg taccatcggc attttctttt 7740
gcgtttttat ttgttaactg ttaattgtcc ttgttcaagg atgctgtctt tgacaacaga 7800
tgttttcttg cctttgatgt tcagcaggaa gctcggcgca aacgttgatt gtttgtctgc 7860
gtagaatcct ctgtttgtca tatagcttgt aatcacgaca ttgtttcctt tcgcttgagg 7920
tacagcgaag tgtgagtaag taaaggttac atcgttagga tcaagatcca tttttaacac 7980
aaggccagtt ttgttcagcg gcttgtatgg gccagttaaa gaattagaaa cataaccaag 8040
catgtaaata tcgttagacg taatgccgtc aatcgtcatt tttgatccgc gggagtcagt 8100
gaacaggtac catttgccgt tcattttaaa gacgttcgcg cgttcaattt catctgttac 8160
tgtgttagat gcaatcagcg gtttcatcac ttttttcagt gtgtaatcat cgtttagctc 8220
aatcataccg agagcgccgt ttgctaactc agccgtgcgt tttttatcgc tttgcagaag 8280
tttttgactt tcttgacgga agaatgatgt gcttttgcca tagtatgctt tgttaaataa 8340
agattcttcg ccttggtagc catcttcagt tccagtgttt gcttcaaata ctaagtattt 8400
gtggccttta tcttctacgt agtgaggatc tctcagcgta tggttgtcgc ctgagctgta 8460
gttgccttca tcgatgaact gctgtacatt ttgatacgtt tttccgtcac cgtcaaagat 8520
tgatttataa tcctctacac cgttgatgtt caaagagctg tctgatgctg atacgttaac 8580
ttgtgcagtt gtcagtgttt gtttgccgta atgtttaccg gagaaatcag tgtagaataa 8640
acggattttt ccgtcagatg taaatgtggc tgaacctgac cattcttgtg tttggtcttt 8700
taggatagaa tcatttgcat cgaatttgtc gctgtcttta aagacgcggc cagcgttttt 8760
ccagctgtca atagaagttt cgccgacttt ttgatagaac atgtaaatcg atgtgtcatc 8820
cgcattttta ggatctccgg ctaatgcaaa gacgatgtgg tagccgtgat agtttgcgac 8880
agtgccgtca gcgttttgta atggccagct gtcccaaacg tccaggcctt ttgcagaaga 8940
gatattttta attgtggacg aatcaaattc agaaacttga tatttttcat ttttttgctg 9000
ttcagggatt tgcagcatat catggcgtgt aatatgggaa atgccgtatg tttccttata 9060
tggcttttgg ttcgtttctt tcgcaaacgc ttgagttgcg cctcctgcca gcagtgcggt 9120
agtaaaggtt aatactgttg cttgttttgc aaactttttg atgttcatcg ttcatgtctc 9180
cttttttatg tactgtgtta gcggtctgct tcttccagcc ctcctgtttg aagatggcaa 9240
gttagttacg cacaataaaa aaagacctaa aatatgtaag gggtgacgcc aaagtataca 9300
ctttgccctt tacacatttt aggtcttgcc tgctttatca gtaacaaacc cgcgcgattt 9360
acttttcgac ctcattctat tagactctcg tttggattgc aactggtcta ttttcctctt 9420
ttgtttgata gaaaatcata aaaggatttg cagactacgg gcctaaagaa ctaaaaaatc 9480
tatctgtttc ttttcattct ctgtattttt tatagtttct gttgcatggg cataaagttg 9540
cctttttaat cacaattcag aaaatatcat aatatctcat ttcactaaat aatagtgaac 9600
ggcaggtata tgtgatgggt taaaaaggat cgatcctcta gcgaacccca gagtcccgct 9660
cagaagaact cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata 9720
ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg 9780
gtagccaacg ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat 9840
ccagaaaagc ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg 9900
acgagatcct cgccgtcggg catccgcgcc ttgagcctgg cgaacagttc ggctggcgcg 9960
agcccctgat gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta 10020
cgtgctcgct cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc 10080
gtatgcagcc gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga 10140
gatgacagga gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca 10200
gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc 10260
gctgcctcgt cttggagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc 10320
gggcgcccct gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt 10380
gcccagtcat agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca 10440
tcttgttcaa tcatgcgaaa cgatcctcat cctgtctctt gatcagatct tgatcccctg 10500
cgccatcaga tccttggcgg caagaaagcc atccagttta ctttgcaggg cttcccaacc 10560
ttaccagagg gcgccccagc tg 10582
<210> 2
<211> 5142
<212> DNA
<213> 人工序列()
<400> 2
gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc 60
ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc 120
gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc 180
tgaaatgagc tgttgacaat taatcatccg gctcgtataa tgtgtggaat tgtgagcgga 240
taacaatttc acacaggaaa cagaccatgg aattcatgcg cgtgcctttt accgagctgc 300
aaagcctgtt gcaggccatt ttccagcgcc atgggtgcag cgaggccgtg gcccgggtgc 360
tggcccacaa ctgcgccagc gcccagcgcg atggcgccca tagccatggg gtgttccgca 420
tgcccggtta tgtctcgacc ttggccagcg gctgggtcga tggccaggcc acgccacagg 480
tcagcgacgt ggccgccggc tatgtgcgcg tcgatgctgc gggcggtttt gcccagccgg 540
cactggcggc ggcccgtgag ctgttggtgg cgaaggcgcg cagcgcaggc attgccgtgc 600
tggcgatcca caactcgcac catttagctg cgttgtggcc ggatgtcgag ccgttcgccg 660
aagagggcct ggtagccctc agcgtggtca acagcatgac ctgcgtggtg ccgcatggtg 720
cacgcaagcc gctgttcggt accaacccca tcgctttcgc tgcgccttgc gccgagcatg 780
acccgatcgt tttcgacatg gccaccagtg ccatggccca tggcgatgtg cagattgccg 840
cacgtgctgg ccagcaattg ccggagggca tgggggtaga tgccgatggc cagccgacta 900
ccgacccgaa ggcgatcctg gaaggcgggg ccttgctgcc atttggcggg cacaagggct 960
cggcgttgtc gatgatggtc gagctgctgg cggcggcgct gaccggcggt catttctcct 1020
gggagttcga ttggtccggg catccggggg cgaaaacgcc atggaccggg cagttgatca 1080
tcgtcatcga cccgggcaag gccgaaggcc agcggtttgc ccagcgcagc cgcgagctgg 1140
tggagcacat gcaggcggtg gggctgacgc gcatgccggg cgagcggcgc taccgtgagc 1200
gcgaggtggc cgaggaggag ggggtggcgg tgaccgagca ggagttgaaa ggcctgaaag 1260
agctgcttgg ctgactgcag gcatgcaagc ttggctgttt tggcggatga gagaagattt 1320
tcagcctgat acagattaaa tcagaacgca gaagcggtct gataaaacag aatttgcctg 1380
gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa ctcagaagtg aaacgccgta 1440
gcgccgatgg tagtgtgggg tctccccatg cgagagtagg gaactgccag gcatcaaata 1500
aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac 1560
gctctcctga gtaggacaaa tccgccggga gcggatttga acgttgcgaa gcaacggccc 1620
ggagggtggc gggcaggacg cccgccataa actgccaggc atcaaattaa gcagaaggcc 1680
atcctgacgg atggcctttt tgcgtttcta caaactcttt ttgtttattt ttctaaatac 1740
attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa 1800
aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat 1860
tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc 1920
agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga 1980
gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg 2040
cggtattatc ccgtgttgac gccgggcaag agcaactcgg tcgccgcata cactattctc 2100
agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag 2160
taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc 2220
tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg 2280
taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg 2340
acaccacgat gcctacagca atggcaacaa cgttgcgcaa actattaact ggcgaactac 2400
ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac 2460
cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg 2520
agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg 2580
tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg 2640
agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac 2700
tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg 2760
ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 2820
tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 2880
aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 2940
tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt 3000
agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 3060
taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 3120
caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 3180
agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 3240
aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 3300
gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 3360
tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 3420
gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 3480
ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct 3540
ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 3600
aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac 3660
accgcatatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata 3720
cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc 3780
tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt 3840
ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagca 3900
gatcaattcg cgcgcgaagg cgaagcggca tgcatttacg ttgacaccat cgaatggtgc 3960
aaaacctttc gcggtatggc atgatagcgc ccggaagaga gtcaattcag ggtggtgaat 4020
gtgaaaccag taacgttata cgatgtcgca gagtatgccg gtgtctctta tcagaccgtt 4080
tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa cgcgggaaaa agtggaagcg 4140
gcgatggcgg agctgaatta cattcccaac cgcgtggcac aacaactggc gggcaaacag 4200
tcgttgctga ttggcgttgc cacctccagt ctggccctgc acgcgccgtc gcaaattgtc 4260
gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg tggtggtgtc gatggtagaa 4320
cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc ttctcgcgca acgcgtcagt 4380
gggctgatca ttaactatcc gctggatgac caggatgcca ttgctgtgga agctgcctgc 4440
actaatgttc cggcgttatt tcttgatgtc tctgaccaga cacccatcaa cagtattatt 4500
ttctcccatg aagacggtac gcgactgggc gtggagcatc tggtcgcatt gggtcaccag 4560
caaatcgcgc tgttagcggg cccattaagt tctgtctcgg cgcgtctgcg tctggctggc 4620
tggcataaat atctcactcg caatcaaatt cagccgatag cggaacggga aggcgactgg 4680
agtgccatgt ccggttttca acaaaccatg caaatgctga atgagggcat cgttcccact 4740
gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa tgcgcgccat taccgagtcc 4800
gggctgcgcg ttggtgcgga tatctcggta gtgggatacg acgataccga agacagctca 4860
tgttatatcc cgccgttaac caccatcaaa caggattttc gcctgctggg gcaaaccagc 4920
gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc 4980
gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata cgcaaaccgc ctctccccgc 5040
gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag 5100
tgagcgcaac gcaattaatg tgagttagcg cgaattgatc tg 5142
<210> 3
<211> 8758
<212> DNA
<213> 人工序列()
<400> 3
gcaattccgg ttcgcttgct gtccataaaa ccgcccagtc tagctatcgc catgtaagcc 60
cactgcaagc tacctgcttt ctctttgcgc ttgcgttttc ccttgtccag atagcccagt 120
agctgacatt catccggggt cagcaccgtt tctgcggact ggctttctac gtgttccgct 180
tcctttagca gcccttgcgc cctgagtgct tgcggcagcg tgaagctagc ttatcgcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttggcacc aatcaaagga 420
cttcttcttg tagcgcttca agattcctga gccgccgtcg tcgaggtatt tgatgcggta 480
gtcatcgttt actgttgggc cggagggtcc gtcgataagc acgtcctttg ctcctagtcc 540
gttttcgacg atgaacagtg gcttctgcca gcgctcccag tagttgttca ggacgatgcg 600
caaaccgagg ggatcaactt gccaacccca ttcggaagcc gcgagagtgg ggctgaccac 660
tccgccgatg atgttaccgc caccggttga gtagttttcg gggttgtggg cttcacatac 720
ggacatgtaa taggagaagg aaatgaaatc gacggtgttt ttttaagatc tcacggtctt 780
catctgtgat gtcgatggtg ataccctttc gcggaatttg tgcagcaagt agcctgggta 840
ttcgccacgg acgtgaatat tgccgaaggc atagtcctcg tgggactttt gctgggcggt 900
aagttgatcg cgtgggtccg gggtaatgcc ataacgagga acggcaataa tcatgcaacc 960
gatctggttt tgtgggtcga tctcatgagc aatcttagtt gccaaagcac ttgctactca 1020
atcatggtaa acagcctggt cgcagtcctt cacgattcaa actttgcctt ccgctacgcc 1080
ttccacctga tcatcataga agacggtgaa gtaacagcag ccggagatcc cacagagatc 1140
gtcactgcgg gactgatcga agaagtctac aacgtcaaag cctgtgcatc ccagaccccg 1200
tgaacagcaa accgatgatc gtgccactgg aaagatctta ggcagccgtg ggattacacc 1260
cttttagagc tagaacagta aaaattcacc caatagcttt caactacgca cacaaagtgg 1320
caacattgag cgggtgacta cagacaagcg caaaacctct aagaccaccg acaccgccaa 1380
caaggctgtg ggcgcggatc aggcttgaca attaatcatc cggctcgtat aatgtgtgga 1440
attgtgagcg gataacaatt tcacacagga aacagaccat ggaattcatg cgcgtgcctt 1500
ttaccgagct gcaaagcctg ttgcaggcca ttttccagcg ccatgggtgc agcgaggccg 1560
tggcccgggt gctggcccac aactgcgcca gcgcccagcg cgatggcgcc catagccatg 1620
gggtgttccg catgcccggt tatgtctcga ccttggccag cggctgggtc gatggccagg 1680
ccacgccaca ggtcagcgac gtggccgccg gctatgtgcg cgtcgatgct gcgggcggtt 1740
ttgcccagcc ggcactggcg gcggcccgtg agctgttggt ggcgaaggcg cgcagcgcag 1800
gcattgccgt gctggcgatc cacaactcgc accatttagc tgcgttgtgg ccggatgtcg 1860
agccgttcgc cgaagagggc ctggtagccc tcagcgtggt caacagcatg acctgcgtgg 1920
tgccgcatgg tgcacgcaag ccgctgttcg gtaccaaccc catcgctttc gctgcgcctt 1980
gcgccgagca tgacccgatc gttttcgaca tggccaccag tgccatggcc catggcgatg 2040
tgcagattgc cgcacgtgct ggccagcaat tgccggaggg catgggggta gatgccgatg 2100
gccagccgac taccgacccg aaggcgatcc tggaaggcgg ggccttgctg ccatttggcg 2160
ggcacaaggg ctcggcgttg tcgatgatgg tcgagctgct ggcggcggcg ctgaccggcg 2220
gtcatttctc ctgggagttc gattggtccg ggcatccggg ggcgaaaacg ccatggaccg 2280
ggcagttgat catcgtcatc gacccgggca aggccgaagg ccagcggttt gcccagcgca 2340
gccgcgagct ggtggagcac atgcaggcgg tggggctgac gcgcatgccg ggcgagcggc 2400
gctaccgtga gcgcgaggtg gccgaggagg agggggtggc ggtgaccgag caggagttga 2460
aaggcctgaa agagctgctt ggctgagcca tgggcatccc agttggaaaa cgcaattgag 2520
cgcctgggta acttcctgtc cacttacaag cagtagtagt tgttaggatt caccacgaat 2580
ctcaggattt ttgagattcg tggtgaattt ttgcgttttc cagtcaggct cctgcaactt 2640
tcggaccgat ttcagagggg cggagctggt ttgtggtgga tccttgaaat ggaacctcgc 2700
aggaagcttt caggaagacc aagttgggcc taggggtggc gggattgcaa aaatccgtcc 2760
ccggttcgcc atgaaatgct gattttgatc gaatctttgc gctaactgta gggcgggttc 2820
agggggtgaa tgcaccacga gcaacccgaa gggtgcgaag tgggcattcg tagaacaatc 2880
ccagaggaaa gccgtacggc tttcctcgac atgatcaatc aaggtatgtc aggtcttgct 2940
gcgtctacag cggtcggggt cagtgaattc accgggcgaa agtgggcgaa ggccgccggg 3000
gtgaaactga cccgcggccc gcgaggtggc aatgcttttg acaccgccga gaaacttgag 3060
attgcagcca gcatgctaga gaaaggatgc ctaccccgag aaatcggcga gtatgtcggc 3120
atgactcggg ccaatatatc cctatggcgc aaacaaggcc cagacaagct tcgccaacgc 3180
gcagccacct tgcgcaccgg caagcgagca gctgaattca tccacgcccc ggtgatgggc 3240
ccttattatg ggccacgcac actccatcaa gtgttgcgtg aggactacac aacactgttt 3300
gacgagttat ctgcgttggg gttgccagca caggtgtgtg gggccttact tcatcttgct 3360
ccaccaccat cattacgctt ttcttatatg tcgtgtgtag tgccgttatt tgctgatgaa 3420
atcaaagtcg taggacaagg cacacgatta tcgttagaag agaaaatgat gatccaacgt 3480
ttccatgaat tcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac 3540
aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt 3600
gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc 3660
gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg 3720
ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt 3780
atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa 3840
gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 3900
gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag 3960
gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt 4020
gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg 4080
aagcgtggcg ctttctcaat gctcacgctg taggtatctc agttcggtgt aggtcgttcg 4140
ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg 4200
taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac 4260
tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg 4320
gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc tgaagccagt 4380
taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg 4440
tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc 4500
tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt 4560
ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttggggtggg cgaagaactc 4620
cagcatgaga tccccgcgct ggaggatcat ccagccctga tagaaacaga agccactgga 4680
gcacctcaaa aacaccatca tacactaaat cagtaagttg gcagcatcac ccgacgcact 4740
ttgcgccgaa taaatacctg tgacggaaga tcacttcgca gaataaataa atcctggtgt 4800
ccctgttgat accgggaagc cctgggccaa cttttggcga aaatgagacg ttgatcggca 4860
cgtaagaggt tccaactttc accataatga aataagatca ctaccgggcg tattttttga 4920
gttatcgaga ttttcaggag ctgatagaaa cagaagccac tggagcacct caaaaacacc 4980
atcatacact aaatcagtaa gttggcagca tcacccgacg cactttgcgc cgaataaata 5040
cctgtgacgg aagatcactt cgcagaataa ataaatcctg gtgtccctgt tgataccggg 5100
aagccctggg ccaacttttg gcgaaaatga gacgttgatc ggcacgtaag aggttccaac 5160
tttcaccata atgaaataag atcactaccg ggcgtatttt ttgagttatc gagattttca 5220
ggagctcttt ggcatcgtct ctcgcctgtc ccctcagttc agtaatttcc tgcatttgcc 5280
tgtttccagt cggtagatat tccacaaaac agcagggaag cagcgctttt ccgctgcata 5340
accctgcttc ggggtcatta tagcgatttt ttcggtatat ccatcctttt tcgcacgata 5400
tacaggattt tgccaaaggg ttcgtgtaga ctttccttgg tgtatccaac ggcgtcagcg 5460
gggcaggata ggtgaagtag gcccacccgc gagcgggtgt tccttcttca ctgtccctta 5520
ttcgcacctg gcggtgctca acgggaatcc tgctctgcga ggctggccgg ctaccgccgg 5580
cgtaacagat gagggcaagc ggatggctga tgaaaccaag ccaaccagga agggcagccc 5640
acctatcaag gtgtactgcc ttccagacga acgaagagcg attgaggaaa aggcggcggc 5700
ggccggcatg agcctgtcgg cctacctgct ggccgtcggc cagggctaca aaatcacggg 5760
cgtcgtggac tatgagcacg tccgcgaggg cgtcccggaa aacgattccg aagcccaacc 5820
tttcatagaa ggcggcggtg gaatcgaaat ctcgtgatgg caggttgggc gtcgcttggt 5880
cggtcatttc gctcggtacc atcggcattt tcttttgcgt ttttatttgt taactgttaa 5940
ttgtccttgt tcaaggatgc tgtctttgac aacagatgtt ttcttgcctt tgatgttcag 6000
caggaagctc ggcgcaaacg ttgattgttt gtctgcgtag aatcctctgt ttgtcatata 6060
gcttgtaatc acgacattgt ttcctttcgc ttgaggtaca gcgaagtgtg agtaagtaaa 6120
ggttacatcg ttaggatcaa gatccatttt taacacaagg ccagttttgt tcagcggctt 6180
gtatgggcca gttaaagaat tagaaacata accaagcatg taaatatcgt tagacgtaat 6240
gccgtcaatc gtcatttttg atccgcggga gtcagtgaac aggtaccatt tgccgttcat 6300
tttaaagacg ttcgcgcgtt caatttcatc tgttactgtg ttagatgcaa tcagcggttt 6360
catcactttt ttcagtgtgt aatcatcgtt tagctcaatc ataccgagag cgccgtttgc 6420
taactcagcc gtgcgttttt tatcgctttg cagaagtttt tgactttctt gacggaagaa 6480
tgatgtgctt ttgccatagt atgctttgtt aaataaagat tcttcgcctt ggtagccatc 6540
ttcagttcca gtgtttgctt caaatactaa gtatttgtgg cctttatctt ctacgtagtg 6600
aggatctctc agcgtatggt tgtcgcctga gctgtagttg ccttcatcga tgaactgctg 6660
tacattttga tacgtttttc cgtcaccgtc aaagattgat ttataatcct ctacaccgtt 6720
gatgttcaaa gagctgtctg atgctgatac gttaacttgt gcagttgtca gtgtttgttt 6780
gccgtaatgt ttaccggaga aatcagtgta gaataaacgg atttttccgt cagatgtaaa 6840
tgtggctgaa cctgaccatt cttgtgtttg gtcttttagg atagaatcat ttgcatcgaa 6900
tttgtcgctg tctttaaaga cgcggccagc gtttttccag ctgtcaatag aagtttcgcc 6960
gactttttga tagaacatgt aaatcgatgt gtcatccgca tttttaggat ctccggctaa 7020
tgcaaagacg atgtggtagc cgtgatagtt tgcgacagtg ccgtcagcgt tttgtaatgg 7080
ccagctgtcc caaacgtcca ggccttttgc agaagagata tttttaattg tggacgaatc 7140
aaattcagaa acttgatatt tttcattttt ttgctgttca gggatttgca gcatatcatg 7200
gcgtgtaata tgggaaatgc cgtatgtttc cttatatggc ttttggttcg tttctttcgc 7260
aaacgcttga gttgcgcctc ctgccagcag tgcggtagta aaggttaata ctgttgcttg 7320
ttttgcaaac tttttgatgt tcatcgttca tgtctccttt tttatgtact gtgttagcgg 7380
tctgcttctt ccagccctcc tgtttgaaga tggcaagtta gttacgcaca ataaaaaaag 7440
acctaaaata tgtaaggggt gacgccaaag tatacacttt gccctttaca cattttaggt 7500
cttgcctgct ttatcagtaa caaacccgcg cgatttactt ttcgacctca ttctattaga 7560
ctctcgtttg gattgcaact ggtctatttt cctcttttgt ttgatagaaa atcataaaag 7620
gatttgcaga ctacgggcct aaagaactaa aaaatctatc tgtttctttt cattctctgt 7680
attttttata gtttctgttg catgggcata aagttgcctt tttaatcaca attcagaaaa 7740
tatcataata tctcatttca ctaaataata gtgaacggca ggtatatgtg atgggttaaa 7800
aaggatcgat cctctagcga accccagagt cccgctcaga agaactcgtc aagaaggcga 7860
tagaaggcga tgcgctgcga atcgggagcg gcgataccgt aaagcacgag gaagcggtca 7920
gcccattcgc cgccaagctc ttcagcaata tcacgggtag ccaacgctat gtcctgatag 7980
cggtccgcca cacccagccg gccacagtcg atgaatccag aaaagcggcc attttccacc 8040
atgatattcg gcaagcaggc atcgccatgg gtcacgacga gatcctcgcc gtcgggcatc 8100
cgcgccttga gcctggcgaa cagttcggct ggcgcgagcc cctgatgctc ttcgtccaga 8160
tcatcctgat cgacaagacc ggcttccatc cgagtacgtg ctcgctcgat gcgatgtttc 8220
gcttggtggt cgaatgggca ggtagccgga tcaagcgtat gcagccgccg cattgcatca 8280
gccatgatgg atactttctc ggcaggagca aggtgagatg acaggagatc ctgccccggc 8340
acttcgccca atagcagcca gtcccttccc gcttcagtga caacgtcgag cacagctgcg 8400
caaggaacgc ccgtcgtggc cagccacgat agccgcgctg cctcgtcttg gagttcattc 8460
agggcaccgg acaggtcggt cttgacaaaa agaaccgggc gcccctgcgc tgacagccgg 8520
aacacggcgg catcagagca gccgattgtc tgttgtgccc agtcatagcc gaatagcctc 8580
tccacccaag cggccggaga acctgcgtgc aatccatctt gttcaatcat gcgaaacgat 8640
cctcatcctg tctcttgatc agatcttgat cccctgcgcc atcagatcct tggcggcaag 8700
aaagccatcc agtttacttt gcagggcttc ccaaccttac cagagggcgc cccagctg 8758
<210> 4
<211> 6660
<212> DNA
<213> 人工序列()
<400> 4
gcaattccgg ttcgcttgct gtccataaaa ccgcccagtc tagctatcgc catgtaagcc 60
cactgcaagc tacctgcttt ctctttgcgc ttgcgttttc ccttgtccag atagcccagt 120
agctgacatt catccggggt cagcaccgtt tctgcggact ggctttctac gtgttccgct 180
tcctttagca gcccttgcgc cctgagtgct tgcggcagcg tgaagctagc ttatcgcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttctgcag accactcgcc 420
catgcggtgt gggaacttct taacaaagtt cttgacagcg attggtgcgc ggcggtcaga 480
gttgccttca cgcagcactg ggttcacagc ggaaccctta acagcgttgt agcgtgcgag 540
gatgtctttt tcctcgtcgg tggtggcgtt atcaggcagt tctgggatgt cgtagccctg 600
gtcctgcagt tccttaatag cagccttgag ctgtggaaca gaagcggaga tgtttggaag 660
cttaatgatg tttgcttcag gagtcttagc aagctcgccg agttctgcga gtgcgttgcc 720
taccttctga tcttcggtga ggcgctctgg gaactgggcg aggatgcgtc cagcgagtga 780
aatgtcccgg gtctcgacct caatgcccgc ggtagcagca aatgcctcga cgaccggctt 840
cagcgagtag gtcgcgagca gcggtgcttc gtcggtgcgg gtccagatga tcttagccac 900
gagtctcctt ggttgatggg ctcttttgtt cagctaccta gaatacagtg ttctactaat 960
tgctggcgcc tatggtggca gtttcagccc cgcatggaaa cgccgtgttt ccaatacccc 1020
atgaatgcca cttgggaacg gtcgaggcct ttctctttca ctagagatcg acgaatgctg 1080
gtcacgacac cactttcgcc agcaatccag tagtagtcgc ctgaacgtgg gttggattct 1140
gtgatctgtt cgccagtggc ggagaacaca ggagtctccc acacaagttc gccttcggag 1200
gtggctgcag cttctgtttc ttcatggtgg agggccaggg tttccatcaa caattcaccg 1260
tgcttgcggc cgttgcgggg cagccatttc acatcgatgg aggaaggtcc ctcgatggca 1320
agtgcatcgt catctgaggg gatttcgatc atggcggttc ctggaatatc ggaatctgcg 1380
aggtcttcga attcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc 1440
acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 1500
gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 1560
tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 1620
cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 1680
gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 1740
aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 1800
gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 1860
aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 1920
gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 1980
ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 2040
cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 2100
ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 2160
actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 2220
tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca 2280
gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 2340
ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 2400
cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 2460
ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttggggtg ggcgaagaac 2520
tccagcatga gatccccgcg ctggaggatc atccagccct gatagaaaca gaagccactg 2580
gagcacctca aaaacaccat catacactaa atcagtaagt tggcagcatc acccgacgca 2640
ctttgcgccg aataaatacc tgtgacggaa gatcacttcg cagaataaat aaatcctggt 2700
gtccctgttg ataccgggaa gccctgggcc aacttttggc gaaaatgaga cgttgatcgg 2760
cacgtaagag gttccaactt tcaccataat gaaataagat cactaccggg cgtatttttt 2820
gagttatcga gattttcagg agctgataga aacagaagcc actggagcac ctcaaaaaca 2880
ccatcataca ctaaatcagt aagttggcag catcacccga cgcactttgc gccgaataaa 2940
tacctgtgac ggaagatcac ttcgcagaat aaataaatcc tggtgtccct gttgataccg 3000
ggaagccctg ggccaacttt tggcgaaaat gagacgttga tcggcacgta agaggttcca 3060
actttcacca taatgaaata agatcactac cgggcgtatt ttttgagtta tcgagatttt 3120
caggagctct ttggcatcgt ctctcgcctg tcccctcagt tcagtaattt cctgcatttg 3180
cctgtttcca gtcggtagat attccacaaa acagcaggga agcagcgctt ttccgctgca 3240
taaccctgct tcggggtcat tatagcgatt ttttcggtat atccatcctt tttcgcacga 3300
tatacaggat tttgccaaag ggttcgtgta gactttcctt ggtgtatcca acggcgtcag 3360
cggggcagga taggtgaagt aggcccaccc gcgagcgggt gttccttctt cactgtccct 3420
tattcgcacc tggcggtgct caacgggaat cctgctctgc gaggctggcc ggctaccgcc 3480
ggcgtaacag atgagggcaa gcggatggct gatgaaacca agccaaccag gaagggcagc 3540
ccacctatca aggtgtactg ccttccagac gaacgaagag cgattgagga aaaggcggcg 3600
gcggccggca tgagcctgtc ggcctacctg ctggccgtcg gccagggcta caaaatcacg 3660
ggcgtcgtgg actatgagca cgtccgcgag ggcgtcccgg aaaacgattc cgaagcccaa 3720
cctttcatag aaggcggcgg tggaatcgaa atctcgtgat ggcaggttgg gcgtcgcttg 3780
gtcggtcatt tcgctcggta ccatcggcat tttcttttgc gtttttattt gttaactgtt 3840
aattgtcctt gttcaaggat gctgtctttg acaacagatg ttttcttgcc tttgatgttc 3900
agcaggaagc tcggcgcaaa cgttgattgt ttgtctgcgt agaatcctct gtttgtcata 3960
tagcttgtaa tcacgacatt gtttcctttc gcttgaggta cagcgaagtg tgagtaagta 4020
aaggttacat cgttaggatc aagatccatt tttaacacaa ggccagtttt gttcagcggc 4080
ttgtatgggc cagttaaaga attagaaaca taaccaagca tgtaaatatc gttagacgta 4140
atgccgtcaa tcgtcatttt tgatccgcgg gagtcagtga acaggtacca tttgccgttc 4200
attttaaaga cgttcgcgcg ttcaatttca tctgttactg tgttagatgc aatcagcggt 4260
ttcatcactt ttttcagtgt gtaatcatcg tttagctcaa tcataccgag agcgccgttt 4320
gctaactcag ccgtgcgttt tttatcgctt tgcagaagtt tttgactttc ttgacggaag 4380
aatgatgtgc ttttgccata gtatgctttg ttaaataaag attcttcgcc ttggtagcca 4440
tcttcagttc cagtgtttgc ttcaaatact aagtatttgt ggcctttatc ttctacgtag 4500
tgaggatctc tcagcgtatg gttgtcgcct gagctgtagt tgccttcatc gatgaactgc 4560
tgtacatttt gatacgtttt tccgtcaccg tcaaagattg atttataatc ctctacaccg 4620
ttgatgttca aagagctgtc tgatgctgat acgttaactt gtgcagttgt cagtgtttgt 4680
ttgccgtaat gtttaccgga gaaatcagtg tagaataaac ggatttttcc gtcagatgta 4740
aatgtggctg aacctgacca ttcttgtgtt tggtctttta ggatagaatc atttgcatcg 4800
aatttgtcgc tgtctttaaa gacgcggcca gcgtttttcc agctgtcaat agaagtttcg 4860
ccgacttttt gatagaacat gtaaatcgat gtgtcatccg catttttagg atctccggct 4920
aatgcaaaga cgatgtggta gccgtgatag tttgcgacag tgccgtcagc gttttgtaat 4980
ggccagctgt cccaaacgtc caggcctttt gcagaagaga tatttttaat tgtggacgaa 5040
tcaaattcag aaacttgata tttttcattt ttttgctgtt cagggatttg cagcatatca 5100
tggcgtgtaa tatgggaaat gccgtatgtt tccttatatg gcttttggtt cgtttctttc 5160
gcaaacgctt gagttgcgcc tcctgccagc agtgcggtag taaaggttaa tactgttgct 5220
tgttttgcaa actttttgat gttcatcgtt catgtctcct tttttatgta ctgtgttagc 5280
ggtctgcttc ttccagccct cctgtttgaa gatggcaagt tagttacgca caataaaaaa 5340
agacctaaaa tatgtaaggg gtgacgccaa agtatacact ttgcccttta cacattttag 5400
gtcttgcctg ctttatcagt aacaaacccg cgcgatttac ttttcgacct cattctatta 5460
gactctcgtt tggattgcaa ctggtctatt ttcctctttt gtttgataga aaatcataaa 5520
aggatttgca gactacgggc ctaaagaact aaaaaatcta tctgtttctt ttcattctct 5580
gtatttttta tagtttctgt tgcatgggca taaagttgcc tttttaatca caattcagaa 5640
aatatcataa tatctcattt cactaaataa tagtgaacgg caggtatatg tgatgggtta 5700
aaaaggatcg atcctctagc gaaccccaga gtcccgctca gaagaactcg tcaagaaggc 5760
gatagaaggc gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg aggaagcggt 5820
cagcccattc gccgccaagc tcttcagcaa tatcacgggt agccaacgct atgtcctgat 5880
agcggtccgc cacacccagc cggccacagt cgatgaatcc agaaaagcgg ccattttcca 5940
ccatgatatt cggcaagcag gcatcgccat gggtcacgac gagatcctcg ccgtcgggca 6000
tccgcgcctt gagcctggcg aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca 6060
gatcatcctg atcgacaaga ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt 6120
tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt atgcagccgc cgcattgcat 6180
cagccatgat ggatactttc tcggcaggag caaggtgaga tgacaggaga tcctgccccg 6240
gcacttcgcc caatagcagc cagtcccttc ccgcttcagt gacaacgtcg agcacagctg 6300
cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc tgcctcgtct tggagttcat 6360
tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc 6420
ggaacacggc ggcatcagag cagccgattg tctgttgtgc ccagtcatag ccgaatagcc 6480
tctccaccca agcggccgga gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg 6540
atcctcatcc tgtctcttga tcagatcttg atcccctgcg ccatcagatc cttggcggca 6600
agaaagccat ccagtttact ttgcagggct tcccaacctt accagagggc gccccagctg 6660
Claims (9)
1.一种构建L-肌氨酸生产菌的方法,其包括以下步骤:
A. 以谷氨酸棒杆菌ATCC13032为出发菌株,在基因组中整合NADPH-NAD+转氢酶的编码基因pntAB,得到过表达pntAB的菌株A;
B. 在菌株A的基因组中整合恶臭假单胞菌KT2440(Pseudomonas putida KT2440)来源的亚胺还原酶突变体DpkAF117L编码基因,得到过表达DpkAF117L的菌株B;
C. 敲除菌株B基因组中的基因glcB和alaT,得到glcB和alaT缺失的菌株C;
D. 将菌株C基因组中的异柠檬酸脱氢酶icd基因起始密码子由ATG变为GTG,筛选阳性克隆,得到L-肌氨酸生产菌,其中
所述基因pntAB是大肠杆菌来源的pntAB。
2.如权利要求1所述的方法,其特征在于,步骤A中基因pntAB的整合和步骤C中基因glcB的敲除包括下述步骤:将核苷酸序列为SEQ ID NO: 1的质粒pK18mobsacB-glc-pntAB导入宿主细胞中;进行SacB蔗糖反筛。
3.如权利要求1所述的方法,其特征在于,步骤B中基因DpkAF117L的整合和步骤C中基因alaT的敲除包括下述步骤:将核苷酸序列为SEQ ID NO: 3的质粒pK18mobsacB- alaT-dpkA导入宿主细胞中;进行SacB蔗糖反筛。
4.如权利要求1所述的方法,其特征在于,步骤D中异柠檬酸脱氢酶icd基因起始密码子由ATG变为GTG的包括下述步骤:将核苷酸序列为SEQ ID NO: 4的质粒pK18mobsacB- icd导入宿主细胞中;进行SacB蔗糖反筛。
5.一种L-肌氨酸生产菌,其特征在于,通过如权利要求1-4中任一项所述的方法构建得到。
6.如权利要求5所述L-肌氨酸生产菌,其为谷氨酸棒杆菌(Corynebacterium glutamicum),保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCCNo. 22265。
7.如权利要求6所述L-肌氨酸生产菌用于生产L-肌氨酸的用途。
8.如权利要求7所述的用途,其特征在于,通过如权利要求6所述L-肌氨酸生产菌CGMCCNo. 22265的发酵来生产L-肌氨酸。
9.如权利要求8所述的用途,其特征在于,发酵培养基组成如下:葡萄糖 100g/L,硫酸镁 100mg/L,酵母粉 35g/L,硫酸铵 20g/L,FeSO4•7H2O 11mg/L,柠檬酸钠 10mg/L,85%H3PO4 250μl/L,消泡剂204 500mg/L,泛酸钙 60mg/L,维生素PP 18mg/L,thiamin HCl15mg/L,生物素 15mg/L,pH 7.0;发酵过程中流加甲胺。
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