CN111235084B - 重组大肠杆菌工程菌及利用其制备s-腺苷甲硫氨酸的方法 - Google Patents

重组大肠杆菌工程菌及利用其制备s-腺苷甲硫氨酸的方法 Download PDF

Info

Publication number
CN111235084B
CN111235084B CN202010250598.5A CN202010250598A CN111235084B CN 111235084 B CN111235084 B CN 111235084B CN 202010250598 A CN202010250598 A CN 202010250598A CN 111235084 B CN111235084 B CN 111235084B
Authority
CN
China
Prior art keywords
val
leu
ala
gly
asp
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202010250598.5A
Other languages
English (en)
Other versions
CN111235084A (zh
Inventor
周晶辉
许岗
黄斌
曾红宇
刘洋
田艳
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hunan Flag Biological Technology Co ltd
Original Assignee
Hunan Flag Biological Technology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hunan Flag Biological Technology Co ltd filed Critical Hunan Flag Biological Technology Co ltd
Priority to CN202010250598.5A priority Critical patent/CN111235084B/zh
Publication of CN111235084A publication Critical patent/CN111235084A/zh
Application granted granted Critical
Publication of CN111235084B publication Critical patent/CN111235084B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1229—Phosphotransferases with a phosphate group as acceptor (2.7.4)
    • 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
    • C12P13/12—Methionine; Cysteine; Cystine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01006—Methionine adenosyltransferase (2.5.1.6), i.e. adenosylmethionine synthetase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/0102—Adenosine kinase (2.7.1.20)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/04—Phosphotransferases with a phosphate group as acceptor (2.7.4)
    • C12Y207/04001—Polyphosphate kinase (2.7.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/04—Phosphotransferases with a phosphate group as acceptor (2.7.4)
    • C12Y207/04003—Adenylate kinase (2.7.4.3)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明属于酶工程技术领域，涉及重组大肠杆菌工程菌及利用其制备S‑腺苷甲硫氨酸的方法。所述的重组大肠杆菌工程菌含有重组表达载体，所述的重组表达载体含有表达S‑腺苷甲硫氨酸合成酶的基因和表达ATP合成酶的基因。相比传统的发酵法和体外酶催化合成方法，利用本发明的重组大肠杆菌工程菌及利用其制备S‑腺苷甲硫氨酸的方法，能够更简单、更低成本、高产率的制备S‑腺苷甲硫氨酸。

Description

重组大肠杆菌工程菌及利用其制备S-腺苷甲硫氨酸的方法
技术领域
本发明属于酶工程技术领域，涉及重组大肠杆菌工程菌及利用其制备S-腺苷甲硫氨酸的方法。
背景技术
S-腺苷甲硫氨酸(S-adenosyl-L-methionine，简称SAM)作为生物体代谢过程的重要中间产物，存在于所有活细胞中，具有转甲基、转硫、转氨丙基作用。SAM是双手性物质，有2种异构体：(R，S)-SAM和(S，S)-SAM，只有(S，S)-SAM才具有生物活性。临床上SAM具有极高的药用价值，主要用于治疗肝内胆汁淤积，还可用于治疗病毒性肝炎、酒精性肝病，改善肝脏功能，缓解抑郁症患者的抑郁症状(具有抗抑郁作用)等。SAM还可与L-多巴联用于治疗帕金森氏病，能提高L-多巴疗效和降低副作用。SAM的市售产品如思美泰。
在生物体内，SAM是由S-腺苷甲硫氨酸合成酶(S-Adenosylmethioninesynthetase，EC 2.5.1.6)催化腺苷三磷酸(ATP)和甲硫氨酸(Met)反应合成，原理见图1。体外S-腺苷甲硫氨酸的制备主要有三种方法：化学合成法、微生物发酵法、酶法催化法。
化学法合成法主要是采用S-同型半胱氨酸和甲基供体合成，存在杂质多、收率低、产物构型复杂等缺陷而未能大规模应用。
微生物发酵法是目前应用最为广泛、成本相对低的一种方法，其主要是利用酵母细胞体内的S-腺苷甲硫氨酸合成酶，通过在培养液中添加前体物质L-甲硫氨酸在酵母细胞体内进行SAM累积合成。该方法的弊端是：SAM积累主要在酵母细胞内，分离纯化过程需要进行细胞壁破碎，工序复杂，同时依然存在着产量低、收率低等问题。
酶法催化法主要是在S-腺苷甲硫氨酸合成酶的催化作用下，利用ATP和L-甲硫氨酸合成SAM。该法主要受限于高催化活性的S-腺苷甲硫氨酸合成酶的获得及ATP的高成本(600-700元/kg)，而且在体外催化过程中，S-腺苷甲硫氨酸合成酶非常不稳定容易失活，因此使得酶法催化合成成本远远高于微生物发酵法，目前还没有该法工业应用的报道。
发明内容
本发明的首要目的是提供一种重组大肠杆菌工程菌，以能够利用其结合微生物发酵法和酶法催化法各自的优点，避免各自的缺点，更简单、低成本、高产率的制备S-腺苷甲硫氨酸。
为实现此目的，在基础的实施方案中，本发明提供一种重组大肠杆菌工程菌，所述的重组大肠杆菌工程菌含有重组表达载体，所述的重组表达载体含有表达S-腺苷甲硫氨酸合成酶的基因和表达ATP合成酶的基因。
在一种优选的实施方案中，本发明提供一种重组大肠杆菌工程菌，其中所述的S-腺苷甲硫氨酸合成酶是SEQ ID NO.1所示氨基酸序列的野生型S-腺苷甲硫氨酸合成酶中突变多个氨基酸位点的S-腺苷甲硫氨酸合成酶突变体，突变的多个氨基酸位点包括C9R与K224R。
在一种更加优选的实施方案中，本发明提供一种重组大肠杆菌工程菌，其中所述的S-腺苷甲硫氨酸合成酶突变体的多个氨基酸突变位点还包括T163S、Q191R、A218T中的一个或多个。
在一种更加优选的实施方案中，本发明提供一种重组大肠杆菌工程菌，其中所述的S-腺苷甲硫氨酸合成酶突变体的氨基酸序列如SEQ ID NO.2-8之一所示。
在一种优选的实施方案中，本发明提供一种重组大肠杆菌工程菌，其中所述的ATP合成酶包括腺苷激酶(EC 2.7.1.20，AK，催化腺苷生成AMP)、腺苷酸激酶(EC 2.7.4.3，ADK，催化AMP与ATP反应生成ADP)和/或多聚磷酸激酶(EC 2.7.4.1，PPK，催化ADP与多聚磷酸反应生成ATP)。
在一种更加优选的实施方案中，本发明提供一种重组大肠杆菌工程菌，其中所述的腺苷激酶的氨基酸序列如SEQ ID NO.9所示，所述的腺苷酸激酶的氨基酸序列如SEQ IDNO.10所示，所述的多聚磷酸激酶的氨基酸序列如SEQ ID NO.11所示。
在一种优选的实施方案中，本发明提供一种重组大肠杆菌工程菌，其中所述的大肠杆菌工程菌选自BL21(DE3)、BL21(DE3)pLysS、Rosetta(DE3)、Arctic Express(DE3)、Origami B(DE3)中的一种。
在一种优选的实施方案中，本发明提供一种重组大肠杆菌工程菌，其中所述的表达载体是质粒载体，选自pET23b、pET24a、pET28a、pET30a、pET32a、pCDFDuet-1、pET15b、pET21a、pETDuet-1、pACYC184、pTXB1、pTYB21。
在一种优选的实施方案中，本发明提供一种重组大肠杆菌工程菌，其中所述的表达S-腺苷甲硫氨酸合成酶的基因和所述的表达ATP合成酶的基因位于同一或不同的重组表达载体内，且当位于不同的重组表达载体内时不同的重组表达载体之间是兼容的。
在一种优选的实施方案中，本发明提供一种重组大肠杆菌工程菌，其中所述的表达S-腺苷甲硫氨酸合成酶的基因位于重组质粒载体pET30a内，所述的表达ATP合成酶的基因位于重组质粒载体pCDFDuet-1内。
本发明的第二个目的是提供一种利用前述重组大肠杆菌工程菌制备S-腺苷甲硫氨酸的方法，以能够利用前述重组大肠杆菌工程菌，结合微生物发酵法和酶法催化法各自的优点，避免各自的缺点，更简单、低成本、高产率的制备S-腺苷甲硫氨酸。
为实现此目的，在基础的实施方案中，本发明提供一种利用前述重组大肠杆菌工程菌制备S-腺苷甲硫氨酸的方法，所述的方法包括如下步骤：
(1)发酵所述的重组大肠杆菌工程菌；
(2)发酵后离心收集菌体并用缓冲液重悬菌体；
(3)在菌体重悬液中先加入ATP合成酶催化反应所需的反应物进行反应，再加入S-腺苷甲硫氨酸合成酶催化反应所需的反应物进行反应。
在一种优选的实施方案中，本发明提供一种利用前述重组大肠杆菌工程菌制备S-腺苷甲硫氨酸的方法，其中步骤(2)中，所述的缓冲液为pH6.0-8.0的磷酸盐缓冲液。
在一种优选的实施方案中，本发明提供一种利用前述重组大肠杆菌工程菌制备S-腺苷甲硫氨酸的方法，其中步骤(2)中，在重悬菌体的同时还加入曲拉通X-100和/或EDTA以对菌体进行通透化处理。
在一种更加优选的实施方案中，本发明提供一种利用前述重组大肠杆菌工程菌制备S-腺苷甲硫氨酸的方法，其中所述的曲拉通X-100、EDTA加入后对于10OD菌体量的浓度分别为5-10mM、2-5mM。
在一种优选的实施方案中，本发明提供一种利用前述重组大肠杆菌工程菌制备S-腺苷甲硫氨酸的方法，其中步骤(3)中，
加入的所述的ATP合成酶催化反应所需的反应物包括腺苷、ATP、ADP、AMP、多聚磷酸盐、镁盐，加入后温度25-30℃、pH 5.0-6.5、搅拌转速150-200r/min下反应30-180min；
加入的所述的S-腺苷甲硫氨酸合成酶催化反应所需的反应物包括L-甲硫氨酸、镁盐、钾盐、磺酸盐，加入后温度25-30℃、pH 7.5-8.0、搅拌转速150-200r/min下继续反应60-240min。
在一种更加优选的实施方案中，本发明提供一种利用前述重组大肠杆菌工程菌制备S-腺苷甲硫氨酸的方法，其中：
所述的腺苷、ATP、ADP、AMP、多聚磷酸盐、镁盐加入后对于20OD菌体量的浓度分别为50-300mM、1-10mM、10-50mM、10-50mM、500-800mM、5-10mM；
所述的L-甲硫氨酸、镁盐、钾盐、磺酸盐加入后对于20OD菌体量的浓度分别为200-500mM、100-250mM、50-100mM、300-500mM。
本发明的有益效果在于，利用本发明的重组大肠杆菌工程菌及利用其制备S-腺苷甲硫氨酸的方法，能够结合微生物发酵法和酶法催化法各自的优点，避免各自的缺点，更简单、低成本、高产率的制备S-腺苷甲硫氨酸。
本发明的重组大肠杆菌工程菌可高效、高酶活的共表达S-腺苷甲硫氨酸合成酶与ATP合成酶，且ATP合成酶的表达使得制备S-腺苷甲硫氨酸无需添加ATP或只需添加少量的ATP，使得ATP成本大大降低，使得制备过程更高效、更低成本、更适用于工业化应用。
利用本发明的重组大肠杆菌工程菌制备S-腺苷甲硫氨酸发酵周期短(<24h)，发酵所得菌体无需破碎，只需进行简单的通透化处理便可利用；反应产物S-腺苷甲硫氨酸能有效分泌至细胞外，便于进一步的检测与分离纯化。
附图说明
图1为S-腺苷甲硫氨酸的合成原理图。
图2为三基因共表达重组质粒载体pCDFDuet-1-AK-ADK-PPK的构建流程图。
具体实施方式
以下结合实施例和附图对本发明的具体实施方式作出进一步的说明。
实施例1：S-腺苷甲硫氨酸合成酶表达菌株的构建
1、表达菌株BL21(DE3)/pET30a-SUMO-MATI的构建
同时下载GenBank中大鼠肝脏来源的S-腺苷甲硫氨酸合成酶MATI的氨基酸序列(本文SEQ ID NO.1，对应GenBank登陆号：NP_036992.2)及小分子泛素样修饰蛋白(SUMO)的氨基酸序列(本文SEQ ID NO.12，对应GenBank登陆号：AQS95516.1)。将SUMO氨基酸序列连接至MATI蛋白氨基酸序列N-端，形成融合蛋白序列，提供给北京擎科生物技术有限公司进行编码核酸的全基因合成(采用大肠杆菌优选密码子)。构建至原核表达载体pET30a中，形成重组质粒载体pET30a-SUMO-MATI。采用热激法转化重组质粒载体pET30a-SUMO-MATI至大肠杆菌菌株BL21(DE3)中，涂布于含有50μg/ml卡那霉素的LB固体培养基平板上，37℃过夜培养，平板上生长出的菌落即为S-腺苷甲硫氨酸合成酶表达菌株BL21(DE3)/pET30a-SUMO-MATI。
2、表达菌株BL21(DE3)/pET30a-SUMO-MATI-1、
BL21(DE3)/pET30a-SUMO-MATI-2A、
BL21(DE3)/pET30a-SUMO-MATI-2B、
BL21(DE3)/pET30a-SUMO-MATI-2C、
BL21(DE3)/pET30a-SUMO-MATI-3A、
BL21(DE3)/pET30a-SUMO-MATI-3B、BL21(DE3)/pET30a-SUMO-MATI-4的构建
以上述重组质粒载体pET30a-SUMO-MATI为模板，选择MATI氨基酸序列第9位、224位，设计定点突变引物，进行全质粒PCR反应，得到重组质粒载体pET30a-SUMO-MATI-1。
以重组质粒载体pET30a-SUMO-MATI-1为模板，选择MATI-1氨基酸序列第163位、191位和/或218位，分别设计定点突变引物，进行全质粒PCR反应，分别得到重组质粒载体pET30a-SUMO-MATI-2A、
pET30a-SUMO-MATI-2B、pET30a-SUMO-MATI-2C、
pET30a-SUMO-MATI-3A、pET30a-SUMO-MATI-3B、
pET30a-SUMO-MATI-4。
MATI-1(在MATI基础上突变C9R，K224R)、MATI-2A(在MATI基础上突变C9R，K224R，T163S)、MATI-2B(在MATI基础上突变C9R，K224R，Q191R)、MATI-2C(在MATI基础上突变C9R，K224R，A218T)、MATI-3A(在MATI基础上突变C9R，K224R，A218T，T163S)、MATI-3B(在MATI基础上突变C9R，K224R，A218T，Q191R)、MATI-4(在MATI基础上突变C9R，K224R，A218T，T163S，Q191R)的氨基酸序列分别如SEQ ID NO.2-8所示。
以下按与如上第1步相类似的方法构建表达菌株
BL21(DE3)/pET30a-SUMO-MATI-1、BL21(DE3)/pET30a-SUMO-MATI-2A、
BL21(DE3)/pET30a-SUMO-MATI-2B、
BL21(DE3)/pET30a-SUMO-MATI-2C、
BL21(DE3)/pET30a-SUMO-MATI-3A、
BL21(DE3)/pET30a-SUMO-MATI-3B、BL21(DE3)/pET30a-SUMO-MATI-4。
实施例2：ATP合成酶重组表达载体的构建
将ATP合成相关酶的氨基酸序列(其中腺苷激酶(EC 2.7.1.20，AK)的氨基酸序列见SEQ ID NO.9；腺苷酸激酶(EC 2.7.4.3，ADK)的氨基酸序列见SEQ ID NO.10；多聚磷酸激酶(EC 2.7.4.1，PPK)的氨基酸序列见SEQ ID NO.11)提供给北京擎科生物技术有限公司进行编码核酸的全基因合成(采用大肠杆菌优选密码子)。构建至原核表达载体pCDFDuet-1中，形成三基因共表达重组质粒载体pCDFDuet-1-AK-ADK-PPK(构建流程图见图2)。在pCDFDuet-1-AK-ADK-PPK中，腺苷激酶AK和腺苷酸激酶ADK的表达基因构建至pCDFDuet-1的同一个阅读框中形成双基因串联表达，多聚磷酸激酶PPK的表达基因则构建至pCDFDuet-1的另一个阅读框中进行表达。
实施例3：S-腺苷甲硫氨酸合成酶与ATP合成酶共表达菌株的构建
以BL21(DE3)/pET30a-SUMO-MATI作为出发菌株，制备化学感受态细胞，采用氯化钙热激转化方法将三基因共表达重组质粒载体pCDFDuet-1-AK-ADK–PPK转入感受态细胞BL21(DE3)/pET30a-SUMO-MATI中，37℃，220r/min，培养60min后，将转化后的细胞涂布于同时添加了硫酸卡那霉素(50μg/mL)和链霉素(25μg/mL)的固体LB培养基中进行阳性克隆筛选，平板中生长出来的菌落即为S-腺苷甲硫氨酸合成酶MATI与ATP合成酶的共表达菌株BL21(DE3)/pET30a-SUMO-MATI/pCDFDuet-1-AK-ADK-PPK。
同理构建：
S-腺苷甲硫氨酸合成酶MATI-1与ATP合成酶的共表达菌株BL21(DE3)/pET30a-SUMO-MATI-1/pCDFDuet-1-AK-ADK-PPK，
S-腺苷甲硫氨酸合成酶MATI-2A与ATP合成酶的共表达菌株BL21(DE3)/pET30a-SUMO-MATI-2A/pCDFDuet-1-AK-ADK-PPK，
S-腺苷甲硫氨酸合成酶MATI-2B与ATP合成酶的共表达菌株BL21(DE3)/pET30a-SUMO-MATI-2B/pCDFDuet-1-AK-ADK-PPK，
S-腺苷甲硫氨酸合成酶MATI-2C与ATP合成酶的共表达菌株BL21(DE3)/pET30a-SUMO-MATI-2C/pCDFDuet-1-AK-ADK-PPK，
S-腺苷甲硫氨酸合成酶MATI-3A与ATP合成酶的共表达菌株BL21(DE3)/pET30a-SUMO-MATI-3A/pCDFDuet-1-AK-ADK-PPK，
S-腺苷甲硫氨酸合成酶MATI-3B与ATP合成酶的共表达菌株BL21(DE3)/pET30a-SUMO-MATI-3B/pCDFDuet-1-AK-ADK-PPK，
S-腺苷甲硫氨酸合成酶MATI-4与ATP合成酶的共表达菌株BL21(DE3)/pET30a-SUMO-MATI-4/pCDFDuet-1-AK-ADK-PPK。
实施例4：S-腺苷甲硫氨酸合成酶与ATP合成酶共表达菌株的发酵与菌体预处理
1、BL21(DE3)/pET30a-SUMO-MATI/pCDFDuet-1-AK-ADK-PPK共表达菌株的发酵
用灭菌枪头在LB固体培养基平板中小心挑取共表达菌株BL21(DE3)/pET30a-SUMO-MATI/pCDFDuet-1-AK-ADK-PPK的单菌落，接种至LB液体培养基中(添加终浓度50μg/mL的硫酸卡那霉素与25μg/mL的链霉素)，37℃，220r/min，振荡培养12-16h。然后按照1-2％接种量将LB培养基中的种子液接种至装有100mL TB液体培养基的发酵摇瓶中(500mL容量)，37℃，220r/min继续培养，并每隔1h检测细胞OD值，待细胞OD＝1.5-2.0时，补加终浓度为1％(m/v)的乳糖，25℃，220r/min继续培养10-12h。10000r/min离心5min收集菌体。
2、BL21(DE3)/pET30a-SUMO-MATI/pCDFDuet-1-AK-ADK-PPK菌体预处理
将收集菌体用含有5-10mM的曲拉通X-100，2-5mM的EDTA的0.1mol/L，pH 8.0磷酸盐缓冲液重悬，使重悬菌浓OD＝10，20-25℃，50-100r/min，振荡30-60min后，10000r/min离心5min，收集菌体备用。
用同样的方法发酵与菌体预处理：
S-腺苷甲硫氨酸合成酶MATI-1与ATP合成酶的共表达菌株BL21(DE3)/pET30a-SUMO-MATI-1/pCDFDuet-1-AK-ADK-PPK，
S-腺苷甲硫氨酸合成酶MATI-2A与ATP合成酶的共表达菌株BL21(DE3)/pET30a-SUMO-MATI-2A/pCDFDuet-1-AK-ADK-PPK，
S-腺苷甲硫氨酸合成酶MATI-2B与ATP合成酶的共表达菌株BL21(DE3)/pET30a-SUMO-MATI-2B/pCDFDuet-1-AK-ADK-PPK，
S-腺苷甲硫氨酸合成酶MATI-2C与ATP合成酶的共表达菌株BL21(DE3)/pET30a-SUMO-MATI-2C/pCDFDuet-1-AK-ADK-PPK，
S-腺苷甲硫氨酸合成酶MATI-3A与ATP合成酶的共表达菌株BL21(DE3)/pET30a-SUMO-MATI-3A/pCDFDuet-1-AK-ADK-PPK，
S-腺苷甲硫氨酸合成酶MATI-3B与ATP合成酶的共表达菌株BL21(DE3)/pET30a-SUMO-MATI-3B/pCDFDuet-1-AK-ADK-PPK，
S-腺苷甲硫氨酸合成酶MATI-4与ATP合成酶的共表达菌株BL21(DE3)/pET30a-SUMO-MATI-4/pCDFDuet-1-AK-ADK-PPK。
实施例5：S-腺苷甲硫氨酸的制备
ATP合成反应：在5L反应罐中，用0.1mol/L，pH 6.3磷酸盐缓冲液重悬菌体，并使菌浓OD＝20。在重悬菌体中加入如下反应物：腺苷、ATP、ADP、AMP、多聚磷酸盐、镁盐，设置温度、反应体系pH(反应过程中维持体系pH稳定)、搅拌转速、反应时间进行反应。
SAM合成反应：进一步加入如下反应物：L-甲硫氨酸、镁盐、钾盐、磺酸盐，设置温度、反应体系pH(反应过程中维持体系pH稳定)、搅拌转速、反应时间进一步进行反应。
反应终止后，离心取上清样用HPLC进行产物S-腺苷甲硫氨酸浓度测定。HPLC具体测定条件如下：
色谱柱：Diamonsil C18(250mm×4.6mm，5μm)
流动相：6.8g KH2PO4溶于1000ml水中，用H3PO4调pH至2.5，取出950ml加入50ml甲醇即得。
标准液：精确称取S-腺苷甲硫氨酸标准品20-25mg，用流动相溶解并定容到100ml容量瓶中，摇匀后过滤。
检测温度：25℃
检测流速：1.0ml/min
检测波长：210nm
进样量：20μl
各总反应具体反应条件(各总反应的ATP合成反应、SAM合成反应的反应体积分别相等，且各总反应的菌体用量相同)及检测结果如下表1-5所示。
表1各S-腺苷甲硫氨酸制备反应的部分反应条件(ATP合成反应，一)
Figure BDA0002435345390000101
表2各S-腺苷甲硫氨酸制备反应的部分反应条件(ATP合成反应，二)
Figure BDA0002435345390000102
Figure BDA0002435345390000111
表3各S-腺苷甲硫氨酸制备反应的部分反应条件(SAM合成反应，一)
Figure BDA0002435345390000112
Figure BDA0002435345390000121
表4各S-腺苷甲硫氨酸制备反应的部分反应条件(SAM合成反应，二)
Figure BDA0002435345390000122
表5各S-腺苷甲硫氨酸制备反应的部分反应条件及检测结果
Figure BDA0002435345390000123
Figure BDA0002435345390000131
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若对本发明的这些修改和变型属于本发明权利要求及其同等技术的范围之内，则本发明也意图包含这些改动和变型在内。上述实施例或实施方式只是对本发明的举例说明，本发明也可以以其它的特定方式或其它的特定形式实施，而不偏离本发明的要旨或本质特征。因此，描述的实施方式从任何方面来看均应视为说明性而非限定性的。本发明的范围应由附加的权利要求说明，任何与权利要求的意图和范围等效的变化也应包含在本发明的范围内。
序列表
<110> 湖南福来格生物技术有限公司
<120> 重组大肠杆菌工程菌及利用其制备S-腺苷甲硫氨酸的方法
<130> -
<141> 2020-04-01
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 398
<212> PRT
<213> E.coli
<400> 1
Met Asn Gly Pro Val Asp Gly Leu Cys Asp His Ser Leu Ser Glu Glu
1 5 10 15
Gly Ala Phe Met Phe Thr Ser Glu Ser Val Gly Glu Gly His Pro Asp
20 25 30
Lys Ile Cys Asp Gln Ile Ser Asp Ala Val Leu Asp Ala His Leu Lys
35 40 45
Gln Asp Pro Asn Ala Lys Val Ala Cys Glu Thr Val Cys Lys Thr Gly
50 55 60
Met Val Leu Leu Cys Gly Glu Ile Thr Ser Met Ala Met Ile Asp Tyr
65 70 75 80
Gln Arg Val Val Arg Asp Thr Ile Lys His Ile Gly Tyr Asp Asp Ser
85 90 95
Ala Lys Gly Phe Asp Phe Lys Thr Cys Asn Val Leu Val Ala Leu Glu
100 105 110
Gln Gln Ser Pro Asp Ile Ala Gln Cys Val His Leu Asp Arg Asn Glu
115 120 125
Glu Asp Val Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr
130 135 140
Asp Glu Thr Glu Glu Cys Met Pro Leu Thr Ile Val Leu Ala His Lys
145 150 155 160
Leu Asn Thr Arg Met Ala Asp Leu Arg Arg Ser Gly Val Leu Pro Trp
165 170 175
Leu Arg Pro Asp Ser Lys Thr Gln Val Thr Val Gln Tyr Val Gln Asp
180 185 190
Asn Gly Ala Val Ile Pro Val Arg Val His Thr Ile Val Ile Ser Val
195 200 205
Gln His Asn Glu Asp Ile Thr Leu Glu Ala Met Arg Glu Ala Leu Lys
210 215 220
Glu Gln Val Ile Lys Ala Val Val Pro Ala Lys Tyr Leu Asp Glu Asp
225 230 235 240
Thr Ile Tyr His Leu Gln Pro Ser Gly Arg Phe Val Ile Gly Gly Pro
245 250 255
Gln Gly Asp Ala Gly Val Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr
260 265 270
Gly Gly Trp Gly Ala His Gly Gly Gly Ala Phe Ser Gly Lys Asp Tyr
275 280 285
Thr Lys Val Asp Arg Ser Ala Ala Tyr Ala Ala Arg Trp Val Ala Lys
290 295 300
Ser Leu Val Lys Ala Gly Leu Cys Arg Arg Val Leu Val Gln Val Ser
305 310 315 320
Tyr Ala Ile Gly Val Ala Glu Pro Leu Ser Ile Ser Ile Phe Thr Tyr
325 330 335
Gly Thr Ser Lys Lys Thr Glu Arg Glu Leu Leu Glu Val Val Asn Lys
340 345 350
Asn Phe Asp Leu Arg Pro Gly Val Ile Val Arg Asp Leu Asp Leu Lys
355 360 365
Lys Pro Ile Tyr Gln Lys Thr Ala Cys Tyr Gly His Phe Gly Arg Ser
370 375 380
Glu Phe Pro Trp Glu Val Pro Lys Lys Leu Val Phe Leu Glu
385 390 395
<210> 2
<211> 398
<212> PRT
<213> Artificial Sequence
<400> 2
Met Asn Gly Pro Val Asp Gly Leu Arg Asp His Ser Leu Ser Glu Glu
1 5 10 15
Gly Ala Phe Met Phe Thr Ser Glu Ser Val Gly Glu Gly His Pro Asp
20 25 30
Lys Ile Cys Asp Gln Ile Ser Asp Ala Val Leu Asp Ala His Leu Lys
35 40 45
Gln Asp Pro Asn Ala Lys Val Ala Cys Glu Thr Val Cys Lys Thr Gly
50 55 60
Met Val Leu Leu Cys Gly Glu Ile Thr Ser Met Ala Met Ile Asp Tyr
65 70 75 80
Gln Arg Val Val Arg Asp Thr Ile Lys His Ile Gly Tyr Asp Asp Ser
85 90 95
Ala Lys Gly Phe Asp Phe Lys Thr Cys Asn Val Leu Val Ala Leu Glu
100 105 110
Gln Gln Ser Pro Asp Ile Ala Gln Cys Val His Leu Asp Arg Asn Glu
115 120 125
Glu Asp Val Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr
130 135 140
Asp Glu Thr Glu Glu Cys Met Pro Leu Thr Ile Val Leu Ala His Lys
145 150 155 160
Leu Asn Thr Arg Met Ala Asp Leu Arg Arg Ser Gly Val Leu Pro Trp
165 170 175
Leu Arg Pro Asp Ser Lys Thr Gln Val Thr Val Gln Tyr Val Gln Asp
180 185 190
Asn Gly Ala Val Ile Pro Val Arg Val His Thr Ile Val Ile Ser Val
195 200 205
Gln His Asn Glu Asp Ile Thr Leu Glu Ala Met Arg Glu Ala Leu Arg
210 215 220
Glu Gln Val Ile Lys Ala Val Val Pro Ala Lys Tyr Leu Asp Glu Asp
225 230 235 240
Thr Ile Tyr His Leu Gln Pro Ser Gly Arg Phe Val Ile Gly Gly Pro
245 250 255
Gln Gly Asp Ala Gly Val Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr
260 265 270
Gly Gly Trp Gly Ala His Gly Gly Gly Ala Phe Ser Gly Lys Asp Tyr
275 280 285
Thr Lys Val Asp Arg Ser Ala Ala Tyr Ala Ala Arg Trp Val Ala Lys
290 295 300
Ser Leu Val Lys Ala Gly Leu Cys Arg Arg Val Leu Val Gln Val Ser
305 310 315 320
Tyr Ala Ile Gly Val Ala Glu Pro Leu Ser Ile Ser Ile Phe Thr Tyr
325 330 335
Gly Thr Ser Lys Lys Thr Glu Arg Glu Leu Leu Glu Val Val Asn Lys
340 345 350
Asn Phe Asp Leu Arg Pro Gly Val Ile Val Arg Asp Leu Asp Leu Lys
355 360 365
Lys Pro Ile Tyr Gln Lys Thr Ala Cys Tyr Gly His Phe Gly Arg Ser
370 375 380
Glu Phe Pro Trp Glu Val Pro Lys Lys Leu Val Phe Leu Glu
385 390 395
<210> 3
<211> 398
<212> PRT
<213> Artificial Sequence
<400> 3
Met Asn Gly Pro Val Asp Gly Leu Arg Asp His Ser Leu Ser Glu Glu
1 5 10 15
Gly Ala Phe Met Phe Thr Ser Glu Ser Val Gly Glu Gly His Pro Asp
20 25 30
Lys Ile Cys Asp Gln Ile Ser Asp Ala Val Leu Asp Ala His Leu Lys
35 40 45
Gln Asp Pro Asn Ala Lys Val Ala Cys Glu Thr Val Cys Lys Thr Gly
50 55 60
Met Val Leu Leu Cys Gly Glu Ile Thr Ser Met Ala Met Ile Asp Tyr
65 70 75 80
Gln Arg Val Val Arg Asp Thr Ile Lys His Ile Gly Tyr Asp Asp Ser
85 90 95
Ala Lys Gly Phe Asp Phe Lys Thr Cys Asn Val Leu Val Ala Leu Glu
100 105 110
Gln Gln Ser Pro Asp Ile Ala Gln Cys Val His Leu Asp Arg Asn Glu
115 120 125
Glu Asp Val Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr
130 135 140
Asp Glu Thr Glu Glu Cys Met Pro Leu Thr Ile Val Leu Ala His Lys
145 150 155 160
Leu Asn Ser Arg Met Ala Asp Leu Arg Arg Ser Gly Val Leu Pro Trp
165 170 175
Leu Arg Pro Asp Ser Lys Thr Gln Val Thr Val Gln Tyr Val Gln Asp
180 185 190
Asn Gly Ala Val Ile Pro Val Arg Val His Thr Ile Val Ile Ser Val
195 200 205
Gln His Asn Glu Asp Ile Thr Leu Glu Ala Met Arg Glu Ala Leu Arg
210 215 220
Glu Gln Val Ile Lys Ala Val Val Pro Ala Lys Tyr Leu Asp Glu Asp
225 230 235 240
Thr Ile Tyr His Leu Gln Pro Ser Gly Arg Phe Val Ile Gly Gly Pro
245 250 255
Gln Gly Asp Ala Gly Val Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr
260 265 270
Gly Gly Trp Gly Ala His Gly Gly Gly Ala Phe Ser Gly Lys Asp Tyr
275 280 285
Thr Lys Val Asp Arg Ser Ala Ala Tyr Ala Ala Arg Trp Val Ala Lys
290 295 300
Ser Leu Val Lys Ala Gly Leu Cys Arg Arg Val Leu Val Gln Val Ser
305 310 315 320
Tyr Ala Ile Gly Val Ala Glu Pro Leu Ser Ile Ser Ile Phe Thr Tyr
325 330 335
Gly Thr Ser Lys Lys Thr Glu Arg Glu Leu Leu Glu Val Val Asn Lys
340 345 350
Asn Phe Asp Leu Arg Pro Gly Val Ile Val Arg Asp Leu Asp Leu Lys
355 360 365
Lys Pro Ile Tyr Gln Lys Thr Ala Cys Tyr Gly His Phe Gly Arg Ser
370 375 380
Glu Phe Pro Trp Glu Val Pro Lys Lys Leu Val Phe Leu Glu
385 390 395
<210> 4
<211> 398
<212> PRT
<213> Artificial Sequence
<400> 4
Met Asn Gly Pro Val Asp Gly Leu Arg Asp His Ser Leu Ser Glu Glu
1 5 10 15
Gly Ala Phe Met Phe Thr Ser Glu Ser Val Gly Glu Gly His Pro Asp
20 25 30
Lys Ile Cys Asp Gln Ile Ser Asp Ala Val Leu Asp Ala His Leu Lys
35 40 45
Gln Asp Pro Asn Ala Lys Val Ala Cys Glu Thr Val Cys Lys Thr Gly
50 55 60
Met Val Leu Leu Cys Gly Glu Ile Thr Ser Met Ala Met Ile Asp Tyr
65 70 75 80
Gln Arg Val Val Arg Asp Thr Ile Lys His Ile Gly Tyr Asp Asp Ser
85 90 95
Ala Lys Gly Phe Asp Phe Lys Thr Cys Asn Val Leu Val Ala Leu Glu
100 105 110
Gln Gln Ser Pro Asp Ile Ala Gln Cys Val His Leu Asp Arg Asn Glu
115 120 125
Glu Asp Val Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr
130 135 140
Asp Glu Thr Glu Glu Cys Met Pro Leu Thr Ile Val Leu Ala His Lys
145 150 155 160
Leu Asn Thr Arg Met Ala Asp Leu Arg Arg Ser Gly Val Leu Pro Trp
165 170 175
Leu Arg Pro Asp Ser Lys Thr Gln Val Thr Val Gln Tyr Val Arg Asp
180 185 190
Asn Gly Ala Val Ile Pro Val Arg Val His Thr Ile Val Ile Ser Val
195 200 205
Gln His Asn Glu Asp Ile Thr Leu Glu Ala Met Arg Glu Ala Leu Arg
210 215 220
Glu Gln Val Ile Lys Ala Val Val Pro Ala Lys Tyr Leu Asp Glu Asp
225 230 235 240
Thr Ile Tyr His Leu Gln Pro Ser Gly Arg Phe Val Ile Gly Gly Pro
245 250 255
Gln Gly Asp Ala Gly Val Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr
260 265 270
Gly Gly Trp Gly Ala His Gly Gly Gly Ala Phe Ser Gly Lys Asp Tyr
275 280 285
Thr Lys Val Asp Arg Ser Ala Ala Tyr Ala Ala Arg Trp Val Ala Lys
290 295 300
Ser Leu Val Lys Ala Gly Leu Cys Arg Arg Val Leu Val Gln Val Ser
305 310 315 320
Tyr Ala Ile Gly Val Ala Glu Pro Leu Ser Ile Ser Ile Phe Thr Tyr
325 330 335
Gly Thr Ser Lys Lys Thr Glu Arg Glu Leu Leu Glu Val Val Asn Lys
340 345 350
Asn Phe Asp Leu Arg Pro Gly Val Ile Val Arg Asp Leu Asp Leu Lys
355 360 365
Lys Pro Ile Tyr Gln Lys Thr Ala Cys Tyr Gly His Phe Gly Arg Ser
370 375 380
Glu Phe Pro Trp Glu Val Pro Lys Lys Leu Val Phe Leu Glu
385 390 395
<210> 5
<211> 398
<212> PRT
<213> Artificial Sequence
<400> 5
Met Asn Gly Pro Val Asp Gly Leu Arg Asp His Ser Leu Ser Glu Glu
1 5 10 15
Gly Ala Phe Met Phe Thr Ser Glu Ser Val Gly Glu Gly His Pro Asp
20 25 30
Lys Ile Cys Asp Gln Ile Ser Asp Ala Val Leu Asp Ala His Leu Lys
35 40 45
Gln Asp Pro Asn Ala Lys Val Ala Cys Glu Thr Val Cys Lys Thr Gly
50 55 60
Met Val Leu Leu Cys Gly Glu Ile Thr Ser Met Ala Met Ile Asp Tyr
65 70 75 80
Gln Arg Val Val Arg Asp Thr Ile Lys His Ile Gly Tyr Asp Asp Ser
85 90 95
Ala Lys Gly Phe Asp Phe Lys Thr Cys Asn Val Leu Val Ala Leu Glu
100 105 110
Gln Gln Ser Pro Asp Ile Ala Gln Cys Val His Leu Asp Arg Asn Glu
115 120 125
Glu Asp Val Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr
130 135 140
Asp Glu Thr Glu Glu Cys Met Pro Leu Thr Ile Val Leu Ala His Lys
145 150 155 160
Leu Asn Thr Arg Met Ala Asp Leu Arg Arg Ser Gly Val Leu Pro Trp
165 170 175
Leu Arg Pro Asp Ser Lys Thr Gln Val Thr Val Gln Tyr Val Gln Asp
180 185 190
Asn Gly Ala Val Ile Pro Val Arg Val His Thr Ile Val Ile Ser Val
195 200 205
Gln His Asn Glu Asp Ile Thr Leu Glu Thr Met Arg Glu Ala Leu Arg
210 215 220
Glu Gln Val Ile Lys Ala Val Val Pro Ala Lys Tyr Leu Asp Glu Asp
225 230 235 240
Thr Ile Tyr His Leu Gln Pro Ser Gly Arg Phe Val Ile Gly Gly Pro
245 250 255
Gln Gly Asp Ala Gly Val Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr
260 265 270
Gly Gly Trp Gly Ala His Gly Gly Gly Ala Phe Ser Gly Lys Asp Tyr
275 280 285
Thr Lys Val Asp Arg Ser Ala Ala Tyr Ala Ala Arg Trp Val Ala Lys
290 295 300
Ser Leu Val Lys Ala Gly Leu Cys Arg Arg Val Leu Val Gln Val Ser
305 310 315 320
Tyr Ala Ile Gly Val Ala Glu Pro Leu Ser Ile Ser Ile Phe Thr Tyr
325 330 335
Gly Thr Ser Lys Lys Thr Glu Arg Glu Leu Leu Glu Val Val Asn Lys
340 345 350
Asn Phe Asp Leu Arg Pro Gly Val Ile Val Arg Asp Leu Asp Leu Lys
355 360 365
Lys Pro Ile Tyr Gln Lys Thr Ala Cys Tyr Gly His Phe Gly Arg Ser
370 375 380
Glu Phe Pro Trp Glu Val Pro Lys Lys Leu Val Phe Leu Glu
385 390 395
<210> 6
<211> 398
<212> PRT
<213> Artificial Sequence
<400> 6
Met Asn Gly Pro Val Asp Gly Leu Arg Asp His Ser Leu Ser Glu Glu
1 5 10 15
Gly Ala Phe Met Phe Thr Ser Glu Ser Val Gly Glu Gly His Pro Asp
20 25 30
Lys Ile Cys Asp Gln Ile Ser Asp Ala Val Leu Asp Ala His Leu Lys
35 40 45
Gln Asp Pro Asn Ala Lys Val Ala Cys Glu Thr Val Cys Lys Thr Gly
50 55 60
Met Val Leu Leu Cys Gly Glu Ile Thr Ser Met Ala Met Ile Asp Tyr
65 70 75 80
Gln Arg Val Val Arg Asp Thr Ile Lys His Ile Gly Tyr Asp Asp Ser
85 90 95
Ala Lys Gly Phe Asp Phe Lys Thr Cys Asn Val Leu Val Ala Leu Glu
100 105 110
Gln Gln Ser Pro Asp Ile Ala Gln Cys Val His Leu Asp Arg Asn Glu
115 120 125
Glu Asp Val Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr
130 135 140
Asp Glu Thr Glu Glu Cys Met Pro Leu Thr Ile Val Leu Ala His Lys
145 150 155 160
Leu Asn Ser Arg Met Ala Asp Leu Arg Arg Ser Gly Val Leu Pro Trp
165 170 175
Leu Arg Pro Asp Ser Lys Thr Gln Val Thr Val Gln Tyr Val Gln Asp
180 185 190
Asn Gly Ala Val Ile Pro Val Arg Val His Thr Ile Val Ile Ser Val
195 200 205
Gln His Asn Glu Asp Ile Thr Leu Glu Thr Met Arg Glu Ala Leu Arg
210 215 220
Glu Gln Val Ile Lys Ala Val Val Pro Ala Lys Tyr Leu Asp Glu Asp
225 230 235 240
Thr Ile Tyr His Leu Gln Pro Ser Gly Arg Phe Val Ile Gly Gly Pro
245 250 255
Gln Gly Asp Ala Gly Val Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr
260 265 270
Gly Gly Trp Gly Ala His Gly Gly Gly Ala Phe Ser Gly Lys Asp Tyr
275 280 285
Thr Lys Val Asp Arg Ser Ala Ala Tyr Ala Ala Arg Trp Val Ala Lys
290 295 300
Ser Leu Val Lys Ala Gly Leu Cys Arg Arg Val Leu Val Gln Val Ser
305 310 315 320
Tyr Ala Ile Gly Val Ala Glu Pro Leu Ser Ile Ser Ile Phe Thr Tyr
325 330 335
Gly Thr Ser Lys Lys Thr Glu Arg Glu Leu Leu Glu Val Val Asn Lys
340 345 350
Asn Phe Asp Leu Arg Pro Gly Val Ile Val Arg Asp Leu Asp Leu Lys
355 360 365
Lys Pro Ile Tyr Gln Lys Thr Ala Cys Tyr Gly His Phe Gly Arg Ser
370 375 380
Glu Phe Pro Trp Glu Val Pro Lys Lys Leu Val Phe Leu Glu
385 390 395
<210> 7
<211> 398
<212> PRT
<213> Artificial Sequence
<400> 7
Met Asn Gly Pro Val Asp Gly Leu Arg Asp His Ser Leu Ser Glu Glu
1 5 10 15
Gly Ala Phe Met Phe Thr Ser Glu Ser Val Gly Glu Gly His Pro Asp
20 25 30
Lys Ile Cys Asp Gln Ile Ser Asp Ala Val Leu Asp Ala His Leu Lys
35 40 45
Gln Asp Pro Asn Ala Lys Val Ala Cys Glu Thr Val Cys Lys Thr Gly
50 55 60
Met Val Leu Leu Cys Gly Glu Ile Thr Ser Met Ala Met Ile Asp Tyr
65 70 75 80
Gln Arg Val Val Arg Asp Thr Ile Lys His Ile Gly Tyr Asp Asp Ser
85 90 95
Ala Lys Gly Phe Asp Phe Lys Thr Cys Asn Val Leu Val Ala Leu Glu
100 105 110
Gln Gln Ser Pro Asp Ile Ala Gln Cys Val His Leu Asp Arg Asn Glu
115 120 125
Glu Asp Val Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr
130 135 140
Asp Glu Thr Glu Glu Cys Met Pro Leu Thr Ile Val Leu Ala His Lys
145 150 155 160
Leu Asn Thr Arg Met Ala Asp Leu Arg Arg Ser Gly Val Leu Pro Trp
165 170 175
Leu Arg Pro Asp Ser Lys Thr Gln Val Thr Val Gln Tyr Val Arg Asp
180 185 190
Asn Gly Ala Val Ile Pro Val Arg Val His Thr Ile Val Ile Ser Val
195 200 205
Gln His Asn Glu Asp Ile Thr Leu Glu Thr Met Arg Glu Ala Leu Arg
210 215 220
Glu Gln Val Ile Lys Ala Val Val Pro Ala Lys Tyr Leu Asp Glu Asp
225 230 235 240
Thr Ile Tyr His Leu Gln Pro Ser Gly Arg Phe Val Ile Gly Gly Pro
245 250 255
Gln Gly Asp Ala Gly Val Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr
260 265 270
Gly Gly Trp Gly Ala His Gly Gly Gly Ala Phe Ser Gly Lys Asp Tyr
275 280 285
Thr Lys Val Asp Arg Ser Ala Ala Tyr Ala Ala Arg Trp Val Ala Lys
290 295 300
Ser Leu Val Lys Ala Gly Leu Cys Arg Arg Val Leu Val Gln Val Ser
305 310 315 320
Tyr Ala Ile Gly Val Ala Glu Pro Leu Ser Ile Ser Ile Phe Thr Tyr
325 330 335
Gly Thr Ser Lys Lys Thr Glu Arg Glu Leu Leu Glu Val Val Asn Lys
340 345 350
Asn Phe Asp Leu Arg Pro Gly Val Ile Val Arg Asp Leu Asp Leu Lys
355 360 365
Lys Pro Ile Tyr Gln Lys Thr Ala Cys Tyr Gly His Phe Gly Arg Ser
370 375 380
Glu Phe Pro Trp Glu Val Pro Lys Lys Leu Val Phe Leu Glu
385 390 395
<210> 8
<211> 398
<212> PRT
<213> Artificial Sequence
<400> 8
Met Asn Gly Pro Val Asp Gly Leu Arg Asp His Ser Leu Ser Glu Glu
1 5 10 15
Gly Ala Phe Met Phe Thr Ser Glu Ser Val Gly Glu Gly His Pro Asp
20 25 30
Lys Ile Cys Asp Gln Ile Ser Asp Ala Val Leu Asp Ala His Leu Lys
35 40 45
Gln Asp Pro Asn Ala Lys Val Ala Cys Glu Thr Val Cys Lys Thr Gly
50 55 60
Met Val Leu Leu Cys Gly Glu Ile Thr Ser Met Ala Met Ile Asp Tyr
65 70 75 80
Gln Arg Val Val Arg Asp Thr Ile Lys His Ile Gly Tyr Asp Asp Ser
85 90 95
Ala Lys Gly Phe Asp Phe Lys Thr Cys Asn Val Leu Val Ala Leu Glu
100 105 110
Gln Gln Ser Pro Asp Ile Ala Gln Cys Val His Leu Asp Arg Asn Glu
115 120 125
Glu Asp Val Gly Ala Gly Asp Gln Gly Leu Met Phe Gly Tyr Ala Thr
130 135 140
Asp Glu Thr Glu Glu Cys Met Pro Leu Thr Ile Val Leu Ala His Lys
145 150 155 160
Leu Asn Ser Arg Met Ala Asp Leu Arg Arg Ser Gly Val Leu Pro Trp
165 170 175
Leu Arg Pro Asp Ser Lys Thr Gln Val Thr Val Gln Tyr Val Arg Asp
180 185 190
Asn Gly Ala Val Ile Pro Val Arg Val His Thr Ile Val Ile Ser Val
195 200 205
Gln His Asn Glu Asp Ile Thr Leu Glu Thr Met Arg Glu Ala Leu Arg
210 215 220
Glu Gln Val Ile Lys Ala Val Val Pro Ala Lys Tyr Leu Asp Glu Asp
225 230 235 240
Thr Ile Tyr His Leu Gln Pro Ser Gly Arg Phe Val Ile Gly Gly Pro
245 250 255
Gln Gly Asp Ala Gly Val Thr Gly Arg Lys Ile Ile Val Asp Thr Tyr
260 265 270
Gly Gly Trp Gly Ala His Gly Gly Gly Ala Phe Ser Gly Lys Asp Tyr
275 280 285
Thr Lys Val Asp Arg Ser Ala Ala Tyr Ala Ala Arg Trp Val Ala Lys
290 295 300
Ser Leu Val Lys Ala Gly Leu Cys Arg Arg Val Leu Val Gln Val Ser
305 310 315 320
Tyr Ala Ile Gly Val Ala Glu Pro Leu Ser Ile Ser Ile Phe Thr Tyr
325 330 335
Gly Thr Ser Lys Lys Thr Glu Arg Glu Leu Leu Glu Val Val Asn Lys
340 345 350
Asn Phe Asp Leu Arg Pro Gly Val Ile Val Arg Asp Leu Asp Leu Lys
355 360 365
Lys Pro Ile Tyr Gln Lys Thr Ala Cys Tyr Gly His Phe Gly Arg Ser
370 375 380
Glu Phe Pro Trp Glu Val Pro Lys Lys Leu Val Phe Leu Glu
385 390 395
<210> 9
<211> 340
<212> PRT
<213> Artificial Sequence
<400> 9
Met Thr Ala Pro Leu Val Val Leu Gly Asn Pro Leu Leu Asp Phe Gln
1 5 10 15
Ala Asp Val Thr Ala Glu Tyr Leu Ala Lys Tyr Ser Leu Lys Glu Asn
20 25 30
Asp Ala Ile Leu Val Asp Ala Lys Ser Gly Asp Ala Lys Met Ala Ile
35 40 45
Phe Asp Glu Leu Leu Gln Met Pro Glu Thr Lys Leu Val Ala Gly Gly
50 55 60
Ala Ala Gln Asn Thr Ala Arg Gly Ala Ala Tyr Val Leu Gly Ala Gly
65 70 75 80
Gln Val Val Tyr Phe Gly Ser Val Gly Lys Asp Lys Phe Ser Glu Arg
85 90 95
Leu Leu Asn Glu Asn Glu Lys Ala Gly Val Lys Ser Met Tyr Gln Val
100 105 110
Gln Asn Asp Ile Gly Thr Gly Lys Cys Ala Ala Leu Ile Thr Gly His
115 120 125
Asn Arg Ser Leu Val Thr Asp Leu Gly Ala Ala Asn Phe Phe Thr Pro
130 135 140
Asp His Leu Asp Lys His Trp Asp Leu Val Glu Ala Ala Lys Leu Phe
145 150 155 160
Tyr Ile Gly Gly Phe His Leu Thr Val Ser Pro Asp Ala Ile Val Lys
165 170 175
Leu Gly Gln His Ala Lys Glu Asn Ser Lys Pro Phe Val Leu Asn Phe
180 185 190
Ser Ala Pro Phe Ile Pro His Val Phe Lys Asp Ala Leu Ala Arg Val
195 200 205
Leu Pro Tyr Ala Thr Val Ile Ile Ala Asn Glu Ser Glu Ala Glu Ala
210 215 220
Phe Cys Asp Ala Phe Gln Leu Asp Cys Ala Asn Thr Asp Leu Glu Ala
225 230 235 240
Ile Ala Gln Arg Ile Val Lys Asp Ser Pro Val Glu Lys Thr Val Ile
245 250 255
Phe Thr His Gly Val Glu Pro Thr Val Val Val Ser Ser Lys Gly Thr
260 265 270
Ser Thr Tyr Pro Val Lys Pro Leu Asp Ser Ser Lys Ile Val Asp Thr
275 280 285
Asn Gly Ala Gly Asp Ala Phe Ala Gly Gly Phe Met Ala Gly Leu Thr
290 295 300
Lys Gly Glu Asp Leu Glu Thr Ser Ile Asp Met Gly Gln Trp Leu Ala
305 310 315 320
Ala Leu Ser Ile Gln Glu Val Gly Pro Ser Tyr Pro Ser Glu Lys Ile
325 330 335
Ser Tyr Ser Lys
340
<210> 10
<211> 345
<212> PRT
<213> Artificial Sequence
<400> 10
Met Ser Ala Leu Pro Gln Leu Tyr Ile Gln Cys Asn Pro Leu Leu Asp
1 5 10 15
Val Ser Ala Pro Val Asp Asp Ala Phe Leu Glu Lys Tyr Lys Val Gln
20 25 30
Lys Thr Ser Ala Cys Leu Met Glu Glu Ile His Lys Gly Ile Phe Glu
35 40 45
Glu Leu Glu Gln His Pro Asn Val Thr Tyr Val Pro Gly Gly Ser Gly
50 55 60
Leu Asn Thr Ala Arg Val Ala Gln Trp Ile Ala Gln Ala Pro Lys Ser
65 70 75 80
Ser Leu Phe Asn Tyr Val Gly Cys Ala Ser Asp Asp Lys Tyr Gly Lys
85 90 95
Ile Leu Lys Glu Ala Ala Glu Lys Asn Gly Val Asn Met His Leu Glu
100 105 110
Tyr Thr Thr Lys Ala Pro Thr Gly Ser Cys Ala Val Cys Ile Ser Gly
115 120 125
Lys Asp Arg Ser Leu Val Ala Asn Leu Ser Ala Ala Asn Leu Leu Ser
130 135 140
Ala Asp His Met His Ser Ser Asp Val Val Glu Thr Leu Lys Gly Cys
145 150 155 160
Gln Leu Tyr Tyr Leu Thr Gly Phe Thr Leu Thr Ile Asp Val Asn Tyr
165 170 175
Val Leu Gln Val Ala Glu Ala Ala Arg Ala Ser Gly Gly Gln Phe Met
180 185 190
Met Asn Leu Ser Ala Pro Phe Val Leu Gln Tyr Phe Thr Glu Ser Phe
195 200 205
Asn Lys Ala Ala Pro Tyr Leu Asp Val Ile Phe Gly Asn Glu Val Glu
210 215 220
Ala Lys Ala Leu Ala Asp Ala Met Lys Trp Asn Pro Ala Ser Thr His
225 230 235 240
Asn Leu Ala Lys Lys Ala Ala Met Glu Leu Pro Tyr Ser Gly Thr Arg
245 250 255
Asp Arg Ile Val Asp Phe Thr Gln Gly Ser Gln Pro Thr Val Tyr Ala
260 265 270
Thr Arg Ser Gly Lys Thr Gly Ser Val Thr Val Gln Pro Ile Ala His
275 280 285
Asp Ile Ile Val Asp Leu Asn Gly Ala Gly Asp Ala Phe Val Gly Gly
290 295 300
Phe Leu Ala Ala Tyr Ala Met Ser Cys Ser Ile Gln Arg Cys Cys Glu
305 310 315 320
Val Gly Asn Tyr Ala Ala Gly Val Ile Ile Gln His Asn Gly Cys Thr
325 330 335
Tyr Pro Glu Lys Pro Ser Ile Ser Pro
340 345
<210> 11
<211> 688
<212> PRT
<213> Artificial Sequence
<400> 11
Met Gly Gln Glu Lys Leu Tyr Ile Glu Lys Glu Leu Ser Trp Leu Ser
1 5 10 15
Phe Asn Glu Arg Val Leu Gln Glu Ala Ala Asp Lys Ser Asn Pro Leu
20 25 30
Ile Glu Arg Met Arg Phe Leu Gly Ile Tyr Ser Asn Asn Leu Asp Glu
35 40 45
Phe Tyr Lys Val Arg Phe Ala Glu Leu Lys Arg Arg Ile Ile Ile Ser
50 55 60
Glu Glu Gln Gly Ser Asn Ser His Ser Arg His Leu Leu Gly Lys Ile
65 70 75 80
Gln Ser Arg Val Leu Lys Ala Asp Gln Glu Phe Asp Gly Leu Tyr Asn
85 90 95
Glu Leu Leu Leu Glu Met Ala Arg Asn Gln Ile Phe Leu Ile Asn Glu
100 105 110
Arg Gln Leu Ser Val Asn Gln Gln Asn Trp Leu Arg His Tyr Phe Lys
115 120 125
Gln Tyr Leu Arg Gln His Ile Thr Pro Ile Leu Ile Asn Pro Asp Thr
130 135 140
Asp Leu Val Gln Phe Leu Lys Asp Asp Tyr Thr Tyr Leu Ala Val Glu
145 150 155 160
Ile Ile Arg Gly Asp Thr Ile Arg Tyr Ala Leu Leu Glu Ile Pro Ser
165 170 175
Asp Lys Val Pro Arg Phe Val Asn Leu Pro Pro Glu Ala Pro Arg Arg
180 185 190
Arg Lys Pro Met Ile Leu Leu Asp Asn Ile Leu Arg Tyr Cys Leu Asp
195 200 205
Asp Ile Phe Lys Gly Phe Phe Asp Tyr Asp Ala Leu Asn Ala Tyr Ser
210 215 220
Met Lys Met Thr Arg Asp Ala Glu Tyr Asp Leu Val His Glu Met Glu
225 230 235 240
Ala Ser Leu Met Glu Leu Met Ser Ser Ser Leu Lys Gln Arg Leu Thr
245 250 255
Ala Glu Pro Val Arg Phe Val Tyr Gln Arg Asp Met Pro Asn Ala Leu
260 265 270
Val Glu Val Leu Arg Glu Lys Leu Thr Ile Ser Arg Tyr Asp Ser Ile
275 280 285
Val Pro Gly Gly Arg Tyr His Asn Phe Lys Asp Phe Ile Asn Phe Pro
290 295 300
Asn Val Gly Lys Ala Asn Leu Val Asn Lys Pro Leu Pro Arg Leu Arg
305 310 315 320
His Ile Trp Phe Asp Lys Ala Gln Phe Arg Asn Gly Phe Asp Ala Ile
325 330 335
Arg Glu Arg Asp Val Leu Leu Tyr Tyr Pro Tyr His Thr Phe Glu His
340 345 350
Val Leu Glu Leu Leu Arg Gln Ala Ser Phe Asp Pro Ser Val Leu Ala
355 360 365
Ile Lys Ile Asn Ile Tyr Arg Val Ala Lys Asp Ser Arg Ile Ile Asp
370 375 380
Ser Met Ile His Ala Ala His Asn Gly Lys Lys Val Thr Val Val Val
385 390 395 400
Glu Leu Gln Ala Arg Phe Asp Glu Glu Ala Asn Ile His Trp Ala Lys
405 410 415
Arg Leu Thr Glu Ala Gly Val His Val Ile Phe Ser Ala Pro Gly Leu
420 425 430
Lys Ile His Ala Lys Leu Phe Leu Ile Ser Arg Lys Glu Asn Gly Glu
435 440 445
Val Val Arg Tyr Ala His Ile Gly Thr Gly Asn Phe Asn Glu Lys Thr
450 455 460
Ala Arg Leu Tyr Thr Asp Tyr Ser Leu Leu Thr Ala Asp Ala Arg Ile
465 470 475 480
Thr Asn Glu Val Arg Arg Val Phe Asn Phe Ile Glu Asn Pro Tyr Arg
485 490 495
Pro Val Thr Phe Asp Tyr Leu Met Val Ser Pro Gln Asn Ser Arg Arg
500 505 510
Leu Leu Tyr Glu Met Val Asp Arg Glu Ile Ala Asn Ala Gln Gln Gly
515 520 525
Leu Pro Ser Gly Ile Thr Leu Lys Leu Asn Asn Leu Val Asp Lys Gly
530 535 540
Leu Val Asp Arg Leu Tyr Ala Ala Ser Ser Ser Gly Val Pro Val Asn
545 550 555 560
Leu Leu Val Arg Gly Met Cys Ser Leu Ile Pro Asn Leu Glu Gly Ile
565 570 575
Ser Asp Asn Ile Arg Ala Ile Ser Ile Val Asp Arg Tyr Leu Glu His
580 585 590
Asp Arg Val Tyr Ile Phe Glu Asn Gly Gly Asp Lys Lys Val Tyr Leu
595 600 605
Ser Ser Ala Asp Trp Met Thr Arg Asn Ile Asp Tyr Arg Ile Glu Val
610 615 620
Ala Thr Pro Leu Leu Asp Pro Arg Leu Lys Gln Arg Val Leu Asp Ile
625 630 635 640
Ile Asp Ile Leu Phe Ser Asp Thr Val Lys Ala Arg Tyr Ile Asp Lys
645 650 655
Glu Leu Ser Asn Arg Tyr Val Pro Arg Gly Asn Arg Arg Lys Val Arg
660 665 670
Ala Gln Leu Ala Ile Tyr Asp Tyr Ile Lys Ser Leu Glu Gln Pro Glu
675 680 685
<210> 12
<211> 98
<212> PRT
<213> Artificial Sequence
<400> 12
Met Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys Pro
1 5 10 15
Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly Ser
20 25 30
Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg Arg Leu
35 40 45
Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser Leu Arg
50 55 60
Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Thr Pro Glu Asp
65 70 75 80
Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu Gln Ile
85 90 95
Gly Gly

Claims (7)

1.一种重组大肠杆菌工程菌，其特征在于：所述的重组大肠杆菌工程菌含有重组表达载体，所述的重组表达载体含有表达S-腺苷甲硫氨酸合成酶的基因和表达ATP合成酶的基因，
所述的S-腺苷甲硫氨酸合成酶的氨基酸序列如SEQ ID NO.2-8之一所示，
所述的ATP合成酶由腺苷激酶、腺苷酸激酶和多聚磷酸激酶组成。
2.根据权利要求1所述的重组大肠杆菌工程菌，其特征在于：
所述的大肠杆菌工程菌选自BL21(DE3)、BL21(DE3) pLysS、Rosetta(DE3)、ArcticExpress(DE3)、Origami B(DE3)中的一种；
所述的表达载体是质粒载体，选自pET23b、pET24a、pET28a、pET30a、pET32a、pCDFDuet-1、pET15b、pET21a、pETDuet-1、pACYC184、pTXB1、pTYB21。
3.根据权利要求1所述的重组大肠杆菌工程菌，其特征在于：所述的表达S-腺苷甲硫氨酸合成酶的基因位于重组质粒载体pET30a内，所述的表达ATP合成酶的基因位于重组质粒载体pCDFDuet-1内。
4.一种利用权利要求1-3之一所述的重组大肠杆菌工程菌制备S-腺苷甲硫氨酸的方法，其特征在于，所述的方法包括如下步骤：
（1）发酵所述的重组大肠杆菌工程菌；
（2）发酵后离心收集菌体并用缓冲液重悬菌体；
（3）在菌体重悬液中先加入ATP合成酶催化反应所需的反应物进行反应，再加入S-腺苷甲硫氨酸合成酶催化反应所需的反应物进行反应。
5.根据权利要求4所述的方法，其特征在于：步骤（2）中，在重悬菌体的同时还加入曲拉通X-100和/或EDTA以对菌体进行通透化处理。
6.根据权利要求4所述的方法，其特征在于：步骤（3）中，
加入的所述的ATP合成酶催化反应所需的反应物包括腺苷、ATP、ADP、AMP、多聚磷酸盐、镁盐，加入后温度25-30℃、pH 5.0-6.5、搅拌转速150-200r/min下反应30-180min；
加入的所述的S-腺苷甲硫氨酸合成酶催化反应所需的反应物包括L-甲硫氨酸、镁盐、钾盐、磺酸盐，加入后温度25-30℃、pH 7.5-8.0、搅拌转速150-200r/min下继续反应60-240min。
7.根据权利要求6所述的方法，其特征在于：
所述的腺苷、ATP、ADP、AMP、多聚磷酸盐、镁盐加入后对于OD=20菌体量的浓度分别为50-300mM、1-10mM、10-50mM、10-50mM、500-800mM、5-10mM；
所述的L-甲硫氨酸、镁盐、钾盐、磺酸盐加入后对于OD=20菌体量的浓度分别为200-500mM、100-250mM、50-100mM、300-500mM。
CN202010250598.5A 2020-04-01 2020-04-01 重组大肠杆菌工程菌及利用其制备s-腺苷甲硫氨酸的方法 Active CN111235084B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010250598.5A CN111235084B (zh) 2020-04-01 2020-04-01 重组大肠杆菌工程菌及利用其制备s-腺苷甲硫氨酸的方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010250598.5A CN111235084B (zh) 2020-04-01 2020-04-01 重组大肠杆菌工程菌及利用其制备s-腺苷甲硫氨酸的方法

Publications (2)

Publication Number Publication Date
CN111235084A CN111235084A (zh) 2020-06-05
CN111235084B true CN111235084B (zh) 2021-08-20

Family

ID=70864428

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010250598.5A Active CN111235084B (zh) 2020-04-01 2020-04-01 重组大肠杆菌工程菌及利用其制备s-腺苷甲硫氨酸的方法

Country Status (1)

Country Link
CN (1) CN111235084B (zh)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1916176A (zh) * 2006-09-11 2007-02-21 中国科学院上海生命科学研究院 一种体外酶催化合成腺苷甲硫氨酸的方法
EP2071022A1 (en) * 2006-08-31 2009-06-17 Bioright Worldwide Company Limited S-adenosylmethionine synthetase mutants, the dnas encoding the same and uses of the mutants
CN101979645A (zh) * 2010-09-30 2011-02-23 北京凯因科技股份有限公司 一种腺苷甲硫氨酸的制备方法
CN109136311A (zh) * 2017-06-15 2019-01-04 深圳市古特新生生物科技有限公司 一种酶法制备s-腺苷甲硫氨酸的方法
CN109136309A (zh) * 2017-06-15 2019-01-04 深圳市古特新生生物科技有限公司 一种利用腺苷代替atp进行酶促反应的生产方法
CN109280680A (zh) * 2017-07-21 2019-01-29 深圳市古特新生生物科技有限公司 一种酶促联产的方法
CN110144336A (zh) * 2019-05-23 2019-08-20 浙江大学 一种s-腺苷甲硫氨酸合成酶突变体及其制备方法
CN110172454A (zh) * 2019-05-23 2019-08-27 浙江大学 一种s-腺苷甲硫氨酸合成酶突变体及其高通量筛选方法

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2071022A1 (en) * 2006-08-31 2009-06-17 Bioright Worldwide Company Limited S-adenosylmethionine synthetase mutants, the dnas encoding the same and uses of the mutants
CN1916176A (zh) * 2006-09-11 2007-02-21 中国科学院上海生命科学研究院 一种体外酶催化合成腺苷甲硫氨酸的方法
CN101979645A (zh) * 2010-09-30 2011-02-23 北京凯因科技股份有限公司 一种腺苷甲硫氨酸的制备方法
CN109136311A (zh) * 2017-06-15 2019-01-04 深圳市古特新生生物科技有限公司 一种酶法制备s-腺苷甲硫氨酸的方法
CN109136309A (zh) * 2017-06-15 2019-01-04 深圳市古特新生生物科技有限公司 一种利用腺苷代替atp进行酶促反应的生产方法
CN109280680A (zh) * 2017-07-21 2019-01-29 深圳市古特新生生物科技有限公司 一种酶促联产的方法
CN110144336A (zh) * 2019-05-23 2019-08-20 浙江大学 一种s-腺苷甲硫氨酸合成酶突变体及其制备方法
CN110172454A (zh) * 2019-05-23 2019-08-27 浙江大学 一种s-腺苷甲硫氨酸合成酶突变体及其高通量筛选方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ATP合成菌株的构建及用于联合生产S-腺苷甲硫氨酸;江林林 等;《生物技术通报》;20190626;第35卷(第6期);第221-226页 *
Site-directed mutagenesis of rat liver S-adenosylmethionine synthetase Identification of a cysteine residue critical for the oligomeric state;Jesus MINGORANCE et al.;《Biochemistry Journal》;19960501;第315卷(第Pt3期);第761-766页 *

Also Published As

Publication number Publication date
CN111235084A (zh) 2020-06-05

Similar Documents

Publication Publication Date Title
KR102652526B1 (ko) 생물 전환으로 타우로우루소데옥시콜산을 제조하는 방법 및 그 응용
CN112280762B (zh) 一种烟酰胺核糖激酶突变体及其编码基因和应用
CN111676204B (zh) 制备烟酰胺单核苷酸的烟酰胺磷酸核糖转移酶、编码基因、载体及应用
CN109750009B (zh) 一种草铵膦脱氢酶突变体及其应用
CN112795606B (zh) 一种β-烟酰胺单核苷酸的酶催化合成方法
CN111235126B (zh) 一种s-腺苷甲硫氨酸合成酶突变体及利用其的制备方法
CN108018252B (zh) 一种中间体2’-脱氧鸟苷的制备方法
CN112980906B (zh) 一种用于制备β-烟酰胺单核苷酸的酶组合物及其应用
CN111996155B (zh) 一种提高l-组氨酸产生菌生产能力的方法
CN111041018A (zh) 支链酮糖的生物合成方法
CN111394289B (zh) 一种基因工程菌及其应用，生产前列腺素e2的方法
CN111235084B (zh) 重组大肠杆菌工程菌及利用其制备s-腺苷甲硫氨酸的方法
CN116376859A (zh) 一种二氢叶酸还原酶及其应用
CN109486780A (zh) 一种催化效率提高的ω-转氨酶突变体
CN107779459B (zh) 葡萄糖脱氢酶dna分子、载体和菌株及应用
CN107287172B (zh) 一种利用大肠杆菌发酵生产胸苷磷酸化酶的方法
CN110004099A (zh) 一种红景天苷的发酵生产方法
CN108265068B (zh) 重组精氨酸脱亚胺酶及其产业化制备方法和应用
CN115058402B (zh) 一种烟酰胺核糖激酶突变体及其编码基因和应用
CN110951717B (zh) 一种l-阿拉伯糖异构酶异构体及其应用
CN110904087B (zh) L-阿拉伯糖差向异构酶突变体及其应用
CN114317631B (zh) 单胺氧化酶在制备托品酮中的应用
CN114875011B (zh) Amp磷酸转移酶突变体、其编码基因及在atp合成中的应用
CN111057698B (zh) L-阿拉伯糖异构酶、突变体及其应用
CN114875003A (zh) 一种短链脱氢酶的突变体、编码基因及编码基因获得方法、突变体的应用

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
PE01 Entry into force of the registration of the contract for pledge of patent right
PE01 Entry into force of the registration of the contract for pledge of patent right

Denomination of invention: Recombinant Escherichia coli engineering strain and method for preparing S-adenosylmethionine using it

Granted publication date: 20210820

Pledgee: Changsha Bank city branch of Limited by Share Ltd.

Pledgor: HUNAN FLAG BIOTECHNOLOGY Co.,Ltd.

Registration number: Y2024980012003