ATP合成菌株的构建及用于联合生产S-腺苷甲硫氨酸

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英文篇名：Construction of ATP Synthetic Strain and Its Application in the Production of S-adenosylmethionine 作者：江林林 ; 吴磊 ; 许海霞 ; 黄坚丽 ; 张永进 ; 徐期 ; 杨勇 英文作者：JIANG Lin-lin;WU Lei;XU Hai-xia;HUANG Jian-li;ZHANG Yong-jin;XU Qi;YANG Yong;Zhejiang Hisun Pharmaceutical Co.Ltd.; 关键词：ATP合成 ; 腺苷激酶 ; 腺苷酸激酶 ; 乙酸激酶 ; S-腺苷甲硫氨酸 英文关键词：ATP synthesis;;adenosine kinase;;adenylate kinase;;acetate kinase;;S-adenosylmethionine 中文刊名：SWJT 英文刊名：Biotechnology Bulletin 机构：浙江海正药业股份有限公司; 出版日期：2019-06-26 出版单位：生物技术通报 年：2019 期：v.35;No.323 语种：中文; 页：SWJT201906030 页数：6 CN：06 ISSN：11-2396/Q 分类号：227-232

摘要

为降低S-腺苷甲硫氨酸的生产成本,构建了同时表达腺苷激酶、腺苷酸激酶和乙酸激酶3种酶的重组大肠杆菌菌株用于ATP的合成,并对ATP的转化条件进行了优化,优化后的反应体系为:腺苷30 mmol/L,乙酰磷酸二锂盐135 mmol/L,硫酸镁5 mmol/L,硼砂50 mmol/L,菌体2 g/L(湿重),反应液初始pH7.5,反应温度为35℃,反应时间为3 h,反应转化率可以达到99%以上。按照上述反应体系进行5 L放大,反应结束后再投入65 mmol/L D,L-甲硫氨酸和50 g/L(湿重)表达腺苷甲硫氨酸合成酶的重组大肠杆菌菌体,并补加15 mmol/L硫酸镁,转化18 h S-腺苷甲硫酸浓度能达到8.7 g/L,转化率达到72.5%。
        In order to achieve ATP production as well as reduce the cost of producing S-adenosylmethionine,a recombinant Escherichia coli strain expressing adenosine kinase,adenylate kinase and acetate kinase simultaneously was constructed for ATP synthesis. The conditions of transforming ATP were optimized,and the optimized reaction system was as :adenosine 30 mmol/L,acetylphosphate dilithium 135 mmol/L,magnesium sulfate 5 mmol/L,borax 50 mmol/L,E. coli strain 2 g/L(wet weight),initial pH 7.5,the reaction temperature 35 ℃,and reacting for 3 h,the conversion rate was over 99%. Based on the above result,the reaction system was enlarged in 5 L. After reaction ended,50 g/L(wet weight)recombinant E. coli strain expressing adenosylmethionine synthase,65 mmol/L D,L-methionine and 15 mmol/L magnesium sulfate were added to the reaction system,the concentration of S-adenosylmethionine was up to 8.7 g/L and the conversion rate reached 72.5% after 18 h transformation.

引文

[1] Xu W, Raetz LB, Wang P, et al. An ATP-dependent ligase catalyzes the fourth ring cyclization in tetracycline biosynthesis[J].Tetrahedron, 2016, 72(25):3599-3604.
    [2]刘桂祯,黄炯威,周华润,等.一种酶催化合成谷胱甘肽的方法:中国, 201610745720. X[P]. 2016-08-29.
    [3] Maruyama A, Fujio T. ATP production from adeine by a self-coupling enzymatic process:high-level accumulation under ammoniumlimited conditions[J]. Biosci Biotechnol Biochem, 2001, 65(3):644-650.
    [4]廖鲜艳,王芸,朱至,等.面包酵母生物合成ATP的影响因素及机理初探[J].食品与生物技术学报, 2007, 26(6):53-57.
    [5]李寅,陈坚,伦世仪. ATP再生系统及其应用[J].食品与发酵工业, 1998, 24(3):60-65.
    [6]颜炳坤.联合葡萄糖激酶与乙酸激酶催化的ATP再生系统酶法合成葡萄糖-6-磷酸[D].上海:华东理工大学, 2014.
    [7]徐扬. S-腺苷甲硫氨酸研究进展[J].国际生物制品学杂志,2009, 32(1):41-44.
    [8]万红贵,郭一丹,蔡恒,等. S-腺苷甲硫氨酸研究进展及市场应用分析[J].现代生物医学进展, 2008, 8(10):1951-1955.
    [9] Yin Chunli, Zheng Tao, Chang Xin. Biosynthesis of S-adenosylmethionine by magnetically immobilized Escherichia coli cells highly expressing a methionine adenosyltransferase variant[J]. Molecules, 2017, 22(8):1-13.
    [10]牛卫宁,左晓佳,顶焰,等.重组大肠杆菌全细胞催化合成S-腺苷甲硫氨酸[J].精细化工, 2009, 26(3):288-292.
    [11]郑计瑞,陈丽芬,等.腺嘌呤合成腺苷甲硫氨酸的工程菌构建及其高产株筛选[J].食品与发酵工程, 2014, 40(9):23-28.