食品与发酵工业 >

2025 , Vol. 51 >Issue 1: 1 - 10

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.038237

研究报告

代谢工程改造大肠杆菌合成甲萘醌-7

  • 何雨锴 ,
  • 曾伟主 ,
  • 徐沙
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  • 1(江南大学 生物工程学院,江苏 无锡,214122)
    2(江南大学,粮食发酵工艺与技术国家工程实验室,江苏 无锡,214122)
    3(江南大学 未来食品科学中心,江苏 无锡,214122)
第一作者:硕士研究生(徐沙副教授为通信作者,E-mail:xusha1984@jiangnan.edu.cn)

收稿日期: 2023-12-12

  修回日期: 2024-03-19

  网络出版日期: 2025-01-23

基金资助

江苏省自然科学基金前沿引领技术基础研究专项(BK20202002)

收起

Metabolic engineering of Escherichia coli for the synthesis of menaquinone-7

  • HE Yukai ,
  • ZENG Weizhu ,
  • XU Sha
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  • 1(School of Biotechnology, Jiangnan University, Wuxi 214122, China)
    2(National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China)
    3(Science Center for Future Foods, Jiangnan University, Wuxi, 214122, China)

Received date: 2023-12-12

  Revised date: 2024-03-19

  Online published: 2025-01-23

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摘要

甲萘醌-7(menaquinone-7,MK-7),作为维生素K2亚型之一,具有治疗骨质疏松,保护心血管健康等作用。常见MK-7生产主要由纳豆等食品发酵而来,但存在产量低,提取复杂等问题,而枯草芽孢杆菌作为MK-7常见生产菌株,也面临发酵周期长,发酵产量低的问题。该研究对一株引入甲羟戊酸途径的大肠杆菌进行改造,首先利用DLKcat模型比较筛选出4个关键酶,包括七聚异戊二烯焦磷酸合酶BtHepS\BtHepT、1, 4-二羟基-2-萘甲酸七烯基转移酶MnMenA、甲萘醌生物合成C-甲基转移酶NtMenG,将其进行表达优化,构建了大肠杆菌内源合成MK-7途径。随后,整合表达异戊烯基焦磷酸异构酶EcIdi提升前体供应,再对得到的菌株进行补料分批发酵条件优化,包括延迟诱导时间,提升溶氧量至60,MK-7产量达到929.58 mg/L,但出现1 217.58 mg/L的去甲基甲萘醌-7(DMK-7)大量堆积的现象。进一步筛选了不同来源的MenG降低DMK-7的堆积,发现Bacillus subtilis 168来源的MenG可以较好地降低DMK-7堆积。最终,在5 L发酵罐上MK-7的产量达到1 090.45 mg/L,DMK-7降低至231.14 mg/L。该研究结果对应用大肠杆菌生产MK-7具有一定的指导意义。

关键词： 甲萘醌-7; 大肠杆菌; DLKcat; 去甲基甲萘醌-7; 发酵优化

本文引用格式

何雨锴 , 曾伟主 , 徐沙 . 代谢工程改造大肠杆菌合成甲萘醌-7[J]. 食品与发酵工业, 2025 , 51(1) : 1 -10 . DOI: 10.13995/j.cnki.11-1802/ts.038237

Abstract

Menaquinone-7 (MK-7), as one of the vitamin K2 subtypes, has the effects of treating osteoporosis and protecting cardiovascular health.Common MK-7 production is mainly fermented by food such as natto, but there are problems such as low yield and complex extraction.Bacillus subtilis, as a common production strain of MK-7, also faces the problems of long fermentation period and low titer.In this study, a strain of Escherichia coli introducing mevalonate pathway was modified.Firstly, four key enzymes were screened by DLKcat model, including heptaisopentadiene pyrophosphate synthase BtHepS\BtHepT, 1,4-dihydroxy-2-naphthoic acid heptaenyl transferase MnMenA, and menadione biosynthesis C-methyltransferase NtMenG.The expression optimization was carried out to construct the endogenous synthesis of MK-7 pathway in E. coli.Subsequently, the prenylated pyrophosphate isomerase EcIdi was integrated and expressed to enhance the precursor supply, and then the fed-batch fermentation conditions of the obtained strains were optimized, including delaying the induction time, increasing the dissolved oxygen to 60. The MK-7 production reached 929.58 mg/L, but there was 1 217.58 mg/L demethylmenaquinone-7 (DMK-7) accumulation.Then, different sources of MenG were further screened to reduce the accumulation of DMK-7, and it was found that MenG from B. subtilis 168 could reduce the accumulation of DMK-7.Finally, the production of MK-7 reached 1 090.45 mg/L and DMK-7 decreased to 231.14 mg/L in a 5 L fermenter.The results of this study have certain guiding significance for the application of E.coli to produce MK-7.

Key words： menaquinone-7; Escherichia coli; DLKcat; demethylmenaquinone-7; fermentation optimization

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