化工学报 ›› 2023, Vol. 74 ›› Issue (5): 2111-2122.DOI: 10.11949/0438-1157.20221582

• 生物化学工程与技术 • 上一篇    下一篇

代谢工程改造大肠杆菌生产软骨素

赵春雷1,2(), 郭亮1,2, 高聪1,2, 宋伟3, 吴静3, 刘佳1,2, 刘立明1,2, 陈修来1,2()   

  1. 1.江南大学食品科学与技术国家重点实验室,江苏 无锡 214122
    2.江南大学食品安全国际合作联合实验室,江苏 无锡 214122
    3.江南大学生命科学与健康工程学院,江苏 无锡 214122
  • 收稿日期:2022-12-08 修回日期:2023-02-17 出版日期:2023-05-05 发布日期:2023-06-29
  • 通讯作者: 陈修来
  • 作者简介:赵春雷(1997—),男,硕士研究生,1972630117@qq.com
  • 基金资助:
    国家重点研发计划项目(2021YFC2103100);江苏省自然科学基金项目(BK20211529)

Metabolic engineering of Escherichia coli for chondroitin production

Chunlei ZHAO1,2(), Liang GUO1,2, Cong GAO1,2, Wei SONG3, Jing WU3, Jia LIU1,2, Liming LIU1,2, Xiulai CHEN1,2()   

  1. 1.State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu, China
    2.International Joint Laboratory on Food Safety, Jiangnan University, Wuxi 214122, Jiangsu, China
    3.School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, Jiangsu, China
  • Received:2022-12-08 Revised:2023-02-17 Online:2023-05-05 Published:2023-06-29
  • Contact: Xiulai CHEN

摘要:

目前生物法合成软骨素的研究策略主要侧重于构建合成路径,缺乏对前体物质供应的精细化调控,从而限制了软骨素的合成效率。为了解决上述问题,本研究借助代谢工程策略,在大肠杆菌(Escherichia coli)中重构与优化了软骨素合成路径,获得了合成软骨素的微生物细胞工厂,实现了发酵法生产软骨素。通过在E. coli BL21 STAR (DE3)中表达UDP-葡萄糖脱氢酶(KfoF)、UDP-氨基葡萄糖异构酶(KfoA)和软骨素聚合酶(KfoC),构建了完整的软骨素合成路径。通过氨基转移酶(GlmS)和磷酸葡萄糖胺变位酶(GlmM)的基因表达水平优化,提高了软骨素合成前体UDP-GalNAc的供给量;优化KfoF的基因表达水平,改善了软骨素合成前体UDP-GlcA的供给效率。在5 L发酵罐上,最优工程菌株E. coli GZ17的软骨素产量达到了2.95 g/L。上述研究策略为硫酸软骨素菌株的构建与应用奠定了基础,也为代谢工程改造生产其他糖胺聚糖提供了借鉴。

关键词: 软骨素, 大肠杆菌, 代谢工程, 路径优化

Abstract:

The current research strategies for biosynthesizing chondroitin mainly focus on the construction of synthetic pathways, lacking the fine regulation of the supply of precursor substances, thus limiting the synthesis efficiency of chondroitin. To solve the above problems, in this study, the biosynthetic pathway for chondroitin synthesis was reconstructed and optimized in Escherichia coli by different metabolic engineering strategies, obtaining a microbial cell factory for chondroitin synthesis by fermentation. A complete synthesis pathway for chondroitin was constructed in E. coli BL21 STAR (DE3) by expressing UDP-glucose dehydrogenase (KfoF), UDP-glucosamine isomerase (KfoA) and chondroitin polymerase (KfoC). The supply of chondroitin precursor UDP-GalNAc was improved by optimizing the gene expression levels of aminotransferase (GlmS) and phosphoglucosamine mutase (GlmM). The supply efficiency of chondroitin synthesis precursor UDP-GlcA was improved by optimizing the gene expression level of KfoF. Finally, with the optimized strain E. coli GZ17, chondroitin production was increased to 2.95 g/L in a 5 L fermenter. The above research strategy laid the foundation for the construction and application of chondroitin sulfate strains, and also provided a reference for metabolic engineering to produce other glycosaminoglycans.

Key words: chondroitin, Escherichia coli, metabolic engineering, pathway optimization

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