食品与发酵工业 >

2023 , Vol. 49 >Issue 23: 16 - 24

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.035600

研究报告

代谢改造大肠杆菌合成8-异戊烯基山奈酚

  • 赵婉莹 ,
  • 周景文 ,
  • 侯颖
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  • 1(天津科技大学 生物工程学院,天津,300000)
    2(江南大学 生物工程学院,江苏 无锡,214122)
第一作者:硕士研究生(侯颖讲师为通信作者,E-mail:houying@tust.edu.cn)

收稿日期: 2023-03-23

  修回日期: 2023-04-14

  网络出版日期: 2024-01-02

基金资助

国家自然科学基金创新研究群体项目(32021005);天津市研究生科研创新项目资助(2022SKYZ092,2022SKYZ094,2022SKYZ099)

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Metabolic engineering of Escherichia coli for 8-prenylkaempferol synthesis

  • ZHAO Wanying ,
  • ZHOU Jingwen ,
  • HOU Ying
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  • 1(College of Bioengineering, Tianjin University of Science and Technology, Tianjin 300000, China)
    2(College of Bioengineering, Jiangnan University, Wuxi 214122, China)

Received date: 2023-03-23

  Revised date: 2023-04-14

  Online published: 2024-01-02

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摘要

8-异戊烯基山奈酚(8-prenylkaempferol, 8-pk)是一种黄酮类化合物,具有抗炎、抗癌等药理活性。目前8-pk的生物合成主要通过植物来源的异戊烯基转移酶(prenyltransferases,PTs)催化山奈酚(kaempferol, Kae)实现。然而植物源的PTs自身含有的信号肽会极大阻碍其在原核生物中异源表达和高效催化,且催化途径中前体物质二甲基烯丙基焦磷酸盐(dimethylallyl pyrophosphate,DMAPP)的供给不足严重限制了8-pk的生物合成产量。该研究以大肠杆菌(Escherichia coli)为底盘细胞,通过强化前体DMAPP供应和截短异戊烯基转移酶信号肽实现了8-pk的高效合成。首先,在E.coli BL21(DE3)中游离表达了淫羊藿来源的异戊烯基转移酶(EkF8DT),并引入异戊烯醇利用途径提升了EkF8DT的前体DMAPP的供应,8-pk产量达到2.14 mg/L。随后,通过AlphaFold2预测了EkF8DT的结构,将EkF8DT N端截短60个氨基酸时8-pk产量最高,达到6.46 mg/L。通过将EkF8DT的N端融合蛋白质标签以及优化发酵条件产量达到24.28 mg/L, 最终在5 L发酵罐中8-pk产量达到44.33 mg/L,这是目前报道的较高水平。该研究为8-pk等类黄酮异戊烯基化的高效生物合成提供了策略。

关键词： 8-异戊烯基山奈酚; 异戊烯基转移酶; 异戊烯醇利用途径; 信号肽截短; 蛋白质标签

本文引用格式

赵婉莹 , 周景文 , 侯颖 . 代谢改造大肠杆菌合成8-异戊烯基山奈酚[J]. 食品与发酵工业, 2023 , 49(23) : 16 -24 . DOI: 10.13995/j.cnki.11-1802/ts.035600

Abstract

8-Prenylkaempferol (8-pk) is a flavonoid with excellent anti-inflammatory and anticancer pharmacological activities. At present, the biosynthesis of 8-pk is mainly obtained from kaempferol (Kae) by the catalysis of plant-derived prenyltransferases (PTs). However, plant-derived PTs contain signal peptides, which greatly hinder their heterologous expression and efficient catalysis in prokaryotes. In addition, the insufficient supply of the precursor molecule dimethylallyl pyrophosphate (DMAPP) in the catalytic pathway severely limits the biosynthesis yield of 8-pk. In this study, Escherichia coli was used as a chassis cell, further enhancing the supply of precursor DMAPP and truncating the signal peptide of prenyltransferase for 8-pk efficient synthesis. Firstly, epimedium derived prenyltransferase (EkF8DT) was expressed in E. coli BL21(DE3) and IUP pathway was introduced to increase the supply of DMAPP, with an 8-pk titer of 2.14 mg/L. Subsequently, the structure of EkF8DT was predicted by AlphaFold2. Subsequently, the structure of EkF8DT was predicted by AlphaFold2 and the highest 8-pk yield of 6.46 mg/L was achieved when the N-end truncation of EkF8DT was truncated by 60 amino acids. By fusing the protein tag to the N-end truncation of EkF8DT and optimizing fermentation conditions a titer of 24.28 mg/L was achieved. Finally, 8-pk titer of 44.33 mg/L was achieved in a 5 L fermenter, which is the higher level reported. This study provides an effective strategy for 8-pk biosynthesis.

Key words： 8-prenylkaempferol; prenyltransferases; IUP pathway; signal peptide truncation; protein tags

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