食品与发酵工业 >

2022 , Vol. 48 >Issue 24: 8 - 15

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.031426

研究报告

合成5-氨基乙酰丙酸谷氨酸棒杆菌的代谢工程构建

  • 李宇虹 ,
  • 李贵荣 ,
  • 杨文君 ,
  • 孟燕 ,
  • 王浚哲 ,
  • 张成林
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  • (天津科技大学 生物工程学院,天津,300457)
第一作者：硕士研究生(张成林教授为通信作者,E-mail:zcl@tust.edu.cn)

收稿日期: 2022-03-07

  修回日期: 2022-03-23

  网络出版日期: 2023-01-06

基金资助

国家自然科学基金青年科学基金项目(32170049);国家重点研发计划(2021YFC2101800);大学生创新创业计划项目(202110057132;202110057314)

收起

Construction of Corynebacterium glutamicum for synthesis of 5-aminolevulinic acid

  • LI Yuhong ,
  • LI Guirong ,
  • YANG Wenjun ,
  • MENG Yan ,
  • WANG Junzhe ,
  • ZHANG Chenglin
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  • (College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China)

Received date: 2022-03-07

  Revised date: 2022-03-23

  Online published: 2023-01-06

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摘要

作为高附加值氨基酸衍生物,5-氨基乙酰丙酸具有重要的生理活性,被广泛应用于医药、农业和畜牧业。为强化5-氨基乙酰丙酸的合成,该研究以前期构建的谷氨酸棒杆菌AL6为出发菌株,首先过表达异柠檬酸脱氢酶编码基因icd强化α-酮戊二酸合成;然后利用弱启动子控制α-酮戊二酸脱氢酶以弱化α-酮戊二酸降解,菌株AL8的5-氨基乙酰丙酸产量达到1.92 g/L。利用动态调控策略调节5-氨基乙酰丙酸的降解,有效降低了副产物胆色素原和血红素的生成。在此基础上证明了谷氨酸棒杆菌苏氨酸输出蛋白ThrE具有输出5-氨基乙酰丙酸的功能,但大肠杆菌苏氨酸输出蛋白RhtA效果更佳,采用基因组整合过表达rhtA的方式效果最好,所构建菌株AL12的5-氨基乙酰丙酸产量为2.69 g/L。该研究可为提升5-氨基乙酰丙酸的发酵合成效率提供参考。

关键词： 谷氨酸棒杆菌; α-酮戊二酸; 5-氨基乙酰丙酸; 动态调控; 血红素

本文引用格式

李宇虹 , 李贵荣 , 杨文君 , 孟燕 , 王浚哲 , 张成林 . 合成5-氨基乙酰丙酸谷氨酸棒杆菌的代谢工程构建[J]. 食品与发酵工业, 2022 , 48(24) : 8 -15 . DOI: 10.13995/j.cnki.11-1802/ts.031426

Abstract

As a value-added amino acid derivative, 5-aminolevulinic acid has been widely used in medicine, agriculture and husbandry due to the excellent activities. In this study, previously constructed C. glutamicum AL6 was used as an original strain to promote the productivity of 5-aminolevulinic acid. The isocitrate dehydrogenase-encoding gene icd was overexpressed to enhance the α-ketoglutarate and NADPH supply, the 5-aminolevulinic acid production by the resulting strain AL7 was increased by 18.3%; then a weak promoter PdapB was employed to downregulate the activity of α-oxoglutarate dehydrogenase complex. As a result, the α-oxoglutarate dehydrogenase complex activity was significantly decreased, and 1.92 g/L of 5-aminolevulinic acid was produced. However, accumulation of porphobilinogen and heme was observed. Dynamic regulation was applied to modulate the degradation of 5-aminolevulinic acid, which significantly decreased the concentration of porphobilinogen and heme. Meanwhile, the 5-aminolevulinic acid was significantly increased to 2.15 g/L. Finally, the effect of overexpressing exporter encoding gene on 5-aminolevulinic acid production was investigated. The efflux of 5-aminolevulinic acid by ThrE (an L-threonine exporter) of C. glutamicum was identified. Overexpression of rhtA resulted in much more improvement of 5-aminolevulinic acid than thrE. Furthermore, the effect of overexpressing rhtA by genome integration was better than by plasmid, and 2.69 g/L of 5-aminolevulinic acid was produced by the final strain AL12. This study could provide reference for improving synthesis of 5-aminolevulinic acid.

Key words： Corynebacterium glutamicum; α-ketoglutarate; 5-aminolevulinic acid; dynamic regulation; heme

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