食品与发酵工业 >

2024 , Vol. 50 >Issue 7: 9 - 16

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.035669

研究报告

在明串珠菌构建高产甘露醇的细胞工厂

  • 刘成川 ,
  • 金红星
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  • (河北工业大学 化工学院,天津,300401)
第一作者:硕士研究生(金红星副教授为通信作者,E-mail:jinhx87@126.com)

收稿日期: 2023-04-02

  修回日期: 2023-05-08

  网络出版日期: 2024-05-09

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The construction of cell factory with high mannitol yield in Leuconostoc

  • LIU Chengchuan ,
  • JIN Hongxing
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  • (School of Chemical Engineering and Technology, Hebei University of Technology, Tianjin 300401, China)

Received date: 2023-04-02

  Revised date: 2023-05-08

  Online published: 2024-05-09

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摘要

甘露醇是一种具有多种功效的六糖醇,被广泛应用于医药、食品、化工和电子等行业。为构建高产甘露醇的明串珠菌,建立了将底盘细胞转化为高产目标产物菌株的理论:细胞工厂的“源-库”学说。通过2次同源重组,打破染色体中基因的操纵子和重叠基因模式,并将甘露醇外排泵蛋白(mannitol efflux pump,MEP)基因序列和再生NADH的酶(甲酸脱氢酶)基因序列定点插入到染色体上。蔗糖为90 g/L时,打破mep基因的操纵子和重叠基因模式的菌株甘露醇产量比原始菌株(CGMCC1.10327)提高了9.5%。原始菌株染色体上敲入一个拷贝mep基因序列的菌株,底物蔗糖能添加到120 g/L,甘露醇产量也达到55.18 g/L。CCTCC M2020762菌株染色体上敲入一个拷贝甲酸脱氢酶基因序列的菌株,底物蔗糖能添加到145 g/L,甘露醇产量也达到101.6 g/L。使细胞工厂具备更强的“源”能(NADH再生等)和更大的“库”容(提高MEP活性),实现目标产物的超高产。

关键词： 细胞工厂; 源-库; 明串珠菌; 甘露醇; 发酵; 合成生物学

本文引用格式

刘成川 , 金红星 . 在明串珠菌构建高产甘露醇的细胞工厂[J]. 食品与发酵工业, 2024 , 50(7) : 9 -16 . DOI: 10.13995/j.cnki.11-1802/ts.035669

Abstract

Mannitol is a hexasugar alcohol with many functions, which is widely used in medicine, food, chemical industry and electronics.In order to construct Leuconostoc with high yield of mannitol, the theory of transforming chassis cells into high-yield target product strain—the “source-sink” theory of cell factory was built.Through two homologous recombination, the operon and overlapping gene patterns were broken, and the mannitol efflux pump (MEP) gene sequence and the NADH-regenerating enzyme (formate dehydrogenase) gene sequence were site-direct inserted into the chromosome.When sucrose was 90 g/L, the mannitol yield of the strain that broke the operon and overlapping gene pattern of mep gene was 9.5% higher than that of the original strain (CGMCC1.10327).A strain with a copy of mep gene expression cassette was knocked into the chromosome of the original strain, and the substrate sucrose could be added to 120 g/L, and the output of mannitol reached 55.18 g/L.A strain with a copy of formate dehydrogenase gene expression cassette was knocked into the chromosome of CCTCC M2020762[Δdts1ΔldhΔpat::mdhΔstpk::mdhΔfk::mdhΔaldh::(mtld-mlp) 0385/0386::ldhA-eeΔadh::mepΔldh(0503)::mepΔldh(0373)::mep], and the substrate sucrose could be added to 145 g/L, and the output of mannitol reached 101.6 g/L.Make the cell factory have stronger “source” energy (NADH regeneration, etc.) and larger “sink” capacity (improve MEP activity), and obtain super-high yield of the target product.

Key words： cell factory; source-sink; Leuconostoc; mannitol; fermentation; synthetic biology

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