代谢工程改造大肠杆菌生产软骨素

Metabolic engineering of Escherichia coli for chondroitin production

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 BL21STAR(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。

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.