重组大肠杆菌1,2,4-丁三醇合成途径的平衡优化

Balanced optimization of the 1,2,4-butanetriol synthesis pathway in recombinant Escherichia coli

1,2,4-丁三醇(1,2,4-butantriol,BT)属于非天然化学品,是军工材料1,2,4-丁三醇三硝酸酯的前体。在重组大肠杆菌中,木糖经脱氢、脱水、脱羧和还原合成BT。但途径各反应不平衡使得中间代谢物积累限制菌体生长和产物合成。本研究首先通过CRISPR/Cas9敲除基因yjh G和yqh D构建无本底表达宿主菌,随后利用不同启动子组合调节BT合成途径中基因xdh、yjh G和yqh D的表达。结果发现,以P_(inv)表达醇脱氢酶基因yqh D使BT产量达到14.4g/L;以P_(tac)表达脱氢酶基因xdh和P_(rrn HP1)表达脱水酶基因yjh G的组合方式,BT产量达到15.6g/L,比对照菌株KXW3009分别增加5.9%和14.7%。本研究通过对中间代谢物木糖酸合成和代谢的组合优化,促进了BT的合成,为后续研究提供了借鉴。

1,2,4-Butantriol (BT) is a non-natural chemical product,which is the precursor of 1,2,4-butantriol trinitrate of military materials.In recombinant E.coli,BT was synthesized from xylose by dehydrogenation,dehydration,decarboxylation and reduction.However,the unbalanced reactions of the pathways lead to the accumulation of intermediate metabolites,which limits bacterial growth and product synthesis.In this study,CRISPR/Cas9 was used to knock out genes yjh G and yqh D to construct a backgroundless expression host bacteria,and then different promoter combinations were used to regulate the expression of genes xdh,yjh G and yqh D in the BT synthesis pathway.It was found that expression of the alcohol dehydrogenase gene yqh D with P_(inv) resulted in BT yield of 14.4g/L and the combination of dehydrogenase gene xdh expressed by P_(tac) and dehydrase gene yjh G expressed by P_(rrnHP1) made BT yield reach 15.6g/L,which was 5.9% and 14.7% higher than that of the control strain KXW3009,respectively.This study facilitated the synthesis of BT through the regulation of upstream and downstream gene expression of intermediate metabolites,which provides a reference for subsequent studies.