Blocking By-product Pathways And Strengthening Key Enzymes Expression Of D-1,2,4-butanetriol Synthesis In Recombinant Escherichia Coli

D-1,2,4-butanetriol (BT) is a sort of non-natural four-carbon polyol widely used in military industry, toiletries, high polymer chemistry and so on. In recent years, a bio-transformation pathway from xylose to BT is established in recombinant Escherichia coli, solving the problems in traditional chemical routes, like environmental pollution, extraction and high cost. But it achieves low production of BT. Based on this synthesis route, knocking out the genes of by-product pathways, overexpressing key enzymes and introducing chaperones are performed to balance cell growth and BT produce, achieving promotion on BT-synthesis ability of recombinant strain.To improve the carbon flux into BT-synthesis, we knocked out xylose isomerase and xylulose kinase genes (xylA and xylB),2-keto-3-deoxy-xylonate (KDX) aldolase genes iyagE and yjhH), together with 2-ketoacid reductase genes (yiaE and ycdW) respectively. Fermentation results showed that xylAB-deficient strain led in increased BT titer per cell by 84%; E. coli AyagE or AyjhH harvested a higher BT production per cell by 23% and 8%, meanwhile, double mutant of AyagE AyjhH(E. coli BTW003) achieved more BT titer by 184%. Genes of yiaE and ycdW were respectively or both deleted in E. coli BTW003, and the results showed that single mutant strains hardly improved BT production, while BT titer of double mutant strains (E. coli BTW004) was raised by 238%, up to 2.91 g·L-1. Further more, 490% promotion was detected in BT yield per cell of the recombinant strain deficient of xylose and KDX by-pathways. However, the decreased biomass led to increased BT titer by 53%. Hence, E. coli BTW004 with higher yield was selected as host strain in following research.For accelerating metabolism from xylonate or 3,4-dihydrobutanal to BT, we overexpressed xylonate dehydratase gene (yjhG) and dehydrogenase gene (yqhD) under different promoters. Fermentation results showed that, overexpression of YjhG or YqhD all brought about lower BT yield per cell compared with E. coli BTW004, suggesting enhanced dehydratase or dehydrogenase shows little effect on improving BT titer, so the limiting step probably be oxidation or decarboxylation reactions catalyzed by heterologous enzymes.In order to elevate the catalyzing activity of heterologous enzymes, chaperones (Trigger factor, GroEL-GroES and DnaK-DnaJ-GrpE) were introduced into recombinant E. coli to assist protein folding, improving enzyme activities and BT titer. Results showed that DnaK-DnaJ-GrpE is detrimental to BT synthesis. Besides, introduction of GroESL increased Xdh activity, but repressed MdlC activity, leading to little incensement on BT yield. Co-expressed with Trigger factor in recombinant strain showed improvement of MdlC and Xdh activities by 11% and 74%, while the final BT titer was increased by 293% comparing to E. coli BTW3110, reaching 3.37 g·L-1. This shows decarboxylation is the crucial step of BT biosynthesis, and confirming the feasibility of introducing chaperones to improve yield, and providing a reference for BT produce.