Construction Of Integrated 1,2,4-butanetriol Synthesis Strain And Optimization Of Fermentation Conditions

D-1,2,4-butanetriol?BT?is a non-natural biosynthesis chemical with good application prospects in the fields of cosmetics,military industry and medicine.So far,the expression of the foreign gene to synthesize BT by microbial metabolism still depends on the free plasmid which is instability limits future industrial applications.Therefore,this study uses molecular means to construct a more stable integrated BT synthetic strain,knocking out the xylose branch metabolic pathway,weakening the PTS system to achieve xylose and glucose co-substrate fermentation,intensifying the expression of pentose transporter and blocking the 3,4-dihydroxybutyric acid by-product pathway.Finally,cheap lactose instead of IPTG to induce the expression of foreign genes was optimized,providing reference for subsequent amplification studies.In order to construct an integrated BT synthetic strain and knock out the xylose branch metabolic pathway,the exogenous genes kivD and xdh were knocked into the xylAB site by Red recombination technology which is the key gene of the xylose branching metabolic pathway in Escherichia coli.The enzymatic determination and PCR verification of kivD and xdh showed that the integration and knockout were completed meanwhile.The conversion of xylose to BT was 0.7 g·L^-1,and the conversion rate of xylose was 0.12 mol·mol^-1.It was added 5 g·L^-1glucose that had alleviated the growth restriction of recombinant bacteria under the single carbon source.The biomass of the cells was increased by 36%with the rate of xylose metabolism 33%decrease and the yield of BT was only 0.59 g·L^-1.In order to inhibit the above CCR effect and realize xylose and glucose co-substrate fermentation,the exogenous genes kivD and xdh integrated into the ptsHI site using the same technology.The BT yield was up to 2.8g·L^-1 with increasing the exogenous genes copy number and modifying PTS system.To improve the BT yield of engineering bacteria,explored the enhancement the efficiency of xylose transport by expression of pentose transporter and blocking the 3,4-dihydroxybutyric acid by-product pathway.By overexpressing different pentose transporter genes found that high expression of proton co-transporter protein?XylE?is more conducive to the synthesis of BT.The yield is increased to 4.37 g·L^-1.Recombinant bacteria have knocked out xylose isomerase?xylA,xylB?genes and aldolase genes?yagE,yjhH?in bypass metabolic pathway of intermediate metabolite 2-keto-3-deoxyxylic acid?KDX?to further improve carbon flux of main metabolic pathway.Using the CRISPR-Cas9 gene editing technology knocked out the 3,4-dihydroxybutyric acid by-product pathway.The loss of aldehyde dehydrogenase gene feaB effectively enhanced the carbon flux competitiveness of the main metabolic pathway and the conversion efficiency of xylose.The titer of BT enhanced by 37%and reached 6.0 g·L^-1.For further increasing BT yield and cost reduction,the study investigated optimizing the substrate sugar concentration and used lactose replaced IPTG to induce the foreign genes expression.The researches showed that increasing the substrate xylose and glucose concentration is beneficial to growth of bacteria and BT synthesis.BT at the highest titer of 6.8g·L^-1 after optimization.The non-toxic and inexpensive lactose instead of IPTG was used to induce BT synthesis by engineering bacteria with optimization the conditions of lactose concentration,induction timing and induction temperature.The comparison showed that the yield of BT was up to 4.5 g·L^-1 with the xylose conversion rate at 0.31 mol·mol^-1.Then the engineering bacteria were subjected to a 5 L fermenter amplification test and optimization fermentation conditions.The constant aeration and high agitation speed were beneficial to the bacteria growth.Compared with the shake flask fermentation,the biomass was increased by38%and BT production reached 5.9 g·L^-1.The research has laid the foundation for subsequent amplification experiment on the stability of BT recombinant bacteria and the integration bacteria co-substrate fermentation.