Metabolic Engineering Modification Of Escherichia Coli Co-Production 1,2,4-butanetriol And Polyhydroxybutyrate

1,2,4-butanetriol（BT）is a kind of high value-added dual-use military-civilian product produced by the fermentation of genetically engineered bacteria E.coli.Important chemicals also face the problem of bacterial processing.Polyhydroxyalkanoate（PHB）is an environmentally friendly product that can be synthesized in E.coli cells.Based on the characteristics of E.coli BT fermentation,this study explored the strategy of co-substrate fermentation of glucose and xylose to co-produce extracellular BT and intracellular PHB to solve the above problems.In order to realize this idea,a BT-producing E.coli was constructed.PHB synthesis pathway,metabolic transformation of PHB synthesis pathway,optimization of the culture conditions in the 5 L fermentor,in order to obtain the E.coli co-production technology of BT and PHB,and explore the industrial green environmental protection biosynthetic method to produce BT and PHB technical route.To achieve the co-production of PHB and BT,the PHB synthesis pathway was from construdted by overexpressing phb CAB from Ralstonia eutropha,and the PHB synthesis was verified by GC-MS and Sudan Black B staining.The IPTG induction concentration at the promoter and shake flask levels was optimized.Finally,E.coli KXW3009P was the optimal strain,the co-production of glucose and xylose to BT and PHB was achieved,and the BT and PHB yield were 10.3 g·L^-1 and 8.1 wt.%,respectively.The co-producing strains were further metabolically modified to enhance product synthesis,using strategies such as back-complementing the PTS system and weakening the acetate pathway.Overexpression and co-expression of galactose permease gene gal P and glucokinase gene glk,both effectively increased the biomass by 18%,12%and 17%.The best single overexpression of glk,reaching 11.9 g·L^-1 and 8.4 wt.%for BT and PHB,which increased by 16.3%and 5%,respectively.Both single knockout and double knockout pyruvate decarboxylase（Pox B）and phosphate transacetylase（Pta）,had a weakening effect on acetate,decreasing by 27.2%,88%and 96%,and the yield per unit bacterium of weakened acetate BT was increased,with single knockout pox B optimal,reaching 13.1 g·L^-1 and 20.7wt.%for BT and PHB,which improved by 27.2%and 156%,respectively.Combining the above two strategies,the single knockout and double knockout of PTS with the weakened acetate pathway both promoted PHB,of which only the double knockout promoted BT synthesis,with BT and PHB reaching 12.7 g·L^-1 and 10.8 wt.%,an increase of 5.8%and27.1%.To further improve PHB and BT production,the fermentation of the co-produced strain in a 5 L fermenter was investigated,and it was found that the growth and metabolism of the strain were concentrated in the first 12 h,and then cell growth,xylose utilization and BT synthesis rate were slowed down.After 24 h,the glucose was completely consumed and the growth of the strain was almost stagnant.For the fermentation characteristics,the effects of one-time replenishment and flow-through replenishment on the co-produced strain were investigated.The results showed that one-time replenishment was the best,and the BT and PHB increased by 51.1%and 40.4%,to 8.9 g·L^-1 and 8.92 wt.%.Further,the induction time,fermentation speed and temperature were optimized,and the BT and PHB yields reached 15.0g·L^-1 and 19.0 wt.%after preliminary optimization.The above research shows that constructing a PHB synthesis pathway in BT-producing E.coli,metabolically transforming the PHB synthesis pathway,and optimizing fermentation conditions in a 5 L fermentor can effectively improve the ability of E.coli to co-produce BT and PHB.That is an effective fermentation strategy to reduce bacteria residue.