Construction Of An E. Coli Cell Factory For Efficient Production Of 1,3-Dihydroxyacetone

1,3-Dihydroxyacetone(DHA)is the simplest three-carbon ketone sugar and is widely used in the chemical,pharmaceutical,feed,cosmetic and food industries.Currently,the industrial production of DHA is mainly based on the conversion of glycerol substrate by Gluconobacter oxydans,but there are problems such as high concentration of glycerol substrate and inhibition of bacterial growth by DHA product,by-products and complicated fermentation process.In recent years,with the technological progress in the field of synthetic biology,the construction of efficient microbial cell factories to synthesize DHA using glucose as substrate has gradually become a research trend.In this study,a glucose-based DHA synthesis pathway was developed by(1)introducing a DHAP phosphatase from Corynebacterium glutamicum and inactivating the PTS system;(2)knocking out the key genes of glycerol and DHA metabolic pathways,Glp K and Gld A,and DHA kinase Dha KLM,and blocking the DHAP paralogous metabolic pathway genes such as Tpi A,methylglyoxal synthase and Mgs A.(3)knockdown of glycerol/DHA uptake transporter protein Glp F;(4)inactivation of key genes of the PPP and ED pathways of the 6-phosphoglucose node Zwf and Edd/Eda;(5)introduction of NADH oxidase Nox to reduce by-product glycerol accumulation.However,further studies showed that two limiting factors were the biggest obstacles to improve the productivity of the strain,one was the lack of tolerance of the chassis strain to the product DHA,and the other was the lack of DHAP phosphatase enzyme activity leading to the inhibition of the toxic precursor DHAP by the strain.normal metabolism.In this study,the modified chassis strain was subjected to adaptive evolution and directed evolution of the DHAP phosphatase Cg Hdp A.In this study,the tolerance threshold of the chassis strain was increased to 50 g/L by adaptive evolution,and the yield was increased to 103.12 g/L by fermentation in a 5 L fermenter,and the conversion rate reached the theoretical maximum conversion rate,with a2.5-fold increase in tolerance and a 2.6-fold increase in yield compared to the starting strain.The transcriptome library and whole genome resequencing were used to clarify the mechanism of DHA detoxification,and to identify the targets env Z,nem R,yea E,nem A,sod A,and glo A as regulators of tolerance improvement.In this study,we performed directed evolution of DHAP phosphatase Cg Hdp A,and obtained mutant SF6-11 through six rounds of evolutionary screening,and measured the enzymatic kinetic parameters of SF6-11 and wild-type WT.