The Discovery Of Transporter Enabled Escherichia Coli To Produce 1,3-dihydroxyacetone Efficiently

1,3-dihydroxyacetone(DHA)is an important tricarbon compound that can be used as a biochemical feedstock and synthetic reagent in a variety of industries,including fine chemicals,food,cosmetics,and medicine.The main methods of synthesis of DHA are chemical synthesis and microbial fermentation.Compared with chemical synthesis,microbial fermentation is environmentally friendly and accords with the concept of green development.In this study,DHA was produced by E.coli microbial fermentation.Although much has been done in microbial fermentation for DHA production,few studies have been done to further improve DHA production using transporter proteins.This study was conducted by constructing E.coli ATCC 8739 source transporter protein granule library,using DHAproducing TZ-249 as host bacteria,screened and identified DHA-related transporters:(1)Construction of an E.coli ATCC 8739-derived transporter protein pellet libraryThe transporter information of E.coli ATCC 8739 was collected from the Transporter Classification Database(TCDB),and a library of 399 transporter overexpression plasmids was constructed by seamless cloning and CPEC method.(2)Screening of DHA-related transporter proteinsThe constructed transporter overexpression plasmids were introduced into the host bacterium TZ-249,and the transporter proteins that were useful for improving DHA production were screened by shaking flask fermentation and measuring DHA production.(3)Knockdown and overexpression of DHA-related transporter protein genesThe transporter genes obtained from plasmid library screening were knocked out and overexpressed on the host TZ-249 genome using the two-step λRed homologous recombination method and CRISPR-Cas9 gene editing technology.(4)Fermentation of TZ series strainsThe transformed strains TZ-260,TZ-261,TZ-262,TZ-263,TZ-264,TZ-265,TZ-266,TZ-267,TZ-268,TZ-269,TZ-270 and TZ-271 were subjected to shake flask fermentation to determine the DHA yield.The strain TZ-267 was subjected to 5 L fermentor fermentation culture and DHA yield was determined.In the end,strain TZ-267 overexpressing the transfer protein gene ara J showed the most significant increase in DHA yield in shake flask fermentation compared to the control strain TZ-249,reaching 21.4 g/L at 72 h,an increase of 0.72 times.The DHA yield of strain TZ- 267 reached 121.1 g/L after 32 h of fermentation in a 5 L fermenter.