Optimizing Synthetic Pathways Of Lipid A To Improve Lipopolysaccharide Production In Escherichia Coli

Lipopolysaccharide（LPS）is the main component in the outer membrane of Escherichia coli.It plays an important role in forming the essential permeability barrier,protecting cells from physical damage and maintaining cell morphology.LPS consists of lipid A,core oligosaccharide and O-antigen.Lipid A can induce immune response,and thus is often used as an efficient immune adjuvant.In this study,a series of metabolic engineering strategies were taken to Escherichia coli MG1655 to generate an efficient and non-resistant strain to produce LPS.The main research conclusions are as follows:1)Gene knockout was performed in MG1655 by CRISPR-Cas9 knockout system.Several genes which affect key enzyme activities in lipid A synthesis pathway were selected.Knockdown of gene pts O solved the inhibition of Lpx D activity by Npr.Knockdown of fab F gene reduced the inhibition of Lpx K activity by saturated fatty acids.Knockdown of rap Z partially relieved the negative feedback inhibition of intracellular UDP-Glc NAc,which is the substrate for the first step of LPS synthesis.Compared with MG1655,the yield of the three single knockout mutant strains was increased by 14.6%,27.6%and 25.0%,respectively.Strain MWD001 was constructed by combined knockout of the above three genes,and the LPS production was 30.6%higher than that of MG1655 and reached 10.10 mg·g^-1 DCW.2)After transcriptional analysis of key genes,lpx D、lpx A and lpx B which located in the upstream of lipid A synthetic pathways were enhanced to further enhance LPS production along with msbA.The MWD001/p WSK29-DA and MWD001/p WSK29-msbA were constructed.The plasmid p WSK29-DA contained the lpx D-fab Z-lpx A-lpx B gene cluster while p WSK29-msbA contained msbA.However,the lipopolysaccharide yield of the two strains did not increase.3)Transcriptional level of key genes was analyzed in MWD001/p WSK29-DA.The strain MWD002 was constructed by inserting the inducible promoter P_BAD into the lpx D-fab Z-lpx A-lpx B gene cluster in MWD001 genome.Promoter replacement in MWD001further improved the yield of LPS at a proper concentration of inducer.The LPS yield was improved to 21.3%and reached 12.24 mg·g^-1 DCW.Finally,A series of E.coli with increased LPS production were obtained by optimizing the lipid A synthesis pathway.