Effects Of σ⁵⁴,Anti-σ²⁸ And σ²⁸ On Colanic Acid Synthesis In Escherichia Coli

Colanic acid（CA）is a kind of extracellular polysaccharides（EPS）secreted by Escherichia coli.The regulation of CA biosynthesis is complex and involves multiple direct and indirect regulators.Currently,the reported high-yielding CA strains are based on the metabolic modification of the starting strain MG1655Δ（L-Q）,which can achieve high CA yield by deleting the negatively regulated genes hns and lon and overexpressing the positively regulated transcription factor Rcs A,etc.The large number of synthetic pathway genes involved makes it difficult to precisely study the effect of transcription factors on clathrate.In this thesis,the effects of inactivating transcription factorσ⁵⁴（Rpo N）,anti-σ²⁸（Flg M）and overexpressing transcription factorσ²⁸（Fli A）on CA synthesis were investigated for E.coli W3110 to further explore the mechanism of CA regulation and to obtain engineered strains with high CA production,with the following main findings:（1）Construction of mutant strains and their effect on clathrate.The mutantΔrpo N,Δflg M,Δrpo NΔflg M were constructed.The comparison of CA production showed that the culture conditions used for CA production were identified as 28℃and M9 fermentation medium.The CA yield ofΔrpo NΔflg M under this condition reached 3,387.29 mg·L^-1.Because CA is a mucilaginous EPS,high CA-producing strains showed slow growth rate and significant expansion of colonies.Growth characteristics showed that the growth rate and maximum biomass ofΔrpo NΔflg M were significantly lower than other strains.The bacterial motility assay showed that the colony diameter ofΔrpo NΔflg M strain cultured for 24 h was 11.20 mm.In addition,theΔrpo NΔflg M mutant strain could be observed with highly significant green fluorescence under CLSM,confirming the production of EPS by the bacteria.（2）The effect of overexpression of fli A on the synthesis of colanic acid.The p Bad33-fli A overexpression plasmid was constructed,and the recombinant strainsΔflg M/p Bad33-fli A,Δrpo N/p Bad33-fli A,Δrpo NΔflg M/p Bad33-fli A and W3110/p Bad33-fli A were constructed.The comparison of CA yield revealed thatΔrpo NΔflg M/p Bad33-fli A yield was 11,976.34 mg·L^-1after 72 h,while CA yield reached 16,175.67 mg·L^-1 after extending the fermentation time to120 h.Bacterial motility experiments revealed thatΔrpo NΔflg M/p Bad33-fli A colonies reached14.30 mm in diameter,and the phenomena observed by AFM were consistent with motility,with a significant increase in the number ofΔrpo NΔflg M/p Bad33-fli A flagella.（3）Differential transcriptomic analysis ofΔrpo NΔflg M mutant.The results showed that compared with W3110,122 genes were up-regulated and 60 genes were down-regulated inΔrpo NΔflg M,19 genes were up-regulated and 1 gene was down-regulated in colanic acid synthesis pathway.In addition,differential analysis of synthetic pathways showed thatΔrpo NΔflg M exhibited significant differences in 11 KEGG items,including sugar metabolism,biofilm formation,etc.Changes in the abundance of the carrageenan synthesis gene cluster showed that the key genes encoding nucleotide sugars,glycosyltransferase,and invertase were all significantly up-regulated.The results showed that the combined deletion of transcription factorsσ⁵⁴ and anti-σ²⁸ could significantly promote the up-regulation of CA synthesis gene transcription,which is one of the key reasons for CA synthesis.