Metabolic Engineering For Overproduction Of Colanic Acid In Escherichia Coli Mutant WQM001 With Short Lipopolysaccharide

Colanic acid（CA）is a protective exopolysaccharide secreted by cells when exposed to extreme environment or facing with envelope stress.It is widely found in Enterobacteriaceae,Aerobacter,Salmonella and Klebsiella.CA contains about 30%fucose,it might have great biological and physiological functions such as anti-cancer,anti-tumor,anti-inflammatory bioactivities.Besides,CA may be used as water hydrogel due to its porous structure,which also provides a new choice for cosmetic industry,food and pharmaceutical industries.However,there are few studies targeting the overproduction of CA in microbial cell factories presently.In this study,a series of rational metabolic engineering strategies were conducted in WQM001strain with truncated lipopolysaccharide structure to improve CA production,which was also for the good of structural and functional studies.The main research conclusions are as follows:1)The deletion of ack A gene improved CA production.WQM001 was a CA overproducing strain from our lab,constructed by knocking out waa L,waa U,waa Z,waa Y,waa R,waa O,waa B,waa P,waa G,waa Q and lon genes from the chromosome of Escherichia coli K-12MG1655 before this research.The fermentation characteristics of WQM001 strain were analyzed.WQM001 produced 8.57 g·L^-1 citric acid and 6.04 g·L^-1 acetate,the p H of fermentation broth decreased to 5.24 at the end of fermentation which limited the continuous biosynthesis of CA.The ack A gene,pox B gene and acs gene were knocked out from the chromosome of WQM001 respectively,generating WZM001,WZM002 and WZM003 strains.and the combinatorial knockout of ack A,pox B and acs genes in WQM001 led to WZM004strain.CA production by WZM001,WZM002,WZM003,and WZM004 strains in shake flask fermentation were 2.01,0.82,1.17,and 1.1 times that of the control strain,respectively,and the acetate production decreased by 83.9%,62%,11.6%,and 86.6%.Among the three genes,the deletion of ack A gene promoted CA production to the most extent,and CA production by WZM001 strain reached 6.63 g·L^-1.2)The knockout of ack A gene led to the redistribution of carbon flow,promoting the synthesis of CA.The knockout of ack A gene in WZM001 could lead to the accumulation of acetyl-phosphate.WZM005 strain was constructed by knocking out pta gene and ack A gene based on WQM001 to investigate the effect of acetyl-phosphate on CA production,CA production by WZM005 decreased by 36%compared with WZM001 strain.Besides,the transcriptional level of wca gene cluster in WZM005 was lower than that of WZM001according to the RT-PCR data of WQM001,WZM001 and WZM005.Accumulated acetyl-phosphate facilitated the biosynthesis of CA by up-regulating the transcriptional level of wca gene cluster.The whole transcriptional analysis showed significantly up-regulated transcriptional level of genes related to TCA cycle,fatty acid metabolism and butanoate metabolism,indicating accumulated acetyl-Co A in the organism.Low Co A pool could inhibit the enzymatic activity of pyruvate dehydrogenase complex,leading to the accumulation of pyruvate in turn.The altered ratio of phosphoenolpyruvate to pyruvate might lead to a decreased carbon flux through glycolysis.Meanwhile,the transcriptional level of genes involved in the biosynthesis of O-antigen was down-regulated,the carbon flow might be diverted into CA biosynthesis pathway.3)The knockout of genes responsible for synthesizing O-antigen on the basis of WZM001strain furtherly improved CA production.O-antigen synthesis pathway and Enterobacterial Common Antigen（ECA）synthesis pathway compete with the biosynthetic pathway of CA for UDP-galactose,1-phosphate-glucose,UDP-glucose and C₅₅ lipid carrier.The O-antigen synthesis pathway was deleted based on WZM001 strain to construct WZM008 strain,and the ECA biosynthesis pathway was deleted from WZM008 to generate WZM009 strain.Compared with the WZM001 strain,the CA production by WZM008 strain increased by 19%,and the CA production was 7.09 g·L^-1.CA production by WZM009 strain decreased by 76.3%compared to WZM008 strain and RT-PCR results showed down-regulated transcriptional level of wca gene cluster in strain with the ECA gene cluster knocked out.4)Enhancing the supply of C₅₅ lipid carrier in WZM008 strain can further enhance CA synthesis.Plasmid p Trc S was constructed to overexpress upp S gene to provide Und-P.p Trc S was transformed into WZM008 strain to construct WZM008/p Trc S,the overexpression of upp S increased CA production by 13%compared to WZM008/p Trc11.Although the CA production of WZM008/p Trc S(6.51 g·L^-1)is less than that of WZM008(7.09 g·L^-1),CA/OD₆₀₀ of WZM008/p Trc S(2.50 g·L^-1)was 1.42 times that of WZM008(1.76 g·L^-1)at the end of fermentation.WZM008/p Trc S was chosen for fed-batch fermentation.And the CA production of WZM008/p Trc S in 2.0 L bioreactor was 11.68 g·L^-1,which was 3.54 times of colanic acid production by WQM001 strain.In this study,CA production increased by 254%by knocking out ack A gene,knocking out genes responsible for synthesizing O-antigen and overexpressing upp S gene using WQM001 as initial strain.Besides,the mechanism of increased CA production caused by ack A deletion was investigated.All the work might provide preliminary insights into metabolic engineering strategies for CA overproduction.