Construction Of E.coli K5 △pta Strain And Analysis Of Its Capsular Polysaccharides

Heparin,the most widely used anticoagulant drug in clinical practice,has the effects of lowering blood fat,anti-allergic,anti-inflammatory,anti-tumor and anti-virus.Heparosan,the capsular polysaccharide of E.coli K5,has a similar structure to heparin.And it can be catalyzed to heparin through chemical methods or enzymatic methods.Thus,a large number of heparosan may be preparation by E.coli K5,and turned into heparin to meet market demand.Before this study,we had researched the growth of E.coli and found that,acetate would be accumulated when the substrate was glucose.The large accumulation of acetate severely inhibit the growth of cells and is not conducive to the synthesis of heparosan.So,after analyzing the acetate metabolism pathway of E.coli K5 genome,the phosphotransacetylase was found to catalyze the conversion of acetyl-CoA to acetyl phosphate.It was encoded by pta gene which is a key gene in the metabolism of acetic acid.Thus,the pta gene was knocked out to construct E.coli K5Δpta by CRISPR/Cas9.After cultured in glucose medium for 24 hour,the content of acetate in the mutant culture was 0.132 g/L,while it was 2.86 g/L in the wild culture.It showed the knockout of pta successfully reduced the accumulation of acetate.However,the cell density of the mutant was similar to that of the wild.And the pH of the mutant culture was still between 5 and 6,that was similar to the wild.This may be due to the production of other organic acids after blocked acetate metabolic pathway.And organic acids inhibit the growth of cells.The capsular polysaccharides produced by fermentation of the two strains were characterized.It was found that the yield of heparosan from two strains was similar.This showed knockout of pta didn’t affect the yield of heparosan.What is interesting is that we found the heparosan weight of the mutant is reduced after analyzing by GPC and polysaccharide gel electrophoresis.The more low molecular weight heparosan may be more beneficial to the preparation of low molecular weight heparin.