Fermentation Of Heparoan And Gene Knockout Of RpoS In E.Coli K5

Heparin is a polysaccharide anticoagulant that is widely used in clinical treatment of thromboembolism,miocrdil infrction and hemodialysis and so on.At present almost all the heparin is extracted from pig intestine and animal-sourced heparin is easily to be tainted,so reasearch on a safer and more reliable way to produce heparosan is vital important.Heparosan is an acidic polysaccharide,biosynthesized as the polysaccharide capsule in Escherichia coli K5.Due to its similar carbonbone structure to heparin and heparan sulfate,heparosan could be utilized as the precursor polysaccharide to biosynthesis of heparin and heparan sulfate via bioengineering technology.This study aimed to improve E.coli K5 biomass and heparosan productivity via the optimization of culture media,conditions,and fed-batch strategies.Furthermore,the rpoS gene in E.coli K5,which is the critical gene for the biosynthesis of acetic acid in E.coli,was knocked out via Red homologous recombination technique.Meanwhile,to directly determine the heparosan content in the E.coli K5 fermentation supernatant,an improved enzymatic method(Heparin lyase III)was established.The carbon source,nitrogen source,K2HPO4 and inoculum were optimized with one-factor experiment.After comparing three different fed-batch strategies,it was found that pseudo-exponential feeding method with ammonium hydroxide was the best feeding strategy for E.coli K5 fermentation,with which the highest productivity of 5.4 g DCW/L biomass and 1.3 g/L heparosan was achieved.Heparin lyase III(Hep III)method is previuoly estabalished for quantitation of heparosan based on the specific reaction of Hep III catalyzed depolymerization of heparosan.However,the previous method could only be utilized to detect the heparosan as crude extract.Thus,the conditions of Hep III method were improved to determine the content of heparosan in the fermentation supernatant directly.RpoS gene of E.coli K5 was successfully eliminated by using λ-Red recombination system.Comparing the growth conditions of wide type and rpoS mutant through flask fermentataion in glucose synthetic medium,acetic acid was less produced in rpoS mutant,while the biomass and heparosan productivity was not increased.