Effects Of Interrupting Branch Pathways Of Glycolysis On Heterologous Protein Expression In Escherichia Coli

Escherichia coli is one of the most widely used hosts for the production of heterologousproteins. However, rapid growth under aerobic condition results in formation of byproductsthrough glycolytic pathway, which was considered to affect cell viability; however, little wasknown about that. In this article, disturbance of glycolytic pathways by gene deletions inEscherichia coli B0013was carried out for evaluation of their effects on recombinant proteinproductivity in various mutants.Firstly, D-lactate dehydrogenase (ldhA), acetate kinase-phosphotransacetylase (ackA-pta),phosphoenolpyruvate synthase (pps), pyruvate formate lyase (pflB), pyruvate oxidase (poxB),alcohol dehydrogenase (adhE), and fumarate reductase (frdA) were inactivated respectively.The resulting7mutants were used as hosts for evaluating expression of heterologous proteins.As a reported gene, β-mannanase gene (man) was cloned from Bacillus amyloliquefaciens andtransformed into above hosts and recombinant strains were obtained. The growth pattern andmetabolites were characterized and β-mannanase activity were detected.The results showedthat gene deletions were helpful in decreasing byproducts, but had different effects on proteinexpression. Mutant strain with deletion of gene ldhA stopped accumulating lactate, andshowed a derease in the yield of formate and acetate from glucose by13.2%and17.3%,besides, biomass yield and the specific activity increased by7.8%and11.5%. Mutant strainwith deletion of gene pflB stopped accumulating formate, showed a decrease by10.6%in theyield of acetate from glucose, and an increase by8%and11.8%in biomass yield and thespecific activity. Deletion of gene pps caused an increase by8.1%and7.3%in biomass yieldand the specific activity. Deletion of poxB caused a decrease by25.7%in the yield of acetatefrom glucose, and biomass yield and the specific activity was7.4%and9.5%higher than thatof the wild strain. Mutant strain with deletion of gene ackA-pta showed a decrease in the yieldof acetate from glucose by64.2%compared with the wild strain; although biomass yield andthe specific activity were increased by6.5%and13%, the glucose consumption and biomassyield was decreased by18.9%and13.7%, and the β-mannanase activity of the cells permilliliter of fermentation broth was17.5%lower than that of the wild strain. However,deletion of gene adhE and frdA had no effect on cell growth or expression of heterologousproteins.Multiple genes deletion was also studied with β-mannanase gene (man) as the reportergene. The result showed that cumulative inactivation of genes ldhA, pflB, pps and poxBresulted in an increase by7.2%,5.8%,5.6%and5.2%in biomass yield and11.1%,8%,5.5%and8.9%in the specific activity. Compared with the wild strain, the mutant inactivated of allthe four genes stopped accumulating lactate and formate, showed a decrease by33%in theyield of acetate from glucose, and an increase by25.9%and37.8%in biomass yield and thespecific activity, which demonstrated that blocking multiple metabolic pathways in glycolysisbenefits increasing carbon flow towards biomass and heterologous proteins synthesis.With a debranching enzyme gene (isa) as a reporter gene, the mutant strain B0013-213(ldhA pflB pps poxB) was futher studied on its protein expression. Compared with thewild strain, this mutant didn’t accumulate lactate and formate, and the yield of acetate fromglucose decreased by37.3%. Biomass yield and the specific activity increased by22%and 57.2%, which demenstrated that inactivition of the four genes (ldhA, pflB, pps and poxB) canalso promote the debranching enzyme expression in E. coli.