Synthesis Of Lactate And Polyhydroxyalkan-oate By Escherichia Coli From Acetate

Acetate is an important chemical product,which can be obtained at low cost from lignocellulose hydrolysate or synthetic gas fermentation,and has potential advantages as a raw material for biosynthesis.A large amount of L-lactate is used as food additive,and D-lactate is mainly used for the production of polylactate.Polyhydroxyalkanoate(PHA)is a class of biosynthetic,fully degradable polymer materials.In addition to being used as a degradable substitute for non-degradable plastics made from petroleum,PHA can also be used as biomedical materials.Its excellent performance and huge application prospects make PHA more and more a hot spot in biosynthesis research..In this study,new metabolic pathways were constructed in Escherichia coli,using acetate as a carbon source to synthesize lactate and polyhydroxyalkanoates.The main resuts are as follows:(1)The lactate dehydrogenase gene ldh2,which can biosynthesize L-lactate,was screened from Lactobacillus rhamnosus,and its lactate production function was verified in lactate metabolism-deficient strains.And in E.coli K12(DE3),an attempt was made to construct a metabolic pathway that uses acetate as a substrate to synthesize lactate.The resulting strain accumulates pyruvate by aerobic fermentation in a minimal medium with acetate as the sole carbon source.0.61 g·L-1,and enhancement of acetate kinase and phosphoacetyltransferase and the addition of the intermediate metabolite malate acid increased the accumulation of pyruvate by 90%and 125%.(2)In E.coli JM109SG,the genes poxB,pflB,and aceEF that encode pyruvate flow to acetate and acetyl-CoA were deleted,and a defective strain E.coli JM109SG3 that relied on acetate growth was constructed.Constructed to express coenzyme A transferase(Pct),?-ketothiolase(PhaA),acetoacetyl-coenzyme A reductase(PhaB)and PHA polymerase(PhaC)to synthesizecopolyesters of lactate and 3-hydroxybutyrate P(LA-co-3HB)approach,the resulting strain accumulated P(LA-co-3HB)in acetate or acetate and glucose mixed medium.When the concentration of glucose in the medium increases,the molar ratio of lactate in PHA also increases.The strain was cultured in a minimal medium containing glucose and acetic acid for 48 h,the highest cell dry weight was 4.36 g·L-1,the P(LA-co-3HB)yield was 2.29 g·L-1,and the molar content of lactic acid was 7.12 mol%.The high expression of acetate kinase and phosphoacetyltransferase can obviously promote the metabolic utilization of acetate.In addition,an attempt was made to construct a copolymer P(LA-co-3HB-co-4HB)which contain lactate,3-hydroxybutyrate and 4-hydroxybutyrate,as well as the metabolic pathway of the copolymer P(LA-co-GA-co-3HB)which contain lactate,glycolate and 4-hydroxybutyrate.