Succinic Acid Production By Metabolic Escherichia Coli Controlled By Anaerobically-induced NirB Promoter

With worldwide deficiency of energy sources and worsening of ecosystem environment,bio-based production of chemicals,fuels,and polymers from renewable and inexpensive materials has been attracting much attention.Succinic acid,also known as amber acid,is a basic platform compound with great economic value.It also could be used as raw materials for the synthesis of 4-amino butanoic acid,tetrahydrofuran,and degradable biopolymer polybutylene succinate?PBS?.The chemical synthesis of succinic acid has shortcomings for example complex techniques,high cost and serious environmental pollution.However,as its low cost,low environmental pollution,high purity product and high production rate,microbial fermentation has been favored.In this study,the engineered strains SD133,SD134,HD133 and HD134 was constructed for succinic acid production.The engineered strains SD133,SD134,HD133and HD134 could produce 15.22 g/L,16.30 g/L,15.90 g/L and 17.46 g/L succinic acid.The result demonstrated the engineered strains with co-expression of ecaA and pepc could obtain a higher titer of succinic acid compared with the individual ecaA expression.The succinic acid produced by engineered strains SD133,SD134,HD133and HD134 concentration increased to 16.84 g/L,17.94 g/L,17.42 g/L and 19.32 g/L after 48 h on mixed sugars,which increased by 8.28%,10.06%,9.56%and 10.65%,respectively.The result suggested that the ability to produce succinic acid was facilitated by using mixed sugars as substrate.The sweet potato waste?SPW?hydrolysate was used as a direct carbon resource to synthesize succinic acid.Compared with using glucose as direct carbon resource,the succinic acid produced on SPW hydrolysate by engineered strains SD133,SD134,HD133 and HD134 increased to 16.01.g/L,17.18 g/L,16.73 g/L and 18.65 g/L,which improved production 5.19%,5.40%,5.22%and 6.82%,respectively,and decreased76.08%fermentation cost.This proved that the engineered strains could efficiently use the SPW hydrolysate to produce succinic acid.The anaerobically-induced nirB promoter cloned from Salmonella choleraesuis C500(P_SnirB)was firstly used for the synthesis of succinic acid.Exogenous genes might be expressed by anaerobically-induced P_SnirB promoter that could endogenously response to anaerobical environments.It could omit the cost of exogenous inducers,cut fermentation cost and simplify fermentation operation.Two-step fermentation strategy,which comprises of aerobic culture of cell growth and then anaerobic fermentation,is necessary and often adopted in succinic acid biosynthesis in recent decades.An feasible strategy for the substitute of promotors induced by exogenous inducers might appear to exist in the anaerobically-induced P_SnirB promoter.The results showed,compared with engineered strains SD133 and SD134 under IPTG-induced Trc promoter control,that a higher concentration of succinic acid could be obtained by engineered strains HD133and HD134 under the anaerobically-induced P_SnirB promoter control on glucose,mixed sugar or SPW hydrolysate.The engineered strains HD133 and HD134 could produce16.73 g/L and 18.65 g/L succinic acid,but engineered strains SD133 and SD134 could produce 16.01 g/L and 17.18 g/L succinic acid on SPW hydrolysate,which increased by4.50%and 8.56%and added no expensive IPTG.Compared with the use of glucose and IPTG,using SPW hydrolysate and anaerobically-induced P_SnirB could decrease 80.36%fermentation cost;Compared with the use of glucose and lactose,it could save 76.84%fermentation cost.Convert the SPW to available sugars could realize high-value utilization of biomass,and reduce environmental pollution.It would be a viable option for large-scale industrial production and applied to other industrial products fermentation executed under anaerobic condition.