Effects Of Overexpression Of Key Enzymes Of Lactose Metabolic Pathway On Utilization Efficiency From Lactose In2,3-Butanediol Production Strain

As an important bio-based chemical product,2,3-butanediol has a wide range of applications in many fields, such as chemical, fuel, food and aerospace. As the main by-product of dairy industries, cheese whey is mainly to produce fuel ethanol in the application of chemical industry. Compared with the production of fuel ethanol,2,3-butanediol is a higher value-added product. Enterobacter cloacae CICC10011is widely used to produce2,3-butanediol with higher product concentration and lower fermentation speed in the lactose medium compared to Klebsiella pneumoniae KG1. The purpose of this study is to improve the utilization efficiency of lactose in KG1and CICC10011by enhancing the key enzymes expression level of lactose metabolic pathway, such as lactose permease and P-galactosidase.The main research contents and results were follows:(1)The KG1and10011strains were cultured in lactose fermentation medium containing added exogenous P-galactosidases to analyse the effects of different addition level of enzymes on the lactose uptake rate. With the addition of β-galactosidases increased the lactose uptake rate of both KG1and10011.(2)The bgaB gene encoding β-galactosidases and KlacY gene encoding K. pneumoniae lactose permease of KG1, the ElacY gene encoding E.coli MG1655lactose permease were overexpressed in both KG1and10011using the plasmid pUC18K, forming KG1/pUC18K-bgaB, KG1/pUC18K-ElacY, KG1/pUC18K-KlacY,10011/pUC18K-bgaB,10011/pUC18K-ElacY,10011/pUC18K-KlacY, respectively.(3)The feasibility of bgaB gene overexpression in KG1/pUC18K-bgaB and10011/pUC18K-bgaB were determined by the determination of enzyme activity and SDS-PAGE. The β-galactosidase activities of KG1/pUC18K-bgaB and10011/pUC18K-bgaB were33.08-fold and3.45-fold of the parent strains; respectively. Compared with10011,the lactose fermentation time of10011/pUC18K-bgaB is shortened by12h, resulting in a38.70%increase in specific lactose uptake rate and a slightly higher2,3-butanediol production. The lactose uptake rate of KG1/pUC18K-bgaB decreased. At the end of fermentation, the residual lactose concentration of KG1/pUC18K-bgaB was41.03g/L.(4)The experimental results revealed that, the lactose permease activities of KG1/pUC18K-ElacY and10011/pUC18K-ElacY were2.01-fold and1.75-fold of those in the parent strains, respectively. The fermentation speed of both KG1/pUC18K-ElacY and10011/pUC18K-ElacY were increased by25.0%and12.5%and the specific lactose uptake rate were increased by66.2%and46.7%, respectively. Our results suggested that the overexpression of the ElacY gene promoted the lactose uptake rate for the mutants and the2,3-butanediol production were unaffected. (5)The experimental results revealed that, the lactose permease activities of KG1/pUC18K-KlacY and10011/pUC18K-KlacY were1.92-fold and1.73-fold of those in the parent strains, respectively. Results indicated that overexpression of K. pneumoniae lactose permease in KG1/pUC18K-KlacY and10011/pUC18K-KlacY individually did not improve the fermentation speed and the2,3-butanediol production were unaffected.