Construction And Preliminary Application Of Constitutive Expression Vectors For Lactobacillus Brevis

Lactobacillus brevis,a generally recognized as safe?GRAS?food-grade microorganism,is an important member of the genus Lactobacillus.It has been widely used in food,medical and aquaculture industries.Research on the function and application of L.brevis has attracted the attention of many researchers,but due to the low electrotransformation efficiency,the difficulty of foreign gene expression and the lack of available expression vectors in L.brevis,there are few reports about its genetic modification.If a suitable expression vector for L.brevis can be constructed,and we modify it by genetic engineering methods,it will be beneficial to make the full use of L.brevis.In this study,based on the intracellular protein expression vector p MG36e of Lactococcus lactis,the vector's P₃₂promoter the endogenous promoters of L.brevis CICC6239(P_{slp A},P_slp,P_ldha,P_gntk,P_pyk)were screened for exogenous gene expression in L.brevis,using green fluorescent protein gene as a reporter gene.The results indicated that the promoter of the pyruvate kinase gene(P_pyk)could successfully initiate GFP expression in L.brevis CICC6239and the novel constructed vector p MG36B-P_pyk could be used as a constitutive expression vector for L.brevis.In this study,the glycerol-3-phosphatase dehydrogenase genes?Sc GPD1 and Sc GPD2?and glycerol-3-phosphatase genes?Sc GPP1 and Sc GPP2?derived from Saccharomyces cerevisiae were respectively cloned into the vector p MG36B-P_pyk and transferred into L.brevis CICC6239 to exam their expression.The results showed that only p MG36B-P_pyk-Sc GPD2 and p MG36B-P_pyk-Sc GPP2 were successfully transferred into the host cells.The GPD2 was successfully expressed with the enzyme activity of 19.8 m U/m L,while the enzyme activity of GPP2 could not be detected.In addition,in this study,another vector(p MAD-P_pyk)based on the plasmid p MAD was constructed by cloning the P_pyk expression cassette of the p MG36B-P_pyk into the p MAD backbone.Three exogenous genes:GFP,Sc GPD1 and Sc GPP2 were cloned into the p MAD-P_pyk to construct three new plasmids p MAD-P_pyk-GFP,p MAD-P_pyk-Sc GPD1 and p MAD-P_pyk-Sc GPP2.However,the newly constructed p MAD-P_pykserie plasmids failed to be transformed into L.brevis cells.