Heterologous Expression Of The Type? Lantibiotic Biosynthetic Gene Cluster LAH

Lantibiotics have large development value and widely application future because of their good biological activity and difficult to produce drug resistance.In our previous study,a putative type I lantibiotic biosynthetic gene cluster(lah)was found in the whole genome sequence analysis of Streptomyces hygroscopicus sp.SH-62.In this study,the lah cluster was introduced into different host strains for heterologous expression in order to find its corresponding products and explore gene functions of the cluster.Firstly,two commonly used Streptomyces strains,Streptomyces albus and Streptomyces lividans ZAY3,were selected as hosts for heterologous expression,and the BAC plasmids containing the complete lah cluster were introduced by conjugation to construct the heterologous expression strains.However,the expected lantibiotic compound could not be detected in their fermentation products by HPLC analysis,which might be due to silence or low expression level of the lah cluster.Subsequently,five key lantibiotic biosynthetic genes,lah A,lah B,lah C,lah T and lah G,were respectively cloned downstream of the constitutive strong promoters Perm E and Pazi A4 in the Streptomyces self-replicative vector p YH7,giving rise to the gene overexpression plasmid p WRN01.It was transferred into the heterologous expression strains to yield the gene overexpression strains by conjugation.The expected lantibiotic was not detected in the gene overexpression strains by HPLC as well.RT-PCR analysis showed that the lantibiotic propeptide encoding gene lah A was transcribed normally in both heterologous expression strains and overexpression strains.Meanwhile,q RT-PCR data reveled that the transcription level of lah A in the gene overexpression strains was significantly higher than that in the heterologous expression strains,which confirmed the success for construction of the gene overexpression strains.Obviously,methods for detection and identification of the lantibiotic product need to be optimized or improved.Secondly,Escherichia coli BL21(DE3)commonly used for protein expression,was selected as the host strain for heterologous expression.The propeptide encoding gene lah A,the dehydratase gene lah B and the cyclase gene lah C were respectively cloned into the expression vectors p RSFDuet and p ACYCDuet to construct the double-plasmids coexpression system for expression of these three key genes in one cell.The GFP-tag was fused into the N-terminal of Lah A for convenient detection of the target protein and minimizing the toxicity of lantibiotic to cells.The green fluorescence signal of the fusion protein GFP-m Lah A was observed by fluorescence microscope and the purified protein was obtained by affinity chromatography.The GFP tag was removed by WELQ protease to produce the lantibiotic propeptide m Lah A.In addition,the protein overexpression strains for Lah B,Lah C and Lah G were also constructed and the purified Lah B protein was obtained,which laid a foundation for the study of post-translational modification of lantibiotic propeptide in vitro.