Construction of a new bacterial artificial chromosome (BAC) vector for cloning of large DNA fragments and heterologous expression in Streptomyces

Abstract:

Abstract:［Objective］Many natural product biosynthetic gene clusters are too large to be entirely cloned into one cosmid for heterologous expression． Because bacterial artificial chromosome (BAC) vectors are well known for their capacity of cloning large DNA fragments，we constructed a new BAC vector for cloning and heterologous expression of natural product biosynthesis gene clusters in Streptomyces．［Methods］The chloramphenicol resistance gene on the original BAC vector pCUGIBAC1 was substituted with a streptomycin resistance gene via λ RED-mediated PCR-targeting technique．The streptomycin resistance gene was then excised by digestion with NheI and the left gap was filled with the origin of transfer (oriT)，the φC31 integrase gene，the integrating attP site，and an apramycin resistance gene．［Results］We achieved the final BAC vector pMSBBACs． To test the newly established vector，pMSBBACs was used to build up a genomic BAC library of Streptomyces U27． The average size of inserts in the library is about 100kb． A 140 kb BAC plasmid as a representative was successfully introduced into heterologous hosts，S．lividans and S．albus，by either conjugation or protoplast transformation． It demonstrated that the BAC plasmids constructed by pMSBBACs could be integrated into chromosomes via site-specific recombination for heterologous expression．［Conclusion］The newly constructed pMSBBACs was verified to be a good BAC vector for cloning of large DNA fragments and heterologous expression in Streptomyces．

Key words:Keywords: bacterial artificial chromosome (BAC) vector，natural products，biosynthesis gene cluster，Streptomyces，heterologous expression