Abstract
We describe a procedure for the conjugative transfer of phage P1-derived Artificial Chromosome (PAC) library clones containing large natural product gene clusters (≥70 kilobases) to Streptomyces coelicolor strains that have been engineered for improved heterologous production of natural products. This approach is demonstrated using the gene cluster for FK506 (tacrolimus), a clinically important immunosuppressant of high commercial value. The entire 83.5 kb FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488 present in one 130 kb PAC clone was introduced into four different S. coelicolor derivatives and all produced FK506 and smaller amounts of the related compound FK520. FK506 yields were increased by approximately five-fold (from 1.2 mg L-1 to 5.5 mg L-1) in S. coelicolor M1146 containing the FK506 PAC upon over-expression of the FK506 LuxR regulatory gene fkbN. The PAC-based gene cluster conjugation methodology described here provides a tractable means to evaluate and manipulate FK506 biosynthesis and is readily applicable to other large gene clusters encoding natural products of interest to medicine, agriculture and biotechnology.
Highlights
A wealth of microbial gene clusters that encode the biosynthetic pathways to bioactive natural products, known as secondary or specialized metabolites, are being unveiled with the everincreasing number of sequenced bacterial genomes [1]
We provide an expedient, reliable procedure for transfer of large natural product gene clusters using phage P1derived Artificial Chromosomes (PACs) to Streptomyces coelicolor derivatives that have been engineered for improved heterologous expression
To the best of our knowledge, our PAC-based conjugation strategy has afforded the first example of heterologous FK506 production in a more tractable expression host
Summary
A wealth of microbial gene clusters that encode the biosynthetic pathways to bioactive natural products, known as secondary or specialized metabolites, are being unveiled with the everincreasing number of sequenced bacterial genomes [1]. While some clusters correspond to known compounds, a large number (so-called cryptic gene clusters) encode the biosynthesis of previously undiscovered molecules and represent a promising source of new drugs and therapeutics. One of the most useful strategies for advancing the study of natural products is heterologous expression of the genes responsible for biosynthesis in a tractable host organism. This approach has many advantages, including compound yield improvements [4], [5], [6] and the ability to work with a strain that is more amenable to genetic manipulation, e. For quantification of FK506 production in each S. coelicolor strain, PAC20N and pESAC13Dbla integrants were grown in triplicate
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