Monday, July 27, 2009
P118
Rapid Cloning and Heterologous Expression of Meridamycin Biosynthetic Gene Cluster Using a Versatile E. coli - Streptomyces Artificial Chromosome Vector pSBAC
Hongbo Liu and Min He. Natural Products Discovery Research, Chemical and Screening Sciences, Wyeth Research, 401 N. Middletown Road, Pearl River, NY 10965
Expression of biosynthetic pathways in a heterologous host is an emerging approach for production improvement and genetic manipulation of microbial natural products. Here we describe the development of a versatile Escherichia coli-Streptomyces shuttle bacterial artificial chromosomal conjugation vector, pSBAC, to facilitate the cloning, genetic manipulation and heterologous expression of actinomycetes secondary metabolite biosynthetic gene clusters. The utility of pSBAC was demonstrated through the rapid cloning and heterologous expression of one of the largest PKS/NRPS biosynthetic pathways - the meridamycin biosynthesis gene cluster (mer). The entire mer gene cluster (~90 kb) was captured in a single pSBAC clone through a straightforward restriction enzyme digestion and cloning approach and transferred into Streptomyces lividans. The production of meridamycin in the heterologous host was achieved after replacement of the original promoter with an ermE* promoter, and was enhanced by feeding with biosynthesis precursor. The success of heterologous expression of such a giant gene cluster demonstrates the versatility of BAC cloning technology and paves the road for future exploration of expression of the meridamycin biosynthetic pathway in various hosts, including strains that have been optimized for polyketide production.
Web Page: pubs.acs.org/doi/abs/10.1021/np8006149
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