Rapid cloning and heterologous expression of the meridamycin biosynthetic gene cluster using a versatile Escherichia coli-streptomyces artificial chromosome vector, pSBAC.
Expression of biosynthetic pathways in heterologous hosts is an emerging approach to expedite production improvement and biosynthetic modification of natural products derived from microbial secondary metabolites. Herein we describe the development of a versatile Escherichia coli-Streptomyces shuttle Bacterial Artificial Chromosomal (BAC) 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 polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) biosynthetic pathways: the meridamycin biosynthesis gene cluster (mer). The entire mer gene cluster ( approximately 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 (1) in the heterologous host was achieved after replacement of the original promoter with an ermE* promoter and was enhanced by feeding with a biosynthetic 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.
Liu
Hongbo
H
Chemical and Screening Science, Wyeth Research, Pearl River, New York 10965, USA.
Jiang
Hao
H
Haltli
Bradley
B
Kulowski
Kerry
K
Muszynska
Elwira
E
Feng
Xidong
X
Summers
Mia
M
Young
Mairead
M
Graziani
Edmund
E
Koehn
Frank
F
Carter
Guy T
GT
He
Min
M
D022201:Chromosomes, Artificial; D022202:Chromosomes, Artificial, Bacterial / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics*; D005822:Genetic Vectors / Q000138:chemical synthesis / Q000235:genetics; D018942:Macrolides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D048630:Polyketide Synthases / Q000235:genetics / Q000378:metabolism*; D013302:Streptomyces / Q000235:genetics*
C511079:3-normeridamycin; D018942:Macrolides; C548746:meridamycin; D048630:Polyketide Synthases