Increasing the efficiency of heterologous promoters in actinomycetes.
An Escherichia coli-actinomycete shuttle vector, pCJW93, was constructed which places cloned genes under the control of the thiostrepton-inducible tip promoter from Streptomyces lividans. We also constructed expression vectors bearing the actII-ORF4/PactI activator-promoter system of the actinorhodin biosynthetic pathway of Streptomyces coelicolor. With both types of vector, levels of expression varied widely in different actinomycete strains, indicating different levels of the host factors needed for optimal expression. Deletion of the actII-ORF4 activator gene from one such plasmid in Saccharopolyspora erythraea drastically reduced expression from the cognate actI promoter, showing that host factors are required for optimal production of the activator protein itself. However, a low copy number expression vector pWIZ1 for the polyketide synthase DEBS1-TE, in which the promoter for the activator gene was replaced by the strong heterologous ermE* promoter of S. erythraea directed highly efficient production of polyketide synthase protein in Streptomyces cinnamonensis; and the levels of triketide lactone product found were up to 100-fold greater than were produced by the same plasmid in which actII-ORF4 was expressed from its own promoter. Ensuring appropriate expression of a specific activator protein should enable more convenient and consistent heterologous expression of genes in a broad range of actinomycete hosts.
Wilkinson
Christopher J
CJ
Cambridge Centre for Molecular Recognition and Department of Biochemistry, University of Cambridge, UK.
Hughes-Thomas
Zoë A
ZA
Martin
Christine J
CJ
Böhm
Ines
I
Mironenko
Tatiana
T
Deacon
Matthew
M
Wheatcroft
Michael
M
Wirtz
Gabriele
G
Staunton
James
J
Leadlay
Peter F
PF
D039903:Actinobacteria / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D016366:Open Reading Frames; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D013302:Streptomyces / Q000235:genetics*