Characterization of a Large, Stable, High Copy Number Streptomyces Plasmid That Requires Stability and Transfer Functions for Heterologous Polyketide Overproduction.
[Meanwhile, back in the labs at Kosan, one problem solved but another one found].
Appl Environ Microbiol. 2006 Dec 1
Fong R, Hu Z, Hutchinson CR, Huang J, Cohen S, Kao C.
Department of Chemical Engineering, Stanford University, Stanford, California 94305; Kosan Biosciences, Inc., Hayward California 94545; Department of Genetics, Stanford University, Stanford, California 94305.
A major limitation to improving small-molecule pharmaceutical production in streptomycetes is the inability of high copy number plasmids to tolerate large biosynthetic gene cluster inserts. A recent finding has overcome this barrier. In 2003, Hu et al. discovered a stable, high copy number, 81-kb plasmid that significantly elevated production of the polyketide precursor to the antibiotic erythromycin in a heterologous Streptomyces host. Here, we identified mechanisms by which this SCP2*-derived plasmid achieves increased levels of metabolite production and examined how the 45-bp deletion mutation in the plasmid replication origin increased plasmid copy number. A plasmid intramycelial transfer gene, spd, and two partition genes, parAB, enhance metabolite production by increasing the stable inheritance of large plasmids containing biosynthetic genes. Additionally, high product titers required both activator (actII-ORF4) and biosynthetic genes (eryA) at high copy numbers. DNA gel shift experiments revealed that the 45-bp deletion abolished replication protein (RepI) binding to a plasmid site which, in part, supports an iteron model for plasmid replication and copy number control. Using the new information, we constructed a large high copy number plasmid capable of overproducing the polyketide 6-dEB. However, this plasmid was unstable over multiple culture generations suggesting that other SCP2* genes may be required for long-term, stable plasmid inheritance. |
