Characterization of a large, stable, high-copy-number Streptomyces plasmid that requires stability and transfer functions for heterologous polyketide overproduction.
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 (J. Ind. Microbiol. Biotechnol. 30:516-522, 2003). Here, we have 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 a partition gene, 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-deoxyerythronolide B. However, this plasmid was unstable over multiple culture generations, suggesting that other SCP2* genes may be required for long-term, stable plasmid inheritance.
Fong
Ryan
R
Department of Chemical Engineering, Stanford University, CA 94305, USA. ryanfong@gmail.com
Vroom
Jonathan A
JA
Hu
Zhihao
Z
Hutchinson
C Richard
CR
Huang
Jianqiang
J
Cohen
Stanley N
SN
Cohen
Stanley
S
Kao
Camilla M
CM
Kao
Camilla
C
D000900:Anti-Bacterial Agents / Q000096:biosynthesis; D004261:DNA Replication; D018628:Gene Dosage; D018942:Macrolides / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000502:physiology*; D013302:Streptomyces / Q000737:chemistry / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D018942:Macrolides