title: Characterization of a large, stable, high-copy-number Streptomyces plasmid that requires stability and transfer functions for heterologous polyketide overproduction.
abstract: 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.
authors.affiliation: Department of Chemical Engineering, Stanford University, CA 94305, USA. ryanfong@gmail.com
authors.forename: Ryan
authors.initials: R
authors.lastname: Fong
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Vroom
authors.forename: Zhihao
authors.initials: Z
authors.lastname: Hu
authors.forename: C Richard
authors.initials: CR
authors.lastname: Hutchinson
authors.forename: Jianqiang
authors.initials: J
authors.lastname: Huang
authors.forename: Stanley N
authors.initials: SN
authors.lastname: Cohen
authors.forename: Stanley
authors.initials: S
authors.lastname: Cohen
authors.forename: Camilla M
authors.initials: CM
authors.lastname: Kao
authors.forename: Camilla
authors.initials: C
authors.lastname: Kao
mesh_terms: 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*
chemical_list: D000900:Anti-Bacterial Agents; D018942:Macrolides