E-SMDB-4078 - Transcription profiling time series of Streptomyces coelicolor containing a large high-copy-number plasmid capable of overproducing the polyketide 6-deoxyerythronolide B

Status
Submitted on 17 May 2007, released on 12 June 2007, last updated on 4 June 2014
Organism
Streptomyces coelicolor
Samples (0)
Array (1)
Protocols (1)
Description
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.
Experiment types
transcription profiling by array, time series
Contacts
Citation
Characterization of a large, stable, high-copy-number Streptomyces plasmid that requires stability and transfer functions for heterologous polyketide overproduction. Fong R , Hu Z , Hutchinson CR , Huang J , Cohen S , Kao C. Fong R, et al. (2007) Appl Environ Microbiol 73(4):1296-307  (2007-02-01)
MIAME
PlatformsProtocolsVariablesProcessedRaw
Files
Investigation descriptionE-SMDB-4078.idf.txt
Sample and data relationshipE-SMDB-4078.sdrf.txt
Raw data (1)E-SMDB-4078.raw.1.zip
Processed data (1)E-SMDB-4078.processed.1.zip
Array designA-SMDB-772.adf.txt
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