Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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

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.

ORGANISM(S): Streptomyces coelicolor

SUBMITTER: Janos Demeter

PROVIDER: E-SMDB-4078 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: Pamamycins macrodiolides of polyketide origin which form a family of differently large homologues with molecular weights between 579 and 663. They offer promising biological activity against pathogenic fungi and gram-positive bacteria. Admittedly, production titers are still low and pamamycins are typically formed as crude mixture which mainly contains smaller derivatives. Therefore, strategies that enable a more efficient production of pamamycins with increased fractions of the rare large derivatives are highly desired. Here we took a systems biology approach, integrating transcription profiling by RNA sequencing and intracellular metabolite analysis to enhance pamamycin production in the heterologous host S. albus J1074/R2. Results: Supplemented with L-valine, the recombinant pamamycin producer revealed a threefold increased pamamycin titer of about 3.5 mg L-1 and elevated fractions of larger derivatives: Pam 649 was strongly increased, and Pam 663 was newly formed. These beneficial effects were driven by increased availability of intracellular CoA esters, the building blocks for the polyketide, resulting from L-valine catabolism. Unfavorably, the presence of L-valine impaired growth of the strain, repressed genes of mannitol uptake and glycolysis, and suppressed pamamycin formation, despite the biosynthetic gene cluster was activated, restricting production to the post L-valine phase. A null mutant of the transcriptional regulator bkdR, controlling a branched-chain amino acid dehydrogenase complex, revealed decoupled pamamycin biosynthesis and accumulated the polyketide independent of the nutrient status. Supplemented with L-valine, the regulator mutant enabled the biosynthesis of pamamycin mixtures with up to 55% of the heavy derivatives Pam 635, Pam 649, and Pam 663, almost 20-fold more than the wild type. Conclusions: Our findings open the door to provide rare heavy pamamycins at markedly increased efficiency and facilitate studies to assess their specific biological activities and explore this important polyketide further.

Dataset Information

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

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