Abstract
BackgroundPamamycins are 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 very low, and pamamycins are typically formed as crude mixture of mainly smaller derivatives, leaving larger derivatives rather unexplored so far. Therefore, strategies that enable a more efficient production of pamamycins and provide 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.ResultsSupplemented with l-valine, the recombinant producer S. albus J1074/R2 achieved a threefold increased pamamycin titer of 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 thioesters, the building blocks for the polyketide, resulting from l-valine catabolism. Unfavorably, l-valine impaired growth of the strain, repressed genes of mannitol uptake and glycolysis, and suppressed pamamycin formation, despite the biosynthetic gene cluster was transcriptionally activated, restricting production to the post l-valine phase. A deletion mutant of the transcriptional regulator bkdR, controlling a branched-chain amino acid dehydrogenase complex, revealed decoupled pamamycin biosynthesis. The regulator mutant accumulated the polyketide independent of the nutrient status. Supplemented with l-valine, the novel strain 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.ConclusionsOur 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.
Highlights
Pamamycins are macrodiolides of polyketide origin which form a family of differently large homologues with molecular weights between 579 and 663
Following initial transamination of BCAAs into α-keto acids, decarboxylation, and dehydrogenation, catalyzed by the branched-chain amino acid dehydrogenase (BCDH) complex, yield the corresponding acyl-CoA derivatives [14]. These are converted into acetyl-CoA, propionyl-CoA, and succinyl-CoA, inter alia potentially leading to the pamamycin precursors malonyl-CoA, methylmalonyl-CoA, and ethylmalonyl-CoA, respectively [15,16,17]
S. albus and shifts the spectrum to larger derivatives To assess its performance, the pamamycin producer Streptomyces albus J1074/R2 was cultivated in a minimal medium using 10 g L−1 mannitol as sole carbon source
Summary
Pamamycins are 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. Following initial transamination of BCAAs into α-keto acids, decarboxylation, and dehydrogenation, catalyzed by the branched-chain amino acid dehydrogenase (BCDH) complex, yield the corresponding acyl-CoA derivatives [14]. These are converted into acetyl-CoA, propionyl-CoA, and succinyl-CoA, inter alia potentially leading to the pamamycin precursors malonyl-CoA, methylmalonyl-CoA, and ethylmalonyl-CoA, respectively [15,16,17]. The genome of S. albus contains the entire catabolic route for degradation of all three BCAAs [18] and the incorporation of l-valine-derived carbon into pamamycin has been experimentally shown [19]
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