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Activation and identification of five clusters for secondary metabolites in Streptomyces albus J1074.

ABSTRACT: Streptomyces albus J1074 is a streptomycete strain widely used as a host for expression of secondary metabolite gene clusters. Bioinformatic analysis of the genome of this organism predicts the presence of 27 gene clusters for secondary metabolites. We have used three different strategies for the activation of some of these silent/cryptic gene clusters in S.?albus J1074: two hybrid polyketide-non-ribosomal peptides (PK-NRP) (antimycin and 6-epi-alteramides), a type I PK (candicidin), a non-ribosomal peptides (NRP) (indigoidine) and glycosylated compounds (paulomycins). By insertion of a strong and constitutive promoter in front of selected genes of two clusters, production of the blue pigment indigoidine and of two novel members of the polycyclic tetramate macrolactam family (6-epi-alteramides A and B) was activated. Overexpression of positive regulatory genes from the same organism also activated the biosynthesis of 6-epi-alteramides and heterologous expression of the regulatory gene pimM of the pimaricin cluster activated the simultaneous production of candicidins and antimycins, suggesting some kind of cross-regulation between both clusters. A cluster for glycosylated compounds (paulomycins) was also identified by comparison of the high-performance liquid chromatography profiles of the wild-type strain with that of a mutant in which two key enzymes of the cluster were simultaneously deleted.

SUBMITTER: Olano C

PROVIDER: S-EPMC3992020 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Activation and identification of five clusters for secondary metabolites in Streptomyces albus J1074.

Olano Carlos C García Ignacio I González Aranzazu A Rodriguez Miriam M Rozas Daniel D Rubio Julio J Sánchez-Hidalgo Marina M Braña Alfredo F AF Méndez Carmen C Salas José A JA

Microbial biotechnology 20140304 3

Streptomyces albus J1074 is a streptomycete strain widely used as a host for expression of secondary metabolite gene clusters. Bioinformatic analysis of the genome of this organism predicts the presence of 27 gene clusters for secondary metabolites. We have used three different strategies for the activation of some of these silent/cryptic gene clusters in S. albus J1074: two hybrid polyketide-non-ribosomal peptides (PK-NRP) (antimycin and 6-epi-alteramides), a type I PK (candicidin), a non-ribos ...[more]

PMID: 24593309

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Project description:In streptomycetes, autoregulators are important signaling compounds that trigger secondary metabolism, and they are regarded as Streptomyces hormones based on their extremely low effective concentrations (nM) and the involvement of specific receptor proteins. Our previous distribution study revealed that butenolide-type Streptomyces hormones, including avenolide, are a general class of signaling molecules in streptomycetes and that Streptomyces albus strain J1074 may produce butenolide-type Streptomyces hormones. Here, we describe metabolite profiling of a disruptant of the S. albusaco gene, which encodes a key biosynthetic enzyme for butenolide-type Streptomyces hormones, and identify four butenolide compounds from S. albus J1074 that show avenolide activity. The compounds structurally resemble avenolide and show different levels of avenolide activity. A dual-culture assay with imaging mass spectrometry (IMS) analysis for in vivo metabolic profiling demonstrated that the butenolide compounds of S. albus J1074 stimulate avermectin production in another Streptomyces species, Streptomyces avermitilis, illustrating the complex chemical interactions through interspecies signals in streptomycetes.IMPORTANCE Microorganisms produce external and internal signaling molecules to control their complex physiological traits. In actinomycetes, Streptomyces hormones are low-molecular-weight signals that are key to our understanding of the regulatory mechanisms of Streptomyces secondary metabolism. This study reveals that acyl coenzyme A (acyl-CoA) oxidase is a common and essential biosynthetic enzyme for butenolide-type Streptomyces hormones. Moreover, the diffusible butenolide compounds from a donor Streptomyces strain were recognized by the recipient Streptomyces strain of a different species, resulting in the initiation of secondary metabolism in the recipient. This is an interesting report on the chemical interaction between two different streptomycetes via Streptomyces hormones. Information on the metabolite network may provide useful hints not only to clarification of the regulatory mechanism of secondary metabolism, but also to understanding of the chemical communication among streptomycetes to control their physiological traits.

Project description: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.

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

Activation and identification of five clusters for secondary metabolites in Streptomyces albus J1074.

Publications

Activation and identification of five clusters for secondary metabolites in Streptomyces albus J1074.

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