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Properties of Spontaneous rpsL Mutant of Streptomyces albus KO-1297.

ABSTRACT: The Streptomyces albus J1074 strain remains one of the most popular platforms for the discovery of new natural compounds due to the expression of biosynthetic gene clusters (BGCs) from the microorganisms of the Actinobacteria class. Different methods were tested to provide a maximal expression of heterologous BGCs in this strain. However, there is still no description of the properties of spontaneous J1074 mutants in the rpsL gene encoding a ribosomal protein S12. The interest in such mutations in actinobacteria is due to the fact that they provide a considerable increase in the antibiotic activity. In this work, we describe the isolation and characterization of the S. albus KO-1297 strain, which contains a spontaneous missense mutation in the rpsL gene leading to a Lys88Glu substitution in the protein S12. As compared with the initial strain, this mutant exhibits an increased resistance to streptomycin and higher antibiotic productivity. The KO-1297 strain and genetically engineered rpsL ^K88E mutant K88E are not identical in their ability to produce antibiotics. KO-1297 also exhibits a certain level of instability of rpsL mutation. The genomes of KO-1297 and its rpsL ^WT revertant contain the mutations that can cause phenotypic differences between these strains (as well as between them and SAM2 and K88E strains).

SUBMITTER: Shemediuk AL

PROVIDER: S-EPMC8831875 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Properties of Spontaneous rpsL Mutant of Streptomyces albus KO-1297.

Shemediuk A L AL Dolia B S BS Ochi K K Fedorenko V O VO Ostash B O BO

Cytology and genetics 20220211 1

The Streptomyces albus J1074 strain remains one of the most popular platforms for the discovery of new natural compounds due to the expression of biosynthetic gene clusters (BGCs) from the microorganisms of the Actinobacteria class. Different methods were tested to provide a maximal expression of heterologous BGCs in this strain. However, there is still no description of the properties of spontaneous J1074 mutants in the rpsL gene encoding a ribosomal protein S12. The intere ...[more]

PMID: 35194265

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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:Streptomyces sp. are a rich source for natural products with recognized industrial value, explaining the high interest to improve and streamline production in these microbes. Here, we studied the production of pamamycins, macrodiolide homologues with a high activity against multi-resistant pathogenic microbes, using recombinant S. albus J1074/R2. Talc particles of micrometer size added to submerged cultures of the recombinant strain tripled pamamycin production up to 50 mg L-1. Furthermore, they strongly affected morphology, reduced the size of cell pellets, formed by the filamentous microbe during the process, up to six-fold, and shifted the pamamycin spectrum to larger derivatives. Integrated analysis of transcriptome and metabolome of particle-enhanced and control cultures provided detailed insights into the underlying molecular changes. The microparticles affected the expression of 3341 genes (56%), revealing a global and fundamental impact on metabolism. Morphology-associated genes, encoding major regulators such as SsgA, RelA, EshA, Factor C, as well as chaplins and rodlins, were found massively upregulated, indicating that the particles caused a substantially accelerated morphogenesis. In line, the pamamycin cluster was strongly upregulated (up to log2 10-fold). Furthermore, the microparticles perturbed genes encoding for central catabolism and CoA-ester metabolism, which were mainly activated. The altered expression resulted in changes in the availability of intracellular CoA-esters, the building blocks of pamamycin. Notably, the ratio between methylmalonyl CoA and malonyl-CoA was increased four-fold. Both metabolites compete for incorporation into pamamycin so that the altered availability explained the pronounced preference for larger derivatives in the microparticle-enhanced process. Our findings are straightforward to further develop pamamycins into antituberculosis leads. The novel insights into the behavior of S. albus in response to talc appears of general relevance to further explore and upgrade the concept of microparticle enhanced cultivation, widely used for filamentous microbes.

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Properties of Spontaneous rpsL Mutant of Streptomyces albus KO-1297.

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Properties of Spontaneous <i>rpsL</i> Mutant of <i>Streptomyces albus</i> KO-1297.

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