Properties of Multidrug-Resistant Mutants Derived from Heterologous Expression Chassis Strain Streptomyces albidoflavus J1074

Streptomyces albidoflavus J1074 is a popular platform to discover novel natural products via the expression of heterologous biosynthetic gene clusters (BGCs). There is keen interest in improving the ability of this platform to overexpress BGCs and, consequently, enable the purification of specialized metabolites. Mutations within gene rpoB for the β-subunit of RNA polymerase are known to increase rifampicin resistance and augment the metabolic capabilities of streptomycetes. Yet, the effects of rpoB mutations on J1074 remained unstudied, and we decided to address this issue. A target collection of strains that we studied carried spontaneous rpoB mutations introduced in the background of the other drug resistance mutations. The antibiotic resistance spectra, growth, and specialized metabolism of the resulting mutants were interrogated using a set of microbiological and analytical approaches. We isolated 14 different rpoB mutants showing various degrees of rifampicin resistance; one of them (S433W) was isolated for the first time in actinomycetes. The rpoB mutations had a major effect on antibiotic production by J1074, as evident from bioassays and LC-MS data. Our data support the idea that rpoB mutations are useful tools to enhance the ability of J1074 to produce specialized metabolites

Properties of Streptomyces albus J1074 mutant deficient in tRNA(Leu)UAA gene bldA.

Streptomyces albus J1074 is one of the most popular and convenient hosts for heterologous expression of gene clusters directing the biosynthesis of various natural metabolic products, such as antibiotics. This fuels interest in elucidation of genetic mechanisms that may limit secondary metabolism in J1074. Here, we report the generation and initial study of J1074 mutant, deficient in gene bldA for tRNA(Leu)UAA, the only tRNA capable of decoding rare leucyl TTA codon in Streptomyces. The bldA deletion in J1074 resulted in a highly conditional Bld phenotype, with depleted formation of aerial hyphae on certain solid media. In addition, bldA mutant of J1074 was unable to produce endogenous antibacterial compounds and two heterologous antibiotics, moenomycin and aranciamycin, whose biosynthesis is directed by TTA-containing genes. We have employed a new TTA codon-specific β-galactosidase reporter system to provide genetic evidence that J1074 bldA mutant is impaired in translation of TTA. In addition, we have discussed the possible reasons for differences in the phenotypes of bldA mutants described here and in previous studies, providing knowledge to study bldA-based regulation of antibiotic biosynthesis

The adpA-like regulatory gene from Actinoplanes teichomyceticus: in silico analysis and heterologous expression

Ostash B, Yushchuk O, Tistechok S, et al. The adpA-like regulatory gene from Actinoplanes teichomyceticus: in silico analysis and heterologous expression. World Journal of Microbiology and Biotechnology. 2015;31(8):1297-1301.Analysis of the draft sequence of the genome of teicoplanin producer Actinoplanes teichomyceticus (NRRL-B16726) led to identification of several genes encoding AraC-family regulators that resemble AdpA, master regulator of transcription in Streptomyces. We elucidated possible regulatory functions of one of the identified genes, adpA19 (at) , most similar to archetypal adpA from model Streptomyces species, in a series of expression experiments. Introduction of adpA19 (at) under control of its own promoter on moderate copy number vector pKC1139 into NRRL-B16726 had no influence on antibiotic production and sporulation. Introduction of adpA19 (at) into Streptomyces coelicolor M145 and several S. ghanaensis strains had major influence on antibiotic production by these bacteria. Finally, adpA19 (at) expression in a set of soil actinomycete isolates led to induction of synthesis of antibiotic compounds. Our data point to pleiotropic regulatory role of adpA19 (at) , warranting its use as a tool to manipulate secondary metabolome of actinomycetes