Enhancing Ristomycin A Production by Overexpression of ParB-like StrR Family Regulators Controlling the Biosynthetic Genes
Amycolatopsis sp. TNS106 harbors a ristomycin biosynthetic gene cluster ( asr ) in its genome and produces ristomycin A. Deletion of the sole cluster-situated StrR family regulatory gene asrR abolished the ristomycin A production and the transcription of asr genes orfs5 - 39 . Ristomycin A fermentation titer in Amycolatopsis sp. TNS106 was dramatically improved by overexpression of asrR and a heterologous StrR family regulatory gene bbr from the balhimycin BGC utilizing strong promoters and multiple gene copies. Ristomycin A production was improved by approximately 60-fold, resulting in fermentation titer of 4.01 g/L in flask culture, in one of the engineered strains. Overexpression of AsrR and Bbr upregulated transcription of tested asr biosynthetic genes, indicating that these asr genes were positively regulated by AsrR and Bbr. However, only the promoter region of the asrR -operon and the intergenic region upstream of orf12 were bound by AsrR and Bbr in gel retardation assays, suggesting that AsrR and Bbr directly regulated the asrR -operon and probably orfs12 - 14 , but no other asr biosynthetic genes. Further assays with synthetic short probes showed that AsrR and Bbr specifically bound not only probes containing the canonical inverted repeats but also a probe with only one 7-bp element of the inverted repeats in its native context. AsrR and Bbr have an N-terminal ParB-like domain and a central winged helix-turn-helix DNA-binding domain. Site-directed mutations indicated that the N-terminal ParB-like domain was involved in activation of ristomycin A biosynthesis and did not affect the DNA-binding activity of AsrR and Bbr. IMPORTANCE This study showed that overexpression of either native StrR family regulator (AsrR) or heterologous StrR family regulator (Bbr) dramatically improved ristomycin A production through increasing the transcription of biosynthetic genes directly or indirectly. The conserved ParB-like domain of AsrR and Bbr was demonstrated to be involved in the regulation of asr BGC expression. These findings provide new insights into the mechanism of StrR family regulators in the regulation of glycopeptide antibiotics biosynthesis. Furthermore, the regulators overexpression plasmids constructed in this study could serve as valuable tools for future utilization in strain improvement and genome mining for new glycopeptide antibiotics. In addition, ristomycin A is a type III glycopeptide antibiotic clinically used as a diagnostic reagent due to its side effect. The overproduction strains engineered in this study are ideal material for industrial production of ristomycin A.