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

Abstract

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.

Machine Learning Uncovers the Transcriptional Regulatory Network for the Production HostStreptomyces albidoflavus

“…Its small genome, capacity to produce versatile precursors, and dispersed mycelial growth compared to other Streptomyces, make it one of the most convenient Streptomyces production host to scale up in industrial bioreactors (Marín et al, 2017; Myronovskyi & Luzhetskyy, 2019; Zaburannyi et al, 2014). However, the production yields are often low, requiring tedious chassis optimization and/or production processes (Dolya et al, 2023; Gummerlich et al, 2021; Li et al, 2023). Genome-wide studies and characterization of the TRN in other Streptomyces has identified regulators, promoter regions, and transcription start sites, which may improve rational strain design decisions and production yield (Hwang et al, 2019; Jeong et al, 2016; Luo et al, 2018; Yi et al, 2017).…”

“…The production of secondary metabolites appear to mainly stem from the activation of biosynthetic gene clusters (BGCs) through a hierarchical network of transcriptional regulators in response to various internal and external signals, such as nutrient availability, stress, and intercellular communication (Barbuto Ferraiuolo et al, 2021; Chen et al, 2010; Wohlleben et al, 2017; Xia et al, 2020; Zhang et al, 2020). However, many BGCs remain silent or poorly expressed under standard laboratory conditions, limiting the discovery and production yield of desired compounds (Dolya et al, 2023). While several strategies have been developed to activate and optimize BGC expression, such as genome editing, heterologous expression, co-cultivation, and elicitor addition, the TRN of Streptomyces is poorly understood compared to other model bacteria, which limits the potential and applicability of rational strain design (Barbuto Ferraiuolo et al, 2021; Chater, 2016; Chen et al, 2010).…”

“…Streptomyces genomes are often large (6)(7)(8)(9)(10)(11)(12), and bottom-up approaches to characterize the TRN have revealed an intricate hierarchy of regulators -ranging from global to pleiotropic and local -that activate under specific growth conditions and developmental stages, reflecting the complex life cycle of Streptomyces (8)(9)(10)(11)(12)(13). Streptomyces albidoflavus J1074 (previously Streptomyces albus J1074) is a well characterized and widely used heterologous host for studying cryptic Actinomycete BGCs and producing industrially important secondary metabolites (14)(15)(16)(17)(18)(19)(20)(21)(22)(23). S. albidoflavus has one of the smallest Streptomyces genomes of 6.8 Mbp, containing around 23 endogenous BGCs (24).…”