Stepwise increase of fidaxomicin in an engineered heterologous host Streptomyces albus through multi-level metabolic engineering

The anti-Clostridium difficile infection(CDI)drug fidaxomicin is a natural polyketide metabolite mainly produced by Micromonosporaceae,such as Actinoplanes deccanensis,Dactylosporangium aurantiacum,and Micromonospora echinospora.In the present study,we employed a stepwise strategy by combining heterologous expression,chassis construction,promoter engineering,activator and transporters overexpression,and optimization of fermentation media for high-level production of fidaxomicin.The maximum yield of 384 mg/L fidaxomicin was achieved with engineered Streptomyces albus D7-VHb in 5 L-tank bioreactor,and it was approximately 15-fold higher than the native strain Actinoplanes deccanensis YP-1 with higher strain stability and growth rate.This study developed an enhanced chassis strain,and for the first time,achieved the heterologous synthesis of fidaxomicin through a combinatorial metabolic engineering strategy.

Activation of anthrachamycin biosynthesis in Streptomyces chattanoogensis L10 by site-directed mutagenesis of rpoB

Genome sequencing projects revealed massive cryptic gene clusters encoding the undiscovered secondary metabolites in Streptomyces. To investigate the metabolic products of silent gene clusters in Streptomyces chattanoogensis L10(CGMCC 2644), we used site-directed mutagenesis to generate ten mutants with point mutations in the highly conserved region of rpsL(encoding the ribosomal protein S12) or rpoB(encoding the RNA polymerase β-subunit). Among them, L10/RpoB(H437 Y) accumulated a dark pigment on a yeast extract-malt extract-glucose(YMG) plate. This was absent in the wild type. After further investigation, a novel angucycline antibiotic named anthrachamycin was isolated and determined using nuclear magnetic resonance(NMR) spectroscopic techniques. Quantitative real-time polymerase chain reaction(qRT-PCR) analysis and electrophoretic mobility shift assay(EMSA) were performed to investigate the mechanism underlying the activation effect on the anthrachamycin biosynthetic gene cluster. This work indicated that the rpoB-specific missense H437 Y mutation had activated anthrachamycin biosynthesis in S. chattanoogensis L10. This may be helpful in the investigation of the pleiotropic regulation system in Streptomyces.

m4C DNA methylation regulates biosynthesis of daptomycin in Streptomyces roseosporus L30

Despite numerous studies on transcriptional level regulation by single genes in drug producing Actinomyces,the global regulation based on epigenetic modification is not well explored.N4-methylcytosine(m4C),an abundant epigenetic marker in Actinomycetes’genome,but its regulatory mechanism remains unclear.In this study,we identify a m4C methyltransferase(SroLm3)in Streptomyces roseosporus L30 and multi-omics studies were performed and revealed SroLm3 as a global regulator of secondary metabolism.Notably,three BGCs inΔsroLm3 strain exhibited decreased expression compared to wild type.In-frame deletion of sroLm3 in S.roseosporus L30 further revealed its role in enhancing daptomycin production.In summary,we characterized a m4C methyltransferase,revealed the function of m4C in secondary metabolism regulation and biosynthesis of red pigment,and mapped a series of novel regulators for daptomycin biosynthesis dominated by m4C methylation.Our research further indicated that m4C DNA methylation may contribute to a metabolic switch from primary to secondary metabolism in Actinomyces.