Here we describe our efforts to improve the levels of phosphoglycolipid antibiotic nosokomycin A production by Streptomyces ghanaensis ATCC14672 via genome engineering approaches. Introduction of two extra copies of leucyl tRNA (UUA) gene bldA and one copy of moenomycin biosynthesis gene cluster led, on average, to threefold increase in nosokomycin A titers (up to 1.5 mg/ L). Our results validate genome engineering approach as a viable strategy to improve moenomycin production.

Low molecular weight signaling compounds (LMWC) are important players in regulating various aspects of Streptomyces biology. Their exact roles in certain strain will ultimately depend on overall configuration of regulatory network and thus cannot be predicted on basis of in silico studies. Here, we explored S. ghanaensis gene SSFG_07725 (afsA) presumably involved in initial steps of formation of γ-butyrolactone LMWC. Disruption of afsA impaired aerial mycelium formation and increased the transcription of pleiotropic regulatory gene adpA, whereas level of moenomycin production remained virtually unaffected. We provide evidence that morphogenetic deficiency of afsA-minus mutant was caused by inability to produce diffusible LMWC. Possible links between γ-butyrolactone signaling and various aspects of S. ghanaensis biology are discussed.

Streptomyces ghanaensis ATCC14672 is the only genetically characterized natural producer of moenomycin A (MmA), a unique phosphoglycolipid antibiotic capable of direct inhibition of peptidoglycan glycosyltransferases. At the moment there are no drugs on the market that would target this step of peptidoglycan formation. There is much interest in development of MmA-based drugs to combat growing threat of multidrug resistant Grampo sitive infections. Consequently, there is also major interest in development of microbiological approaches towards production of this natural compound via submerged cultivation. Currently available ATCC14672 strains accumulate minute quantities of MmA, which fueled the investigations of genetic control of its biosynthesis over the last decade. Much of our understanding of the regulation of MmA production stems from homology-based approaches. Namely, homologs of known pleiotropic regulators of antibiotic production were searched in ATCC14672 genome and functionally characterized. Although this approach was largely successful, it left out of the focus those regulators of MmA production that share no similarity to the regulators identified in model streptomycetes. In this work we report an attempt to discover novel genes involved in regulation of moenomycin biosynthesis via the use of Streptomyces codon-adapted transposon Tn5, which represents an unbiased way to find genuinely new regualtors. After a primary screening of transposon library we were able to identify five Tn mutants that differed in morphology and/or total antibiotic production. We identified Tn5 insertion sites in these mutants through sequencing and re-analyzed their moenomycin production levels, revealing two strains with increased total antibiotic activity. The genes affected in Tn5 mutants were cloned into expression vectors and introduced back into wild type (ATCC14672) in order to study the effects of their increased dosage on the strain. One of the genes, ssfg_04565, exerted negative effects on antibiotic activity when introduced on the plasmid. Possible functions of the identified genes in the context of secondary metabolism are discussed.