Genetic Engineering of Streptomyces ghanaensis ATCC14672 for Improved Production of Moenomycins

Streptomycetes are soil-dwelling multicellular microorganisms famous for their unprecedented ability to synthesize numerous bioactive natural products (NPs). In addition to their rich arsenal of secondary metabolites, Streptomyces are characterized by complex morphological differentiation. Mostly, industrial production of NPs is done by submerged fermentation, where streptomycetes grow as a vegetative mycelium forming pellets. Often, suboptimal growth peculiarities are the major bottleneck for industrial exploitation. In this work, we employed genetic engineering approaches to improve the production of moenomycins (Mm) in Streptomyces ghanaensis, the only known natural direct inhibitors of bacterial peptidoglycan glycosyltransferses. We showed that in vivo elimination of binding sites for the pleiotropic regulator AdpA in the oriC region strongly influences growth and positively correlates with Mm accumulation. Additionally, a marker- and “scar”-less deletion of moeH5, encoding an amidotransferase from the Mm gene cluster, significantly narrows down the Mm production spectrum. Strikingly, antibiotic titers were strongly enhanced by the elimination of the pleiotropic regulatory gene wblA, involved in the late steps of morphogenesis. Altogether, we generated Mm overproducers with optimized growth parameters, which are useful for further genome engineering and chemoenzymatic generation of novel Mm derivatives. Analogously, such a scheme can be applied to other Streptomyces spp.

Identification and Characterization of Four c-di-GMP-Metabolizing Enzymes from Streptomyces ghanaensis ATCC14672 Involved in the Regulation of Morphogenesis and Moenomycin A Biosynthesis

Diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) are essential enzymes deputed to maintain the intracellular homeostasis of the second messenger cyclic dimeric (3′→5′) GMP (c-di-GMP). Recently, c-di-GMP has emerged as a crucial molecule for the streptomycetes life cycle, governing both morphogenesis and secondary metabolite production. Indeed, in Streptomyces ghanaensis ATCC14672 c-di-GMP was shown to be involved in the regulatory cascade of the peptidoglycan glycosytransferases inhibitor moenomycin A (MmA) biosynthesis. Here, we report the role of four c-di-GMP-metabolizing enzymes on MmA biosynthesis as well as morphological progression in S. ghanaensis. Functional characterization revealed that RmdAgh and CdgAgh are two active PDEs, while CdgEgh is a DGC. In vivo, overexpression of rmdAgh and cdgAgh led to precocious sporulation, whereas overexpression of cdgEgh and cdgDgh (encoding a predicted DGC) caused an arrest of morphological development. Furthermore, we demonstrated that individual deletion of rmdAgh, cdgAgh, and cdgDgh enhances MmA accumulation, whereas deletion of cdgEgh has no impact on antibiotic production. Conversely, an individual deletion of each studied gene does not affect morphogenesis. Altogether, our results show that manipulation of c-di-GMP-metabolizing enzymes represent a useful approach to improving MmA production titers in S. ghanaensis.

Microbe-plant interactions between Streptomyces and model agricultural plants – Hordeum vulgare and Lycopersicon esculentum (Microtom)

Aim. Microbe-plant interactions (MPI) constitute an important aspect of ecology because of their significant influence on plant’s ability to withstand abiotic stress and infection. In comparison to proteobacteria and bacilli, the roles of streptomycetes in MPI remain poorly studied. Here, we elucidate some aspects of MPI between two model plant species, Hordeum vulgare and Lycopersicon esculentum, and several strains of Streptomyces lividans 1326 and S. ghanaensis ATCC14672. Methods. Microbiology, microscopy and molecular genetics were combined to reveal the MPI. Results. We demonstrate the colonization of H. vulgare and L. esculentum roots by different strains of S. ghanaensis deficient in production of either the antibiotic moenomycin or signaling molecule of the γ-butyrolactone type. The treatment of H. vulgare seeds with S. lividans spores increased the root biomass. Plants treated with 1,4-butyrolactone had no positive influence on plants, at milimolar concentrations this compound inhibited the root and shoot growth of L. esculentum. Conclusions. Roots of two mono- and dicot plants are colonized by Streptomyces; reporter gene uidA is useful to monitor the colonization. Under our experimental conditions the ability to colonize plants by streptomycetes was not affected by the deficiency in antibiotic or butenolide production. Keywords: Streptomyces ghanaensis, moenomycinA, low-molecular weight signal compounds, root colonization.