Semi-rational mutagenesis of an industrial Streptomyces fungicidicus strain for improved enduracidin productivity.

The biosynthesis of the valuable antibiotic enduracidin by Streptomyces fungicidicus TXX3120 is a complex multistep process. To identify the rate-limiting step of the entire biosynthetic process, we carried out a deep RNA sequencing towards the mycelia of TXX3120 at different fermentation stages. Comparative RNA-seq analysis indicated that the expression level of the endC gene during the enduracidin production phase was evidently lower than that of the other relevant genes to enduracidin biosynthesis. This result was further confirmed by quantitative RT-PCR, and the giant non-ribosomal peptide synthase (NRPS) encoded by endC was predicated to be the rate-limiting enzyme in enduracidin biosynthesis. To increase the expression of endC during the enduracidin production phase, a reporter-based selection system was developed by genetically replacing the initial part of the endC gene with a thiostrepton resistance gene (tsr), which will then act as a selectable marker to report the expression level of the rate-limiting gene endC, thereby facilitating the selection of enduracidin-overproducing mutants following random mutagenesis. After one round of mutagenesis, thiostrepton resistance selection, and restoration of the endC gene, three mutant strains with improved endC expression levels were obtained. Their highest enduracidin titers reached 9780.54, 9272.46, and 8849.06 U/mL, respectively representing 2.31-, 2.19-, and 2.09-fold of the initial industrial strain TXX3120. Our research provides a useful strategy for the rational breeding of industrial strains that synthesize complex natural products.

Discovery, gene modification, and optimization of fermentation of an enduracidin-producing strain.

Enduracidin significantly inhibits Gram-positive bacteria and had been widely used in many fields. However, as the poor technology for production of enduracidin and its scarcity, identification of novel strategies for production of enduracidin is important. Our group developed two methods to improve the yield of the production of enduracidin. The yield of enduracidin was increased by three- to fivefold. The highest yields of enduracidin A and enduracidin B achieved were 63.7 and 82.13 mg/ml. Thus, our results might provide a new reference method for the industrial production of enduracidin.

Investigation of halogenation during the biosynthesis of ramoplanin in Actinoplanes sp. ATCC33076.

Ramoplanin and enduracidin are lipopeptide antibiotics effective against Gram-positive pathogens, which share close similarity in structure and biosynthetic pathway. Both compounds have chlorine atoms attached to 4-hydroxyphenylglycine (Hpg) but with different chlorinating sites and levels. Here, to probe the factor affecting the site and level of halogenation, gene inactivation and heterologous expression were carried out in Actinoplanes sp. ATCC33076 by homologous recombination. Metabolite analysis confirmed that ram20 encodes the only halogenase in ramoplanin biosynthetic pathway, and enduracidin halogenase End30 could heterologously complement the ram20-deficient mutant. Additionally, the mannosyltransferase-deficient mutant produces a dichlorinated ramoplanin aglycone with the halogenation site at Hpg(13). This study has refined our understanding of how halogenation occurs in ramoplanin biosynthetic pathway, and lays the foundation for further exploitation of ramoplanin and enduracidin halogenase in combinatorial biosynthesis.