Regulation of Secondary Metabolite Production in Streptomyces

Abstract

Among the myriad of natural products are pseudosugar-containing secondary metabolites, e.g., the C7-pseudosugar-containing oligosaccharide acarbose, the C6- pseudosugar-containing aminoglycoside antibiotics, and the five-membered ring pseudosugar-containing antibiotic pactamycin. The C7-pseudosugars are normally derived from sedoheptulose 7-phosphate catalyzed by a family of enzymes called sedoheptulose 7-phosphate cyclases (SH7PCs). This family of enzymes includes 2- epi-5-epi-valiolone synthase (EEVS), 2-epi-valiolone synthase (EVS), and demethyl4-deoxygadusol synthase (DDGS). While DDGS is exclusively involved in the biosynthesis of the sunscreen compounds mycosporine-like amino acids, EEVS and EVS (less common) are involved in the biosynthesis of a wide variety of natural products, including the C7-pseudosugar-containing oligosaccharides (aka pseudooligosaccharides). In pseudooligosaccharide biosynthesis, coupling between the C7-pseudosugar unit with other sugar molecules is catalyzed by a family of enzymes called pseudoglycosyltransferases (PsGTs). PsGTs are believed to haveevolved from glycosyltransferases and gained the capability of accepting NDPpseudosugars as “sugar-donors”. Our preliminary bioinformatic studies revealed that many bacteria contain an EEVS gene as part of a biosynthetic gene cluster (BGC) in their genomes. Despite its wide distribution in the bacterial kingdom, the products of the BGCs and their functions in nature are largely unknown. As part of our effort to identify novel C7-pseudosugarcontaining natural products and to understand their functions, we screen the NCBI genome sequence database for novel BGCs that contain EEVS and putative PsGT genes. This genome mining study resulted in hundreds of novel BGCs in a broad range of bacteria. Among them, Streptomyces clavuligerus ATCC 27064 was selected to be investigated further, as in addition to an EEVS gene, the BGC contains a putative PsGT-2 gene, which is rarely seen in other EEVS gene-containing BGCs. Inactivation of the EEVS and PsGT genes in S. clavuligerus resulted in mutant strains that surprisingly produced many more secondary metabolites than the wild-type. Direct involvement of the genes in this unusual phenotype was confirmed by genetic and chemical complementation experiments. Isolation and structure characterization of the secondary metabolites whose production was augmented in the mutants revealed their identities as maculosine cyclo-(D-proline-L-tyrosine), and nocardamine. As these compounds are most likely derived from different biosynthetic pathways, the products of the putative EEVS-PsGT cluster (most likely pseudosugars) may function as global regulators. Intriguingly, these novel regulators are not only function in S. clavuligerus but are also active in other species ofStreptomyces. Addition of the aqueous fraction of the culture broth from S. clavuligerus to the cultures of Streptomyces pactum ATCC 27456 significantly affected the metabolic profile of the latter bacterium. S. pactum ATCC 27456 is the producer of the antitumor antibiotic pactamycin. Structurally, pactamycin contains a five-membered pseudosugar (aka aminocyclopentitols), which is derived from N-acetylglucosamine. While pactamycin and its congeners have a wide range of biological activities, e.g., antibacterial, antitumor, antiviral, and antiplasmodial activities, their development as pharmaceutical lead compounds was hampered by the relatively low production yields. To understanding how pactamycin biosynthesis is regulated and to engineer mutant strains with improved production yields, we investigated both the pathway specific regulatory genes, ptmF and ptmE, and the pleiotropic regulatory system PhoP-PhoR in S. pactum. Gene inactivation, genetic complementation, and RT-PCR studies revealed that the PtmF-PtmE system cont