Biosynthetic Studies and Modulations of Secondary Metabolite Production in Streptomyces pactum

Abstract

The genus Streptomyces comprises filamentous, spore-forming, and Gram-positive bacteria from the Actinobacteria phylum and is widely distributed in the environment. Streptomyces are well known to produce a wide range of secondary metabolites, e.g., polyketides, non-ribosomal peptides, terpenes, and aminocyclitols, with important biological activities. Some of them have been used in medicine, food industries, agriculture, etc. In Streptomyces, the production of secondary metabolites is

usually controlled by global and pathway-specific regulators, whose functions may be influenced by nutrients, pH, temperature, osmotic pressure, and cell density. Furthermore, the availability of biosynthetic building blocks and cofactors may affect the production of these secondary metabolites. Many secondary metabolites are usually produced in low titer or the biosynthetic gene clusters (BGCs) that are responsible for their production are silent under normal laboratory conditions. Recently, the Mahmud group and collaborators sequenced the whole genome of Streptomyces pactum ATCC 27456, a producer of the antitumor antibiotic pactamycin. In silico analysis of the genome sequence revealed the presence of 31 putative BGCs in the genome, 9 of which contain polyketide synthase genes (PKS). Despite the presence of many BGCs in this strain, only four classes of bioactive compounds (the pactamycins, the NFAT-133s, the conglobatins, and the piericidins) have been identified from this strain. While the biosynthetic pathways to pactamycin, conglobatin, and piericidin have been extensively studied, the pathway to NFAT-133, which is an inhibitor of the nuclear factor of activated T cells, was unknown. Recently, we reported the BGC of NFAT-133 in S. pactum ATCC 27456 and proposed the biosynthetic pathway to this natural product. However, many details of the pathway remained unclear. Therefore, the first part of this dissertation aimed at investigating the biosynthesis pathway that leads to the formation of NFAT-133. The NFAT-133 BGC is conspicuous by its highly disordered noncollinear type I modular polyketide synthase (PKS) genes that encode PKSs with one module more than those expected for the heptaketide NFAT-133 biosynthesis. Thus, the major metabolite NFAT-133 was proposed to derive from an octaketide analogue, TM-123.

However, further bioinformatic analysis and gene inactivation studies suggest that NFAT-133 is not derived from TM-123 but rather a product of programmed KS7 extension skipping of a nascent heptaketide from the PKS assembly line that produces TM-123. Furthermore, the identification of NFAT-133/TM-123 analogues from mutants of the ATCC 27456 strain suggests that NftN (a putative dehydrogenase), NftE (a cytochrome P450), and NftG (a putative hydrolase/decarboxylase) function “in trans” during the polyketide chain assembly processes. The gene inactivation studies also resulted in the production of two new NFAT-133 analogues, TM-136 and TM- 137. The second part of this dissertation aimed at modulating the production of secondary metabolites in S. pactum by decompetition of biosynthetic pathways in the organism. First, we inactivated three of the four major biosynthetic pathways, the NFAT-133, the conglobatin, and the piericidin BGCs. We found that inactivation of these major BGCs significantly increased the production of pactamycin. Subsequently, we inactivated all four major BGCs and analyzed the phenotype. We found that the mutant is no longer able to produce those compounds but showed enhanced production of minor or previously undetected secondary metabolites. Metabolomic studies of this mutant showed the production of several new compounds that are not produced in the wild type. Isolation and structure characterization of the compounds led to the identification of three known cyclic dipeptides and four new polyketides, which are derived from the truncated piericidin PKS machinery. Therefore, systematic decompetition of biosynthetic pathways in actinomycetes may be used to modulate secondary metabolite production and to access natural products from cryptic BGCs that

are normally not expressed under normal laboratory conditions. In addition, LC- MS/MS and molecular networking analyses revealed the presence of several other compounds in the extracts whose structures have not been determined. This warrants a follow-up investigation of their chemical structures and biological activities.