Dessecting Role of type iii Polyketide Synthase, Methyltransferase and Sulfatransferase in the Biology of Corynebacterineae

Abstract

The present thesis provides a thorough insight into type III polyketide synthases (PKSs) from microorganisms belonging to sub-order corynebacterineae. Type III PKSs are versatile enzymes making variable products with different bioactivities. M. marinum (Mmar) has four kinds of type III polyketide synthases (PKSs) belonging to three types of clusters. The PKSs produce a spectrum of polyketides and polyphenolics. MMAR_2470 and MMAR_2474 belong to the first cluster. The orthologous genes are found to be present in pathogenic bacteria from 27 species of mycobacteria. Whereas, MMAR_2190, from the second cluster, belongs to nine genomes of pathogenic corynebacterineae. Our biochemical characterization of MMAR_2190 revealed unprecedented cyclization potential of an enzyme to form four classes of non-methylated products using a common substrate pool through a single catalytic site. The structural study of protein depicts the presence of three active site residues cysteine (Cys), Histidine (His) and Asparagine (Asn) similar to other type III PKSs but with unique features, i.e. malonate bound to active site Cys and constriction in the active site cavity. It provides a continuous channel for the access of substrate, which is further regulated through the orientation of gatekeeper residues. Leu266, Leu348, Ile319 and Leu223 regulate the binding of substrate to the substrate-binding tunnel, and different catalytic pockets orient type III PKS by the substrate. We further attempted to identify the physiological roles through over-expression of different type III PKSs from Mmar and NFIII, an ortholog of MMAR_2190 from N. farcinica in Δpks10 M. smegmatis strain. This type III PKS knockout strain shows defect in biofilm formation. An acetamide inducible pMyNT vector was utilized to over-express different type III PKSs and generate different Δpks10 Msmeg/MyNT-type III PKS strains. These over-expressing strains displayed interesting 1physiological features. The Δpks10 Msmeg/pMyNT_MMAR_2190 strain showed complete biofilm recovery, similar to wildtype M. smegmatis and M. marium biofilms. Whereas, Δpks10 Msmeg/pMyNT_MMAR_2474 showed partial recovery of biofilm. Δpks10 Msmeg/pMyNT_MMAR_2470 and Δpks10 Msmeg/pMyNT_NFIII strains could not revive biofilm growth. Scanning Electron Microscopy (SEM) also showed thick interconnecting pigmented cells in the biofilm of Δpks10Msmeg/pMyNT_MMAR_2190 strain. This study showed metabolic diversity of polyketide molecules in over-expressed strains along with the presence of a series of quinone like molecules in over-expressed pMyNT strains. Possibly, the polyketide molecules help in remodelling the cell wall as inferred from the consistent cell envelope observed through the Transmission Electron Microscopy (TEM) of planktonic cultures of pMyNT_MMAR_2190. We found acyl phloroglucinols in MMAR_2190 overexpressed mycobacterial strain. We observed polyketide modification by two groups of enzymes, methyltransferase and sulfotransferase from MMAR_2190 cluster. O-methylation seemed essential in biofilm formation and was also observed in a biofilm of wildtype M. marinum. MMAR_2190 genomic cluster contains a methyltransferase (MMAR_2193) and two sulfotransferases (MMAR_2191 and MMAR_2192). Our biochemical analysis of these Mmar proteins revealed unprecedented potential of the enzymes to modify all available hydroxyl groups in polyphenolic compounds. In an interesting experiment, type III polyketide products produced by cognate type III PKSs could be directly methylated by MMAR_2193 to generate multi-methylated polyketides. Collectively, these studies implicate the genomic cluster of type III PKSs to aid mycobacterial growth and survival. Additionally, this thesis unravels novel biosynthetic potentials of the Mmar type III PKSs and modifying enzymes that could be utilized for production of divergent chemical scaffolds with unique bioactive potentials. 2