Identification and characterization of type III polyketide synthase genes from culturable endophytes of ethnomedicinal plants.

Endophytic fungi provide benefits to host plants by producing a diverse class of secondary metabolites (natural products). Arrays of polyketide natural products are synthesized by specific classes of polyketide synthases (PKS I, II and III) in host organisms. In the present study, we attempt to screen and identify type III PKSs in culturable fungal endophytes isolated from the ethno medicinal plants including Arbus precatorius, Bacopa monnieri,Citrus aurantifolia and Datura metel to detect the genetic potential of endophytic fungi in producing bioactive compounds. A total of seventeen endophytic fungal strains belonging to eight genera were identified using fungal morphology and rDNA-ITS phylogenetic analyses. A CODEHOP-PCR based strategy was followed to design degenerate primers for the screening of type III PKS genes from fungal endophytes. We had successfully amplified partial PKS genes from eight endophytes. The amplified PKS sequences showed 60-99% identity to already characterized/putative PKS genes. From the partial sequence of FiPKS from Fusarium incarnatum BMER1, a full-length gene was amplified, cloned and characterized. FiPKScDNA was cloned and expressed in E. coli Lemo21 (DE3) and the purified protein was shown to produce pyrones and resorcinols using acyl-CoA thioesters as substrates. FiPKS showed the highest catalytic efficiency of 7.6 × 104 s-1 M-1 with stearoyl CoA as a starter unit. This study reports the identification and characterization of type III PKS from endophytes of medicinal plants by CODEHOP PCR.

Molecular characterization of two alkylresorcylic acid synthases from Sordariomycetes fungi.

Two putative type III polyketide synthase genes (PKS) were identified from Sordariomycetes fungi. These two type III PKS genes from Sordaria macrospora (SmPKS) and Chaetomium thermophilum (CtPKS), shared 59.8% sequence identity. Both, full-length and truncated versions of type III PKSs were successfully cloned and overexpressed in a bacterial host, Escherichia Coli BL21 (DE3) using a N-terminus hexa-histidine tag. The full-length and the truncated construct of PKSs showed similar activity profiles, suggesting that additional amino acid residues at the C-terminal of both SmPKS and CtPKS may not be involved in catalytic functions. We demonstrate that these two recombinant polyketide synthases could efficiently synthesize tri- and tetraketide pyrones, resorcinols and resorcylic acids using various acyl-CoAs (C4-C20) as starter units. The truncated S. macrospora polyketide synthases (TrSmPKS) showed a maximum of 7.0 × 104 s-1 M-1 catalytic efficiency towards stearoyl-CoA.Whereas, truncated C. thermophilum polyketide synthases (TrCtPKS) preferred the long-chain acyl-CoA starter arachidoyl-CoA, to produce pentaketide and hexaketide resorcinols with a high catalytic efficiency of 6.2 × 104 s-1 M-1. Homology model and substrate docking analyses suggest a shorter distance between sulfur of catalytic Cys152 and thioester carbonyl group of arachidoyl-CoA as well as stronger imidazolium-thiolate ion pair distance in TrCtPKS between catalytic Cys152-His309 compared to TrSmPKS- arachidoyl CoA complex. Enhanced binding interactions of CtPKS residues forming intermolecular contacts at the active site could be attributed to its high specificity towards arachidoyl-CoA. This study reports the functional characterization of two fungal type III polyketide synthases, SmPKS and CtPKS with high catalytic efficiency from S. macrospora and C. thermophilum respectively. Furthermore, the results suggested that the both SmPKS and CtPKS could be attractive targets for protein engineering to discern the unique substrate specificity and catalytic efficiency.