Type III polyketide synthases (PKSs) produce an array of metabolites with diverse functions. In this study, we have cloned the complete reading frame encoding type III PKS (SbPKS) from a brown seaweed, Sargassum binderi, and characterized the activity of its recombinant protein biochemically. The deduced amino acid sequence of SbPKS is 414 residues in length, sharing a higher sequence similarity with bacterial PKSs (38% identity) than with plant PKSs. The Cys-His-Asn catalytic triad of PKS is conserved in SbPKS with differences in some of the residues lining the active and CoA binding sites. The wild-type SbPKS displayed broad starter substrate specificity to aliphatic long-chain acyl-CoAs (C(6)-C(14)) to produce tri- and tetraketide pyrones. Mutations at H(331) and N(364) caused complete loss of its activity, thus suggesting that these two residues are the catalytic residues for SbPKS as in other type III PKSs. Furthermore, H227G, H227G/L366V substitutions resulted in increased tetraketide-forming activity, while wild-type SbPKS produces triketide α-pyrone as a major product. On the other hand, mutant H227G/L366V/F93A/V95A demonstrated a dramatic decrease of tetraketide pyrone formation. These observations suggest that His(227) and Leu(366) play an important role for the polyketide elongation reaction in SbPKS. The conformational changes in protein structure especially the cavity of the active site may have more significant effect to the activity of SbPKS compared with changes in individual residues.
The Sfp-type 4'-phosphopantetheinyl transferase Ppt1 is required for activation of nonribosomal peptide synthetases, including α-aminoadipate reductase (AAR) for lysine biosynthesis and polyketide synthases, enzymes that biosynthesize peptide and polyketide secondary metabolites, respectively. Deletion of the PPT1 gene, from the maize pathogen Cochliobolus heterostrophus and the rice pathogen Cochliobolus miyabeanus, yielded strains that were significantly reduced in virulence to their hosts. In addition, ppt1 mutants of C. heterostrophus race T and Cochliobolus victoriae were unable to biosynthesize the host-selective toxins (HST) T-toxin and victorin, respectively, as judged by bioassays. Interestingly, ppt1 mutants of C. miyabeanus were shown to produce tenfold higher levels of the sesterterpene-type non-HST ophiobolin A, as compared with the wild-type strain. The ppt1 strains of all species were also reduced in tolerance to oxidative stress and iron depletion; both phenotypes are associated with inability to produce extracellular siderophores biosynthesized by the nonribosomal peptide synthetase Nps6. Colony surfaces were hydrophilic, a trait previously associated with absence of C. heterostrophus Nps4. Mutants were decreased in asexual sporulation and C. heterostrophus strains were female-sterile in sexual crosses; the latter phenotype was observed previously with mutants lacking Nps2, which produces an intracellular siderophore. As expected, mutants were albino, since they cannot produce the polyketide melanin and were auxotrophic for lysine because they lack an AAR.
There is an urgent need to search for new antibiotics to counter the growing number of antibiotic-resistant bacterial strains, one of which is methicillin-resistant Staphylococcus aureus (MRSA). Herein, we report a Streptomyces sp. strain MUSC 125 from mangrove soil in Malaysia which was identified using 16S rRNA phylogenetic and phenotypic analysis. The methanolic extract of strain MUSC 125 showed anti-MRSA, anti-biofilm and antioxidant activities. Strain MUSC 125 was further screened for the presence of secondary metabolite biosynthetic genes. Our results indicated that both polyketide synthase (pks) gene clusters, pksI and pksII, were detected in strain MUSC 125 by PCR amplification. In addition, gas chromatography-mass spectroscopy (GC-MS) detected the presence of different chemicals in the methanolic extract. Based on the GC-MS analysis, eight known compounds were detected suggesting their contribution towards the anti-MRSA and anti-biofilm activities observed. Overall, the study bolsters the potential of strain MUSC 125 as a promising source of anti-MRSA and antibiofilm compounds and warrants further investigation.