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Fungi involved in lichen symbioses produce a large array of secondary metabolites that are often diagnostic in the taxonomic delimitation of lichens. The most common lichen secondary metabolites-polyketides-are synthesized by polyketide synthases, particularly by Type I PKS (TI-PKS). Here, we present a comparative genomic analysis of the TI-PKS gene content of 23 lichen-forming fungal genomes from Ascomycota, including the de novo sequenced genome of Bacidia rubella. Firstly, we identify a putative atranorin cluster in B. rubella. Secondly, we provide an overview of TI-PKS gene diversity in lichen-forming fungi, and the most comprehensive Type I PKS phylogeny of lichen-forming fungi to date, including 624 sequences. We reveal a high number of biosynthetic gene clusters and examine their domain composition in the context of previously characterized genes, confirming that PKS genes outnumber known secondary substances. Moreover, two novel groups of reducing PKSs were identified. Although many PKSs remain without functional assignments, our findings highlight that genes from lichen-forming fungi represent an untapped source of novel polyketide compounds.

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BACKGROUND: Streptomycetes from the rhizospheric soils are a rich resource of novel secondary metabolites with various biological activities. However, there is still little information related to the isolation, antimicrobial activity and biosynthetic potential for polyketide and non-ribosomal peptide discovery associated with the rhizospheric streptomycetes of Panax notoginseng. Thus, the aims of the present study are to (i) identify culturable streptomycetes from the rhizospheric soil of P. notoginseng by 16S rRNA gene, (ii) evaluate the antimicrobial activities of isolates and analyze the biosynthetic gene encoding polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) of isolates, (iii) detect the bioactive secondary metabolites from selected streptomycetes, (iv) study the influence of the selected isolate on the growth of P. notoginseng in the continuous cropping field. This study would provide a preliminary basis for the further discovery of the secondary metabolites from streptomycetes isolated from the rhizospheric soil of P. notoginseng and their further utilization for biocontrol of plants. RESULTS: A total of 42 strains representing 42 species of the genus Streptomyces were isolated from 12 rhizospheric soil samples in the cultivation field of P. notoginseng and were analyzed by 16S rRNA gene sequencing. Overall, 40 crude cell extracts out of 42 under two culture conditions showed antibacterial and antifungal activities. Also, the presence of biosynthesis genes encoding type I and II polyketide synthase (PKS I and PKS II) and nonribosomal peptide synthetases (NRPSs) in 42 strains were established. Based on characteristic chemical profiles screening by High Performance Liquid Chromatography-Diode Array Detector (HPLC-DAD), the secondary metabolite profiles of strain SYP-A7257 were evaluated by High Performance Liquid Chromatography-High Resolution Mass Spectrometry (HPLC-HRMS). Finally, four compounds actinomycin X2 (F1), fungichromin (F2), thailandin B (F7) and antifungalmycin (F8) were isolated from strain SYP-A7257 by using chromatography techniques, UV, HR-ESI-MS and NMR, and their antimicrobial activities against the test bacteria and fungus were also evaluated. In the farm experiments, Streptomyces sp. SYP-A7257 showed healthy growth promotion and survival rate improvement of P. notoginseng in the continuous cropping field. CONCLUSIONS: We demonstrated the P. notoginseng rhizospheric soil-derived Streptomyces spp. distribution and diversity with respect to their metabolic potential for polyketides and non-ribosomal peptides, as well as the presence of biosynthesis genes PKS I, PKS II and NRPSs. Our results showed that cultivatable Streptomyces isolates from the rhizospheric soils of P. notoginseng have the ability to produce bioactive secondary metabolites. The farm experiments suggested that the rhizospheric soil Streptomyces sp. SYP-A7257 may be a potential biological control agent for healthy growth promotion and survival rate improvement of P. notoginseng in the continuous cropping field.

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Marine microalgae are photosynthetic microorganisms at the base of the marine food webs. They are characterized by huge taxonomic and metabolic diversity and several species have been shown to have bioactivities useful for the treatment of human pathologies. However, the compounds and the metabolic pathways responsible for bioactive compound synthesis are often still unknown. In this study, we aimed at analysing the microalgal transcriptomes available in the Marine Microbial Eukaryotic Transcriptome Sequencing Project (MMETSP) database for an in silico search of polyketide synthase type III homologs and, in particular, chalcone synthase (CHS) and stilbene synthase (STS), which are often referred to as the CHS/STS family. These enzymes were selected because they are known to produce compounds with biological properties useful for human health, such as cancer chemopreventive, anti-inflammatory, antioxidant, anti-angiogenic, anti-viral and anti-diabetic. In addition, we also searched for 4-Coumarate: CoA ligase, an upstream enzyme in the synthesis of chalcones and stilbenes. This study reports for the first time the occurrence of these enzymes in specific microalgal taxa, confirming the importance for microalgae of these pathways and giving new insights into microalgal physiology and possible biotechnological applications for the production of bioactive compounds.

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Type III polyketide synthases (PKS IIIs) contribute to the synthesis of many economically important natural products, most of which are currently produced by direct extraction from plants or through chemical synthesis. Olivetolic acid (OLA) is a plant secondary metabolite sourced from PKS III catalysis, which along with its prenylated derivatives has various pharmacological activities. To demonstrate the potential for microbial cell factories to circumvent limitations of plant extraction or chemical synthesis for OLA, here we utilize a synthetic approach to engineer Escherichia coli for the production of OLA. In vitro characterization of polyketide synthase and cyclase enzymes, OLA synthase and OLA cyclase, respectively, validated their requirement as enzymatic components of the OLA pathway and confirmed the ability for these eukaryotic enzymes to be functionally expressed in E. coli. This served as a platform for the combinatorial expression of these enzymes with auxiliary enzymes aimed at increasing the supply of hexanoyl-CoA and malonyl-CoA as starting and extender units, respectively. Through combining OLA synthase and OLA cyclase expression with the required modules of a ß-oxidation reversal for hexanoyl-CoA generation, we demonstrate the in vivo synthesis of olivetolic acid from a single carbon source. The integration of additional auxiliary enzymes to increase hexanoyl-CoA and malonyl-CoA, along with evaluation of varying fermentation conditions enabled the synthesis of 80 mg/L OLA. This is the first report of OLA production in E. coli, adding a new example to the repertoire of valuable compounds synthesized in this industrial workhorse.

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Type I polyketides are natural products with diverse functions that are important for medical and agricultural applications. Manipulation of large biosynthetic gene clusters containing type I polyketide synthases (PKS) for heterologous expression is difficult due to the existence of conservative sequences of PKS in multiple modules. Red/ET mediated recombination has permitted rapid manipulation of large fragments; however, it requires insertion of antibiotic selection marker in the cassette, raising the problem of interference of expression by leaving "scar" sequence. Here, we report a method for precise seamless stitching of large polyketide biosynthetic gene cluster using a 48.4kb fragment containing type I PKS involved in fostriecin biosynthesis as an example. rpsL counter-selection was used to assist seamless stitching of large fragments, where we have overcome both the size limitations and the restriction on endonuclease sites during the Red/ET recombination. The compatibility and stability of the co-existing vectors (p184 and pMT) which respectively accommodate 16kb and 32.4kb inserted fragments were demonstrated. The procedure described here is efficient for manipulation of large DNA fragments for heterologous expression.

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Polyketides are very large group of natural products with functional and structural diversity.Most of them are produced by microor- ganism and have medicinal activities,including antibiotic,anticancer,antifungal and antiparasitic properties.The researchs in this area have progressed greatly.More and more polyketides are discovered,on the other hand the mechanisms of biosynthesis of those various polyketides are researched more deeply and clearly.The article reviewed the progress of the research in the diversity of polyketide synthases and the mechanisms of polyketide biosynthesise.