Polyketide Synthase Gene Research Articles

Six type-I PKS classes and highly conserved melanin and elsinochrome gene clusters found in diverse Elsinoë species

Elsinoë species are phytopathogenic fungi that cause serious scab diseases on economically important plants. The disease symptoms arise from the effects of a group of phytotoxins known as elsinochromes, produced via a type-I polyketide synthase (PKS) biosynthetic pathway. The elsinochrome gene cluster was first annotated in Elsinoë fawcettii where the main type-I PKS gene was characterized as EfPKS1. A later study showed that this gene and the associated cluster had not been correctly annotated, and that EfPKS1 was actually the anchor gene of the melanin biosynthetic pathway. A new type-I PKS gene EfETB1 associated with elsinochrome production was also identified. The aim of this study was to identify all type-I PKS genes in the genomes of seven Elsinoë species with the goal of independently verifying the PKS containing clusters for both melanin and elsinochrome production. A total of six type-I PKS classes were identified, although there was variation between the species in the number and type of classes present. Genes similar to the E. fawcettii EfPKS1 and EfETB1 type-I PKS genes were associated with melanin and elsinochrome production respectively in all species. The complete melanin and elsinochrome PKS containing clusters were subsequently annotated in all the species with high levels of synteny across Elsinoë species. This study provides a genus-level overview of type-I PKS distribution in Elsinoë species, including an additional line of support for the annotation of the melanin and elsinochrome PKS containing clusters in these important plant pathogens.

CRISPR-Cas9/Cas12a systems for efficient genome editing and large genomic fragment deletions in Aspergillus niger.

CRISPR technology has revolutionized fungal genetic engineering by accelerating the pace and expanding the feasible scope of experiments in this field. Among various CRISPR-Cas systems, Cas9 and Cas12a are widely used in genetic and metabolic engineering. In filamentous fungi, both Cas9 and Cas12a have been utilized as CRISPR nucleases. In this work we first compared efficacies and types of genetic edits for CRISPR-Cas9 and -Cas12a systems at the polyketide synthase (albA) gene locus in Aspergillus niger. By employing a tRNA-based gRNA polycistronic cassette, both Cas9 and Cas12a have demonstrated equally remarkable editing efficacy. Cas12a showed potential superiority over Cas9 protein when one gRNA was used for targeting, achieving an editing efficiency of 86.5% compared to 31.7% for Cas9. Moreover, when employing two gRNAs for targeting, both systems achieved up to 100% editing efficiency for single gene editing. In addition, the CRISPR-Cas9 system has been reported to induce large genomic deletions in various species. However, its use for engineering large chromosomal segments deletions in filamentous fungi still requires optimization. Here, we engineered Cas9 and -Cas12a-induced large genomic fragment deletions by targeting various genomic regions of A. niger ranging from 3.5kb to 40kb. Our findings demonstrate that targeted engineering of large chromosomal segments can be achieved, with deletions of up to 69.1% efficiency. Furthermore, by targeting a secondary metabolite gene cluster, we show that fragments over 100kb can be efficiently and specifically deleted using the CRISPR-Cas9 or -Cas12a system. Overall, in this paper, we present an efficient multi-gRNA genome editing system utilizing Cas9 or Cas12a that enables highly efficient targeted editing of genes and large chromosomal regions in A. niger.

RNA silencing is a key regulatory mechanism in the biocontrol fungus Clonostachys rosea-wheat interactions

BackgroundSmall RNA (sRNAs)- mediated RNA silencing is emerging as a key player in host-microbe interactions. However, its role in fungus-plant interactions relevant to biocontrol of plant diseases is yet to be explored. This study aimed to investigate Dicer (DCL)-mediated endogenous and cross-kingdom gene expression regulation in the biocontrol fungus Clonostachys rosea and wheat roots during interactions.ResultsC. rosea Δdcl2 strain exhibited significantly higher root colonization than the WT, whereas no significant differences were observed for Δdcl1 strains. Dual RNA-seq revealed the upregulation of CAZymes, membrane transporters, and effector coding genes in C. rosea, whereas wheat roots responded with the upregulation of stress-related genes and the downregulation of growth-related genes. The expression of many of these genes was downregulated in wheat during the interaction with DCL deletion strains, underscoring the influence of fungal DCL genes on wheat defense response. sRNA sequencing identified 18 wheat miRNAs responsive to C. rosea, and three were predicted to target the C. rosea polyketide synthase gene pks29. Two of these miRNAs (mir_17532_x1 and mir_12061_x13) were observed to enter C. rosea from wheat roots with fluorescence analyses and to downregulate the expression of pks29, showing plausible cross-kingdom RNA silencing of the C. rosea gene by wheat miRNAs.ConclusionsWe provide insights into the mechanisms underlying the interaction between biocontrol fungi and plant roots. Moreover, the study sheds light on the role of sRNA-mediated gene expression regulation in C. rosea-wheat interactions and provides preliminary evidence of cross-kingdom RNA silencing between plants and biocontrol fungi.

Transcription Factor Engineering in Aspergillus nidulans Leads to the Discovery of an Orsellinaldehyde Derivative Produced via an Unlinked Polyketide Synthase Gene.

Secondary metabolites are generally produced by enzymes encoded by genes within a biosynthetic gene cluster. Transcription factor genes are frequently located within these gene clusters. These transcription factors often drive expression of the other genes of the biosynthetic gene cluster, and overexpression of the transcription factor provides a facile approach to express all genes within a gene cluster, resulting in production of downstream metabolite(s). Unfortunately this approach is not always successful, leading us to engineer more effective hybrid transcription factors. Herein, we attempted to activate a putative cryptic biosynthetic gene cluster in Aspergillus nidulans using a combination of transcription factor engineering and overexpression approaches. This resulted in the discovery of a novel secondary metabolite we term triorsellinaldehyde. Surprisingly, deletion of the polyketide synthase gene within the gene cluster did not prevent triorsellinaldehyde production. However, targeted deletion of a polyketide synthase gene elsewhere in the genome revealed its role in triorsellinaldehyde biosynthesis.

Two Polyketide Synthase Genes, VpPKS10 and VpPKS33, Regulated by VpLaeA Are Essential to the Virulence of Valsa pyri.

Valsa pyri, the causal agent of pear canker disease, typically induces cankers on the bark of infected trees and even leads to tree mortality. Secondary metabolites produced by pathogenic fungi play a crucial role in the pathogenic process. In this study, secondary metabolic regulator VpLaeA was identified in V. pyri. VpLaeA was found to strongly affect the pathogenicity, fruiting body formation, and toxicity of secondary metabolites of V. pyri. Additionally, VpLaeA was found to be required for the response of V. pyri to some abiotic stresses. Transcriptome data analysis revealed that many of differentially expressed genes were involved in the secondary metabolite biosynthesis. Among them, about one third of secondary metabolite biosynthesis core genes were regulated by VpLaeA at different periods. Seven differentially expressed secondary metabolite biosynthesis core genes (VpPKS9, VpPKS10, VpPKS33, VpNRPS6, VpNRPS7, VpNRPS16, and VpNRPS17) were selected for knockout. Two modular polyketide synthase genes (VpPKS10 and VpPKS33) that were closely related to the virulence of V. pyri from the above seven genes were identified. Notably, VpPKS10 and VpPKS33 also affected the production of fruiting body of V. pyri but did not participate in the resistance of V. pyri to abiotic stresses. Overall, this study demonstrates the multifaceted biological functions of VpLaeA in V. pyri and identifies two toxicity-associated polyketide synthase genes in Valsa species fungi for the first time.

Differential gene expression in leaves and roots of Hydrangea serrata treated with aluminium chloride.

Hydrangea serrata, also knowen as the Japanese tea hortensia, is known for its sweet taste and health properties of bevarages produced from this plant. The H. serrata 3,4-dihydroisocoumarins, hydrangenol and phyllodulcin harbour a variety of biological activities and pharmacological properties. Therefore, a detailed understanding of dihydroisocoumarin biosynthesis in H. serrata is of major interest. Their biosynthesis is assumed to be enhanced by elicitors and mediated by polyketide synthases like in cases of phenylpropanoid derived phytoalexins. A de-novo transcriptome assembly of leaves and roots from the aluminium chloride treatment group versus the control group alongside with annotation was generated. Secondary plant metabolites were analysed by LC-MS. It revealed that a terpene synthase and a triterpenoid synthase gene as well as lignin biosynthesis encoding genes were upregulated in roots. Many genes for transporters, glycosyl, and other transferases as well as glycosylases were found to be differentially expressed in both organs. As no differentially expressed polyketide synthase gene homolog was found, the relative leaf and root 3,4-dihydroisocoumarin content was analysed by LC-MS measurement. Although Hydrangea species are known for their aluminium detoxification using phenylpropanoid-derived compounds, the levels of 3,4- dihydroisocoumarins were not enhanced. In this metabolite analysis, an organ- specific accumulation profile of hydrangenol, phyllodulcin, hydrangeic acid and their mono- and di-glycosides was figured out.

Antibiotic properties of seaweed-associated heterotrophic Bacilli against drug-resistant pathogens

The increasing prevalence in antibiotic resistance has driven the search for novel bioactive compounds in previously unexplored marine habitats. Seaweed-associated symbionts have emerged as significant sources of novel antibacterials against nosocomial pathogens. Herein, eighty-one bacterial isolates were obtained through culture-dependent isolation of seaweed symbionts from the southern coast of India. Bacillus velezensis SK54 represented the largest share (54 %), followed by Bacillus siamensis SK53 (33 %), Bacillus subtilis SK20 (8 %), and Bacillus filamentous SK55 (5 %). B. siamensis SK53 and B. velezensis SK54, which were separated from seaweeds Turbinaria conoides and Dictyota cervicornis, respectively, demonstrated clearance zones over 25 mm on spot-over-lawn assay against multiple clinical pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Staphylococcus aureus (VRSA). Selected isolates were sensitive to commercially available antibiotics, and no pathogenicity was observed. The isolates did not amplify pore-forming non-haemolytic haemolysin BL (hbl) and enterotoxin (nhe) genes, and haemolysis on blood agar confirmed their non-pathogenic nature. The bacterial extracts exhibited significant antibacterial activity against the tested pathogens, including MRSA and VRSA, with a minimum inhibitory concentration (MIC) range of 6.25–12.5 μg/mL. The 700 bp type-I polyketide synthase (pks) genes (OQ657454, OQ706631, OQ737799, and OQ834957) were amplified from the heterotrophic B. siamensis SK53 and B. velezensis SK54, with a 99 % identity in the BLAST search. The substantial antibacterial potential against drug-resistant bacteria, along with the presence of genes encoding bioactive lead molecules, suggests that these marine symbiotic bacteria, B. siamensis SK53 and B. velezensis SK54, could be effective in combating emerging antibiotic resistance.

Reprogramming of the Aurantinin Polyketide Assembly Line to Synthesize Auritriacids by Excising an Atypical Enoyl-CoA Hydratase Domain.

Modular polyketide synthases (PKSs) are capable of synthesizing diverse natural products with fascinating bioactivities. Canonical enoyl-CoA hydratases (ECHs) are components of the β-branching cassette that modifies the polyketide chain by adding a β-methyl branch. Herein, it is demonstrated that the deletion of an atypical ECHQ domain (featuring a Q280 residue) of Art21, a didomain protein contains an ECHQ domain and a thioesterase (TE) domain, reprograms the polyketide assembly line from synthesizing tetracyclic aurantinins (ARTs) to bicyclic auritriacids (ATAs) with much lower antibacterial activities. Genes encoding the ECHQ-TE didomain proteins distribute in many PKS gene clusters from different bacteria. Significantly, the ART PKS machinery can be directed to make ARTs, ATAs, or both of them by employing appropriate ECHQ-TE proteins, implying a great potential for using this reprogramming strategy in polyketide structure diversification.

Effective enhancement of the ability of Monascus pilosus to produce lipid-lowering compound Monacolin K via perturbation of metabolic flux and histone acetylation modification

Monacolin K (MK), also known as lovastatin, is a polyketide compound with the ability to reduce plasma cholesterol levels and many other bio-activities. Red yeast rice (also named Hongqu) rich in MK derived from Monascus fermentation has attracted widespread attention due to its excellent performance in reducing blood lipids. However, industrial Monascus fermentation suffers from the limitations such as low yield of MK, long fermentation period, and susceptibility to contamination. In this study, we firstly blocked the competitive pathway of MK biosynthesis to create polyketide synthase gene pigA (the key gene responsible for the biosynthesis of Monascus azaphilone pigments) deficient strain A1. Then, based on the strategies to increase precursor supply for MK biosynthesis, acetyl-CoA carboxylase gene acc overexpression strains C1 and C2 were constructed with WT and A1 as the parent, respectively. Finally, histone deacetylase gene hos2 overexpression strain H1 was constructed by perturbation of histone acetylation modification. HPLC detection revealed all these four strains significantly increased their abilities to produce MK. After 14 days of solid-state fermentation, the MK yields of strains A1, C1, C2, and H1 reached 2.03 g/100 g, 1.81 g/100 g, 2.45 g/100 g and 2.52 g/100 g, which increased by 28.5 %, 14.7 %, 43.9 % and 36.1 % compared to WT, respectively. RT-qPCR results showed that overexpression of hos2 significantly increased the expression level of almost all genes responsible for MK biosynthesis after 5-day growth. Overall, the abilities of these strains to produce MK has been greatly improved, and MK production period has been shortened to 14 days from 20 days, providing new approaches for efficient production of Hongqu rich in MK.

Giant polyketide synthase enzymes in the biosynthesis of giant marine polyether toxins.

Prymnesium parvum are harmful haptophyte algae that cause massive environmental fish kills. Their polyketide polyether toxins, the prymnesins, are among the largest nonpolymeric compounds in nature and have biosynthetic origins that have remained enigmatic for more than 40 years. In this work, we report the "PKZILLAs," massive P. parvum polyketide synthase (PKS) genes that have evaded previous detection. PKZILLA-1 and -2 encode giant protein products of 4.7 and 3.2 megadaltons that have 140 and 99 enzyme domains. Their predicted polyene product matches the proposed pre-prymnesin precursor of the 90-carbon-backbone A-type prymnesins. We further characterize the variant PKZILLA-B1, which is responsible for the shorter B-type analog prymnesin-B1, from P. parvum RCC3426 and thus establish a general model of haptophyte polyether biosynthetic logic. This work expands expectations of genetic and enzymatic size limits in biology.

Construction of an expression platform for fungal secondary metabolite biosynthesis in Penicillium crustosum

Filamentous fungi are prolific producers of bioactive natural products and play a vital role in drug discovery. Yet, their potential cannot be fully exploited since many biosynthetic genes are silent or cryptic under laboratory culture conditions. Several strategies have been applied to activate these genes, with heterologous expression as one of the most promising approaches. However, successful expression and identification of new products are often hindered by host-dependent factors, such as low gene targeting efficiencies, a high metabolite background, or a lack of selection markers. To overcome these challenges, we have constructed a Penicillium crustosum expression host in a pyrG deficient strain by combining the split-marker strategy and CRISPR-Cas9 technology. Deletion of ligD and pcribo improved gene targeting efficiencies and enabled the use of an additional selection marker in P. crustosum. Furthermore, we reduced the secondary metabolite background by inactivation of two highly expressed gene clusters and abolished the formation of the reactive ortho-quinone methide. Finally, we replaced the P. crustosum pigment gene pcr4401 with the commonly used Aspergillus nidulans wA expression site for convenient use of constructs originally designed for A. nidulans in our P. crustosum host strain. As proof of concept, we successfully expressed a single polyketide synthase gene and an entire gene cluster at the P. crustosum wA locus. Resulting transformants were easily detected by their albino phenotype. With this study, we provide a highly efficient platform for heterologous expression of fungal genes.Key pointsConstruction of a highly efficient Penicillium crustosum heterologous expression hostReduction of secondary metabolite background by genetic dereplication strategyIntegration of wA site to provide an alternative host besides Aspergillus nidulansGraphical

Co-expression of a pair of interdependent regulators coding genes ovmZ and ovmW awakens the production of angucyclinones antibiotics in Streptomyces neyagawaensis

BackgroundMicrobial genome sequencing and analysis revealed the presence of abundant silent secondary metabolites biosynthetic gene clusters (BGCs) in streptomycetes. Activating these BGCs has great significance for discovering new compounds and novel biosynthetic pathways.ResultsIn this study, we found that ovmZ and ovmW homologs, a pair of interdependent transcriptional regulators coding genes, are widespread in actinobacteria and closely associated with the biosynthesis of secondary metabolites. Through co-overexpression of native ovmZ and ovmW in Streptomyces neyagawaensis NRRL B-3092, a silent type II polyketide synthase (PKS) gene cluster was activated to produce gephyromycin A, tetrangomycin and fridamycin E with the yields of 22.3 ± 8.0 mg/L, 4.8 ± 0.5 mg/L and 20.3 ± 4.1 mg/L respectively in the recombinant strain of S.ne/pZnWn. However, expression of either ovmZ or ovmW failed to activate this gene cluster. Interestingly, overexpression of the heterologous ovmZ and ovmW pair from oviedomycin BGC of S. ansochromogenes 7100 also led to awakening of this silent angucyclinone BGC in S. neyagawaensis.ConclusionA silent angucyclinone BGC was activated by overexpressing both ovmZ and ovmW in S. neyagawaensis. Due to the wide distribution of ovmZ and ovmW in the BGCs of actinobacteria, co-overexpression of ovmZ and ovmW could be a strategy for activating silent BGCs, thus stimulating the biosynthesis of secondary metabolites.

Seamless site-directed mutagenesis in complex cloned DNA sequences using the RedEx method.

Seamless site-directed mutagenesis is an important technique for studying protein functions, tuning enzyme catalytic activities and modifying genetic elements in multiple rounds because it can insert, delete or substitute nucleotides, DNA segments or even entire genes at the target site without introducing any unwanted change. To facilitate seamless site-directed mutagenesis in large plasmids and bacterial artificial chromosomes (BACs) with repetitive sequences, we recently developed the RedEx strategy. Compared with previous methods, our approach achieves the recovery of correct recombinants with high accuracy by circumventing unwanted recombination between repetitive sequences. RedEx readily yields more than 80% accuracy in seamless DNA insertion and deletion in large multimodular polyketide synthase gene clusters, which are among the most difficult targets due to the large number of repetitive DNA sequences in modules encoding almost identical enzymes. Here we present the RedEx method by describing in detail the seamless site-directed mutagenesis in a BAC vector. Overall, the process includes three parts: (1) insertion of the RedEx cassette containing the desired mutation together with selection-counterselection markers flanked by unique restriction sites and 20-bp overlapping sequences into the target site by recombineering, (2) removal of the selection-counterselection markers in the BAC by restriction digestion and (3) circularization of the linear BAC by exonuclease-mediated in vitro DNA annealing. This protocol can be performed within 3 weeks and will enable researchers with DNA cloning experience to master seamless site-directed mutagenesis to accelerate their research.

Antibacterial activity and biosynthetic potential of Streptomyces sp. PBR19, isolated from forest rhizosphere soil of Assam.

An Actinomycetia isolate, designated as PBR19, was derived from the rhizosphere soil of Pobitora Wildlife Sanctuary (PWS), Assam, India. The isolate, identified as Streptomyces sp., shares a sequence similarity of 93.96% with its nearest type strain, Streptomyces atrovirens. This finding indicates the potential classification of PBR19 as a new taxon within the Actinomycetota phylum. PBR19 displayed notable antibacterial action against some ESKAPE pathogens. The ethyl acetate extract of PBR19 (EtAc-PBR19) showed the lowest minimum inhibitory concentration (MIC) of ≥ 0.195µg/mL against Acinetobacter baumannii ATCC BAA-1705. A lower MIC indicates higher potency against the tested pathogen. Scanning electron microscope (SEM) findings revealed significant changes in the cytoplasmic membrane structure of the pathogen. This suggests that the antibacterial activity may be linked to the disruption of the microbial membrane. The predominant chemical compound detected in the EtAc-PBR19 was identified as phenol, 3,5-bis(1,1-dimethylethyl), comprising 48.59% of the area percentage. Additionally, PBR19 was found to contain the type II polyketide synthases (PKS type II) gene associated with antibiotic synthesis. The predicted gene product of PKSII was identified as the macrolide antibiotic Megalomicin A. The taxonomic distinctiveness, potent antibacterial effects, and the presence of a gene associated with antibiotic synthesis suggest that PBR19 could be a valuable candidate for further exploration in drug development and synthetic biology. The study contributes to the broader understanding of microbial diversity and the potential for discovering bioactive compounds in less-explored environments.

Development of a CRISPR/Cas9-mediated gene knockout method for functional genomics of the barley spot blotch pathogen Bipolaris sorokiniana

Bipolaris sorokiniana (=Cochliobolus sativus) is an important fungal pathogen that causes spot blotch, common root rot and kernel blight in barley and wheat. In this study, we aimed to develop a CRISPR/Cas9-mediated approach for efficiently knocking out genes of B. sorokiniana. We first assessed the efficiency of Cas9/sgRNA combined with the split marker system for gene replacement. We designed sgRNAs to target a polyketide synthase gene (PKS1) that is required for melanin biosynthesis of the fungus. When the preassembled Cas9/gRNA ribonucleoproteins (RNPs) and the split hygromycin B resistance gene (HygB) fragments each harboring 449 bp upstream and 595 bp downstream flanking sequences of PKS1 were co-transformed into the protoplasts of B. sorokiniana, the number of transformants with the PKS1 gene replaced by the selection marker were significantly increased compared to the control without RNPs. We then used the RNPs with PCR-amplified HygB gene cassette carrying 40 bp or 60 bp arms homologous to the flanking sequences of the PKS1 target site for fungal transformation and showed that the RNPs significantly enhanced gene disruption efficiency through the short-homology recombination, and more gene knockout mutants were obtained with longer (60 bp) homologous arms than the shorter (40 bp) ones. Finally, we disrupted an uncharacterized non-ribosomal peptide synthetase (NRPS) gene (NPSx) in B. sorokiniana strain ND90Pr using the RNP-mediated gene knockout approach and showed the mutants lost virulence on barley cv. Bowman. The Cas9/sgRNA-mediated gene knockout method developed will facilitate large-scale functional genomics studies of B. sorokiniana.

The haplotype-resolved Prymnesium parvum (type B) microalga genome reveals the genetic basis of its fish-killing toxins

The catastrophic loss of aquatic life in the Central European Oder River in 2022, caused by a toxic bloom of the haptophyte microalga Prymnesium parvum (in a wide sense, s.l.), underscores the need to improve our understanding of the genomic basis of the toxin. Previous morphological, phylogenetic, and genomic studies have revealed cryptic diversity within P.parvum s.l. and uncovered three clade-specific (types A, B, and C) prymnesin toxins. Here, we used state-of-the-art long-read sequencing and assembled the first haplotype-resolved diploid genome of a P.parvum type B from the strain responsible for the Oder disaster. Comparative analyses with type A genomes uncovered a genome-size expansion driven by repetitive elements in type B. We also found conserved synteny but divergent evolution in several polyketide synthase (PKS) genes, which are known to underlie toxin production in combination with environmental cues. We identified an approximately 20-kbp deletion in the largest PKS gene of type B that we link to differences in the chemical structure of types A and B prymnesins. Flow cytometry and electron microscopy analyses confirmed diploidy in the Oder River strain and revealed differences to closely related strains in both ploidy and morphology. Our results provide unprecedented resolution of strain diversity in P.parvum s.l. and a better understanding of the genomic basis of toxin variability in haptophytes. The reference-quality genome will enable us to better understand changes in microbial diversity in the face of increasing environmental pressures and provides a basis for strain-level monitoring of invasive Prymnesium in the future.

Bacillus Paralicheniformis: A potential candidate for natural antibacterial agents from marine macro-algae associated bacteria.

Antibiotic resistance poses a major global health threat, necessitating new approaches to treat bacterial infections. We isolated bacteria from the brown macroalgae Sargassum swartzii collected along the Indian coast. Of 279 isolates, 47.6% showed antibacterial activity against Staphylococcus aureus and Vibrio parahaemolyticus when screened. Ten potent isolates underwent analysis of biosynthetic gene clusters and 16S rRNA sequencing. Isolate MP-99 carried polyketide synthase (pks) and non-ribosomal peptide synthetase (nrps) genes and exhibited antibacterial effects.

Дослідження криптичного спеціалізованого метаболому Streptomyces cyanogenus S136

Streptomyces cyanogenus S136 is known to produce landomycin family antibiotics, particularly its largest congener, landomycin A. Except for landomycins and polyene lucensomycin, no other specialized metabolites were sourced from S136. Nevertheless, S136 genome sequencing revealed over 40 biosynthetic gene clusters (BGCs), implying underappreciated potential of this strain for the production of novel bioactive natural compounds. We set out to gain deeper insight into the specialized metabolome of this strain. First, trans­criptomic analysis of S136 grown under landomycin production conditions has been carried out, revealing that most of them are expressed at a basal level. This, likely, leads to a phenotypic silence of most of the BGCs. Nevertheless, several notable exceptions have been spotted. First of all, landomycin BGC is expressed at high level (at least 100 Transcripts Per Million mapped reads (TPM); and around 1000 TPM for minimal polyketide synthase genes lanFABC). Similarly, high levels of expression showed BGCs # 2, 4, 7 and 33, of which #2, encoding unknown saccharide, is the most dissimilar to the described precedents. RNAseq data also allowed us to delineate better the borders of several presumed BGCs. In the next phase of the work we singled out a few BGCs within S136 that appeared to be promising. First, these BGCs exhibited low similarity to the other gene clusters directing the production of known natural products. Second, the BGCs harbored cluster-situated regulatory genes that can be employed in the attempts to activate the expression of cryptic pathways. For one such BGC we constructed two plasmids for expression of several such regulatory genes and introduced them into S136 and its derivative deficient in production of landomycin A. Bioassays showed no differences in bioactivity of the recombinant strains as compared to the initial strains. Liquid chromatography coupled to mass spectrometry (LC-MS) analysis of several S. cyanogenus samples revealed the effects of genotype, growth conditions and extraction on specialized metabolome of this species, setting reference point for further stu­dies.

Elucidation of Palmarumycin Spirobisnaphthalene Biosynthesis Reveals a Set of Previously Unrecognized Oxidases and Reductases

AbstractSpirobisnaphthalenes (SBNs) are a class of highly oxygenated, fungal bisnaphthalenes containing a unique spiroketal bridge, that displayed diverse bioactivities. Among the reported SBNs, palmarumycins are the major type, which are precursors for the other type of SBNs structurally. However, the biosynthesis of SBNs is unclear. In this study, we elucidated the biosynthesis of palmarumycins, using gene disruption, heterologous expression, and substrate feeding experiments. The biosynthetic gene cluster for palmarumycins was identified to be distant from the polyketide synthase gene cluster, and included two cytochrome P450s (PalA and PalB), and one short chain dehydrogenase/reductase (PalC) encoding genes as key structural genes. PalA is an unusual, multifunctional P450 that catalyzes the oxidative dimerization of 1,8‐dihydroxynaphthalene to generate the spiroketal linkage and 2,3‐epoxy group. Chemical synthesis of key intermediate and in vitro biochemical assays proved that the oxidative dimerization proceeded via a binaphthyl ether. PalB installs the C‐5 hydroxy group, widely found in SBNs. PalC catalyzes 1‐keto reduction, the reverse 1‐dehydrogenation, and 2,3‐epoxide reduction. Moreover, an FAD‐dependent oxidoreductase, encoded by palD, which locates outside the cluster, functions as a 1‐dehydrogenase. These results provided the first genetic and biochemical evidence for the biosynthesis of palmarumycin SBNs.

Elucidation of Palmarumycin Spirobisnaphthalene Biosynthesis Reveals a Set of Previously Unrecognized Oxidases and Reductases.

Spirobisnaphthalenes (SBNs) are a class of highly oxygenated, fungal bisnaphthalenes containing a unique spiroketal bridge, that displayed diverse bioactivities. Among the reported SBNs, palmarumycins are the major type, which are precursors for the other type of SBNs structurally. However, the biosynthesis of SBNs is unclear. In this study, we elucidated the biosynthesis of palmarumycins, using gene disruption, heterologous expression, and substrate feeding experiments. The biosynthetic gene cluster for palmarumycins was identified to be distant from the polyketide synthase gene cluster, and included two cytochrome P450s (PalA and PalB), and one short chain dehydrogenase/reductase (PalC) encoding genes as key structural genes. PalA is an unusual, multifunctional P450 that catalyzes the oxidative dimerization of 1,8-dihydroxynaphthalene to generate the spiroketal linkage and 2,3-epoxy group. Chemical synthesis of key intermediate and in vitro biochemical assays proved that the oxidative dimerization proceeded via a binaphthyl ether. PalB installs the C-5 hydroxy group, widely found in SBNs. PalC catalyzes 1-keto reduction, the reverse 1-dehydrogenation, and 2,3-epoxide reduction. Moreover, an FAD-dependent oxidoreductase, encoded by palD, which locates outside the cluster, functions as a 1-dehydrogenase. These results provided the first genetic and biochemical evidence for the biosynthesis of palmarumycin SBNs.