Polyketide Synthase Gene Cluster Research Articles

Discovery of the 2,4'-Dihydroxy-3'-methoxypropiophenone Biosynthesis Genes in Aspergillus oryzae.

The filamentous fungus Aspergillus oryzae has 27 putative iterative type I polyketide synthase (PKS) gene clusters, but the secondary metabolites produced by them are mostly unknown. Here, we focused on eight clusters that were reported to be expressed at relatively high levels in a transcriptome analysis. By comparing metabolites between an octuple-deletion mutant of these eight PKS gene clusters and its parent strain, we found that A. oryzae produced 2,4'-dihydroxy-3'-methoxypropiophenone (1) and its precursor, 4'-hydroxy-3'-methoxypropiophenone (3) in a specific liquid medium. Furthermore, an iterative type I PKS (PpsB) encoded by AO090102000166 and an acetyl-CoA ligase (PpsA) encoded downstream from ppsB were shown to be essential for their biosynthesis. PpsC, encoded upstream from ppsB, was shown to have 3-binding activity (Kd =26.0±6.2 μM) and is suggested to be involved in the conversion of 3 to 1. This study deepens our understanding of cryptic secondary metabolism in A. oryzae.

Isolation, phylogenetic analysis and antimicrobial activity of halophilic actinomycetes from different saline environments located near Çorum province

The aim of this study was to isolate and characterize halophilic actinomycetes. Halophilic actinomycetes isolates were obtained from soil samples from Iskilip-Kekec, Tayrak, Yerlikoy and Sungurlu-Akpinar, Akcakoyunlu, Alibaba, Boncuk salterns located near Corum province with a dilution plate technique on four selective media with 3%, 10% and 15% NaCl. PCR amplification belonging to 16S rRNA gene region of the isolates was realized out with primers of 27F and 1525R. Phylogenetic trees were formed with the three different algorithms in MEGA 7.0 software. All fifteen microorganisms were isolated on four selective media with different salt proportions and based on 16S rRNA gene sequence analysis of 15 isolates, eleven Streptomyces isolates, two Nocardiopsis isolates, two Saccharopolyspora isolates were determined. 7 of the strains exhibited only antibacterial activity, 3 only antifungal activity, and 1 both antibacterial and antifungal activities In addition, NRPS, PKS-I ve PKS-II gene regions of isolates with antimicrobial activity were investigated with related primers. According to the results of the work done, of the 11 actinomycete isolates, 4 were found to have PKS-I, 5 were PKS-II and 8 had NRPS gene regions. When the results were evaluated, it was observed that the isolates contained more nonribozomal peptide synthetase (NRPS) gene clusters than type I-II polyketide synthase (PKS-I-II) gene clusters. In addition, it was determined that 1 isolate contain three gene clusters together. As a result, it was found that actinomycetes isolated from saline soils have the capacity to produce secondary metabolites.

Genome-wide analysis and prediction of genes involved in the biosynthesis of polysaccharides and bioactive secondary metabolites in high-temperature-tolerant wild Flammulina filiformis

BackgroundFlammulina filiformis (previously known as Asian F. velutipes) is a popular commercial edible mushroom. Many bioactive compounds with medicinal effects, such as polysaccharides and sesquiterpenoids, have been isolated and identified from F. filiformis, but their biosynthesis and regulation at the molecular level remains unclear. In this study, we sequenced the genome of the wild strain F. filiformis Liu355, predicted its biosynthetic gene clusters (BGCs) and profiled the expression of these genes in wild and cultivar strains and in different developmental stages of the wild F. filiformis strain by a comparative transcriptomic analysis.ResultsWe found that the genome of the F. filiformis was 35.01 Mb in length and harbored 10,396 gene models. Thirteen putative terpenoid gene clusters were predicted and 12 sesquiterpene synthase genes belonging to four different groups and two type I polyketide synthase gene clusters were identified in the F. filiformis genome. The number of genes related to terpenoid biosynthesis was higher in the wild strain (119 genes) than in the cultivar strain (81 genes). Most terpenoid biosynthesis genes were upregulated in the primordium and fruiting body of the wild strain, while the polyketide synthase genes were generally upregulated in the mycelium of the wild strain. Moreover, genes encoding UDP-glucose pyrophosphorylase and UDP-glucose dehydrogenase, which are involved in polysaccharide biosynthesis, had relatively high transcript levels both in the mycelium and fruiting body of the wild F. filiformis strain.ConclusionsF. filiformis is enriched in a number of gene clusters involved in the biosynthesis of polysaccharides and terpenoid bioactive compounds and these genes usually display differential expression between wild and cultivar strains, even in different developmental stages. This study expands our knowledge of the biology of F. filiformis and provides valuable data for elucidating the regulation of secondary metabolites in this unique F. filiformis strain.

Screening for Candidate Genes Associated with Biocontrol Mechanisms of Bacillus pumilus DX01 Using Tn5 Transposon Mutagenesis and a 2-DE-Based Comparative Proteomic Analysis.

A total of 1467 mutants of the biocontrol bacterium Bacillus pumilus DX01 were obtained by Tn5 insertional mutagenesis and subjected to the determination of antagonistic capabilities. Compared with the wild-type strain DX01, the mutant M25 was identified to have the most significant reduction in antagonistic capability against the phytopathogen Bipolaris maydis and extracellular proteinase activity. The integration site of the exogenous T-DNA in the genome of mutant M25 was revealed in the coding region of malony CoA-ACP transacylase (MCAT) gene (mcat), which belongs to a polyketide synthase (PKS) gene cluster, DX01pks of B. pumilus DX01. Furthermore, the whole DX01pks gene cluster was cloned using Illumina Solexa sequencing technology, and it has a modular framework different from the other two gene clusters involved in polyketide synthesis in B. amyloliquefaciens FZB42 (pks1) and B. subtilis 168 (pksX). Finally, in order to gain more insights into the molecular mechanisms of biocontrol of B. pumilus DX01, the changes in the relative level of expression of total proteins between the original strain DX01 and the mutant M25 were detected by 2-DE-based proteomic analysis. A total of twenty differentially expressed proteins were identified upon the mcat gene transposition mutagenesis. Of these proteins, seven proteins were up-regulated in expression level and the other proteins were down-regulated. Taken together, the results in this study showed that Tn5 transposon mutagenesis of B. pumilus DX01 can lead to a significant change of antiphytopathogen ability, and the DX01pks gene cluster possibly play a potential role in the biocontrol processes of this bacterium.

Unveiling of Swainsonine Biosynthesis via a Multibranched Pathway in Fungi.

The indolizidine alkaloid swainsonine (SW) is a deadly mycotoxin to livestock that can be produced by different plant-associated fungi, including the endophytic entomopathogenic fungi Metarhizium species. The SW biosynthetic gene cluster has been identified but the genetic mechanism of SW biosynthesis remains obscure. To unveil the SW biosynthetic pathway, we performed gene deletions in M. robertsii, heterologous expression of a core biosynthetic gene, substrate feedings, mass spectrometry, and bioassay analyses in this study. It was unveiled that SW is produced via a multibranched pathway by the hybrid nonribosomal peptide-polyketide synthase (NRPS-PKS) gene cluster in M. robertsii. The precursor pipecolic acid can be converted from lysine by both the SW biosynthetic cluster and the unclustered genes such as lysine cyclodeaminase. The hybrid NRPS-PKS enzyme produces three intermediates with and without domain skipping. Intriguingly, the biosynthetic process is coupled with the cis to trans nonenzymatic epimerization of C1-OH for both hydroxyl- and dihydroxyl-indolizidine intermediates. We also found that SW production was dispensable for fungal colonization of plants and infection of insect hosts. Functional characterization of the SW biosynthetic genes in this study may benefit the safe use of Metarhizium fungi as insect biocontrol agents and the management of livestock pastures from SW contamination by genetic manipulation of the toxin-producing fungi.

Streptomyces sp. Strain MUSC 125 from Mangrove Soil in Malaysia with Anti-MRSA, Anti-Biofilm and Antioxidant Activities.

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.

The Wolfiporia cocos Genome and Transcriptome Shed Light on the Formation of Its Edible and Medicinal Sclerotium

Wolfiporia cocos (F. A. Wolf) has been praised as a food delicacy and medicine for centuries in China. Here, we present the genome and transcriptome of the Chinese strain CGMCC5.78 of W. cocos. High-confidence functional prediction was made for 9277 genes among the 10,908 total predicted gene models in the W. cocos genome. Up to 2838 differentially expressed genes (DEGs) were identified to be related to sclerotial development by comparing the transcriptomes of mycelial and sclerotial tissues. These DEGs are involved in mating processes, differentiation of fruiting body tissues, and metabolic pathways. A number of genes encoding enzymes and regulatory factors related to polysaccharide and triterpenoid production were strikingly regulated. A potential triterpenoid gene cluster including the signature lanosterol synthase (LSS) gene and its modified components were annotated. In addition, five nonribosomal peptide synthase (NRPS)-like gene clusters, eight polyketide synthase (PKS) gene clusters, and 15 terpene gene clusters were discovered in the genome. The differential expression of the velevt family proteins, transcription factors, carbohydrate-active enzymes, and signaling components indicated their essential roles in the regulation of fungal development and secondary metabolism in W. cocos. These genomic and transcriptomic resources will be valuable for further investigations of the molecular mechanisms controlling sclerotial formation and for its improved medicinal applications.

Novel Fredericamycin Variant Overproduced by a Streptomycin-resistant Streptomyces albus subsp. chlorinus Strain.

Streptomycetes are an important source of natural products potentially applicable in the pharmaceutical industry. Many of these drugs are secondary metabolites whose biosynthetic genes are very often poorly expressed under laboratory cultivation conditions. In many cases, antibiotic-resistant mutants exhibit increased production of natural drugs, which facilitates the identification and isolation of new substances. In this study, we report the induction of a type II polyketide synthase gene cluster in the marine strain Streptomyces albus subsp. chlorinus through the selection of streptomycin-resistant mutants, resulting in overproduction of the novel compound fredericamycin C2 (1). Fredericamycin C2 (1) is structurally related to the potent antitumor drug lead fredericamycin A.

Transcriptomic and Exometabolomic Profiling Reveals Antagonistic and Defensive Modes of Clonostachys rosea Action Against Fusarium graminearum.

The mycoparasite Clonostachys rosea ACM941 is under development as a biocontrol organism against Fusarium graminearum, the causative agent of Fusarium head blight in cereals. To identify molecular factors associated with this interaction, the transcriptomic and exometabolomic profiles of C. rosea and F. graminearum GZ3639 were compared during coculture. Prior to physical contact, the antagonistic activity of C. rosea correlated with a response heavily dominated by upregulation of polyketide synthase gene clusters, consistent with the detected accumulation of corresponding secondary metabolite products. Similarly, prior to contact, trichothecene gene clusters were upregulated in F. graminearum, while those responsible for fusarielin and fusarin biosynthesis were downregulated, correlating with an accumulation of trichothecene products in the interaction zone over time. A concomitant increase in 15-acetyl deoxynivalenol-3-glucoside in the interaction zone was also detected, with C. rosea established as the source of this detoxified mycotoxin. After hyphal contact, C. rosea was found to predominantly transcribe genes encoding cell wall-degradation enzymes, major facilitator superfamily sugar transporters, anion:cation symporters, as well as alternative carbon source utilization pathways, together indicative of a transition to necrotropism at this stage. F. graminearum notably activated the transcription of phosphate starvation pathway signature genes at this time. Overall, a number of signature molecular mechanisms likely contributing to antagonistic activity by C. rosea against F. graminearum, as well as its mycotoxin tolerance, are identified in this report, yielding several new testable hypotheses toward understanding the basis of C. rosea as a biocontrol agent for continued agronomic development and application.

Draft Genome Sequence of Saccharomonospora piscinae KCTC 19743T, an Actinobacterium Containing Secondary Metabolite Biosynthetic Gene Clusters.

The draft genome sequence of Saccharomonospora piscinae KCTC 19743T, with a size of 4,897,614 bp, was assembled into 11 scaffolds containing 4,561 open reading frames and a G+C content of 71.0 mol%. Polyketide synthase and nonribosomal peptide synthetase gene clusters, which are responsible for the biosynthesis of several biomolecules, were identified and located in different regions in the genome.

Biosynthesis of depsipeptides with a 3-hydroxybenzoate moiety and selective anticancer activities involves a chorismatase

Neoantimycins are anticancer compounds of 15-membered ring antimycin-type depsipeptides. They are biosynthesized by a hybrid multimodular protein complex of nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS), typically from the starting precursor 3-formamidosalicylate. Examining fermentation extracts of Streptomyces conglobatus, here we discovered four new neoantimycin analogs, unantimycins B–E, in which 3-formamidosalicylates are replaced by an unusual 3-hydroxybenzoate (3-HBA) moiety. Unantimycins B–E exhibited levels of anticancer activities similar to those of the chemotherapeutic drug cisplatin in human lung cancer, colorectal cancer, and melanoma cells. Notably, they mostly displayed no significant toxicity toward noncancerous cells, unlike the serious toxicities generally reported for antimycin-type natural products. Using site-directed mutagenesis and heterologous expression, we found that unantimycin productions are correlated with the activity of a chorismatase homolog, the nat-hyg5 gene, from a type I PKS gene cluster. Biochemical analysis confirmed that the catalytic activity of Nat-hyg5 generates 3-HBA from chorismate. Finally, we achieved selective production of unantimycins B and C by engineering a chassis host. On the basis of these findings, we propose that unantimycin biosynthesis is directed by the neoantimycin-producing NRPS–PKS complex and initiated with the starter unit of 3-HBA. The elucidation of the biosynthetic unantimycin pathway reported here paves the way to improve the yield of these compounds for evaluation in oncotherapeutic applications.

Bioprospecting for Novel Natural Products in Ancient Non‐Actinobacteria

Polyketides are a particularly important category of secondary metabolite, and alone comprise 20% of the top‐selling drugs around the world. 1 Polyaromatic (type II) polyketides incorporate aromatic rings into their structures and are synthesized in vivo by a cohort of enzymes called a synthase. Three enzymes remain constant in all type II polyketide synthases: ketosynthase (KS), chain length factor (CLF), and acyl carrier protein (ACP). These three enzymes are known as the minimal polyketide synthase (PKS) and form the basis for the production of all type II polyketides. The most well‐studied of these polyketides are produced by a phylum known as Actinobacteria. Some of the polyketides that these bacteria produce include tetracycline, erythromycin, and lovastatin. Unfortunately, Actinobacteria are difficult to work with in the lab and researchers have historically been unsuccessful in expressing their KS‐CLF complexes in robust heterologous hosts such as E. coli. In 2015, a study by the Charkoudian and Hillenmeyer labs showed that the gene clusters responsible for the transcription and translation of the minimal PKS are present in some anciently diverged strains of non‐Actinobacteria. 2 These strains are known as orphan strains; the sequences of the genes are known but the enzymes and the product they manufacture remain uncharacterized. Many of the uncharacterized products include those made by non‐Actinobacterial species. Additionally, many of the identified non‐Actinobacterial species originate in phyla such as the Firmicutes and Proteobacteria, which means that their gene products could be expressed in tractable, non‐Actinobacterial heterologous hosts, such as S. pyogenes (a Firmicute) or E. coli (a Proteobacteria). Thus, non‐Actinobacterial ancient strains seemed ripe for bioprospecting.One of the first non‐Actinobacterial species that this research considered was the strain Gloeocapsa sp. PCC 7428, a cyanobacteria whose genome was found to contain a putative PKS gene cluster in the original 2015 study. Herein we show that the Gloeocapsa KS‐CLF can be expressed and purified in E. coli in high titers. Further characterization has shown this KS‐CLF to have several valuable qualities. The successful expression of such a protein may open doors to solutions to the antibiotic resistance problem that were previously closed.Support or Funding Information1. This work was supported by the Arnold & Mabel Beckman Foundation Scholar Award to G.S.H. and NSF CAREER grant number CHE‐1652424 to L.K.C.

Microfluidic automated plasmid library enrichment for biosynthetic gene cluster discovery.

Microbial biosynthetic gene clusters are a valuable source of bioactive molecules. However, because they typically represent a small fraction of genomic material in most metagenomic samples, it remains challenging to deeply sequence them. We present an approach to isolate and sequence gene clusters in metagenomic samples using microfluidic automated plasmid library enrichment. Our approach provides deep coverage of the target gene cluster, facilitating reassembly. We demonstrate the approach by isolating and sequencing type I polyketide synthase gene clusters from an Antarctic soil metagenome. Our method promotes the discovery of functional-related genes and biosynthetic pathways.

Diversity of nonribosomal peptide synthetase and polyketide synthase gene clusters in the genus <i>Acrocarpospora</i>

Acrocarpospora is a rare, recently established actinomycete genus of the family Streptosporangiaceae. In the present study, we sequenced whole genomes of the type strains of Acrocarpospora corrugate, Acrocarpospora macrocephala, and Acrocarpospora pleiomorpha to assess their potency as secondary metabolite producers; we then surveyed their nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) gene clusters. The genome sizes of A. corrugate NBRC 13972T, A. macrocephala NBRC 16266T, and A. pleiomorpha NBRC 16267T were 9.3 Mb, 12.1 Mb, and 11.8 Mb, respectively. Each genome contained 12-17 modular NRPS and PKS gene clusters. Among the 23 kinds of NRPS and PKS gene clusters identified from the three strains, eight clusters were conserved in all the strains, six were shared between A. macrocephala and A. pleiomorpha, and the remaining nine were strain-specific. We predicted the chemical structures of the products synthesized by these gene clusters based on bioinformatic analyses. Since the chemical structures are diverse, Acrocarpospora strains are considered an attractive source of diverse nonribosomal peptide and polyketide compounds.

Draft genome sequence of Actinomadura sp. K4S16 and elucidation of the nonthmicin biosynthetic pathway.

Actinomadura sp. K4S16 (=NBRC 110471) is a producer of a novel tetronate polyether compound nonthmicin. Here, we report the draft genome sequence of this strain together with features of the organism and assembly, annotation and analysis of the genome sequence. The 9.6 Mb genome of Actinomadura sp. K4S16 encoded 9,004 putative ORFs, of which 7,701 were assigned with COG categories. The genome contained four type-I polyketide synthase (PKS) gene clusters, two type-II PKS gene clusters, and three nonribosomal peptide synthetase (NRPS) gene clusters. Among the type-I PKS gene (t1pks) clusters, a large t1pks cluster was annotated to be responsible for nonthmicin synthesis based on bioinformatic analyses. We also performed feeding experiments using labeled precursors and propose the biosynthetic pathway of nonthmicin.

Diversity of PKS and NRPS gene clusters between Streptomyces abyssomicinicus sp. nov. and its taxonomic neighbor.

Streptomyces sp. CHI39, isolated from a rock soil sample, is a producer of abyssomicin I. The taxonomic status was clarified by a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain was closely related to Streptomyces fragilis, with similarity of 99.9%. Strain CHI39 comprised LL-diaminopimelic acid, glutamic acid, glycine, and alanine in its peptidoglycan. The predominant menaquinones were MK-9(H6), and major fatty acids were anteiso-C15:0, anteiso-C17:0, and iso-C16:0. The chemotaxonomic features matched those described for the genus Streptomyces. Genome sequencing was conducted for strain CHI39 and S. fragilis NBRC 12862T. The results of digital DNA-DNA hybridization along with differences in phenotypic characteristics between the strains suggested strain CHI39 to be a novel species, for which Streptomyces abyssomicinicus sp. nov. is proposed; the type strain is CHI39T (=NBRC 110469T). Next, we surveyed polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) gene clusters in genomes of S. abyssomicinicus CHI39T and S. fragilis NBRC 12862T. These strains encoded 9 and 12 clusters, respectively, among which only four clusters were shared between them while the others are specific in each strain. This suggests that strains classified to distinct species each harbor many specific secondary metabolite-biosynthetic pathways even if the strains are taxonomically close.

Mucosal cancer-associated microbes and anastomotic leakage after resection of colorectal carcinoma

BackgroundClinical and experimental evidence suggests that colorectal mucosal microbiota changes during colorectal carcinogenesis and may impair colorectal anastomotic wound healing. Thus, we hypothesized that amounts of colorectal cancer-associated microbes in colorectal tissue might be associated with anastomotic leakage after resection for colorectal carcinoma. MethodsWe analyzed 256 fresh frozen tissues of colorectal cancer from patients who underwent elective colorectal resection and anastomosis. Amounts of colorectal cancer-associated microbes, including Fusobacterium nucleatum, Escherichia coli possessing the polyketide synthase (pks) gene cluster, Enterococcus faecalis, and Bifidobacterium genus, in colorectal cancer tissues were measured by quantitative polymerase chain reaction assay; we equally dichotomized positive cases (high versus low). Multivariable logistic regression analysis was conducted to assess associations of these microbes with anastomotic leakage, adjusting for patient and tumor characteristics, and surgery-related factors. ResultsFusobacterium nucleatum, pks-positive Escherichia coli, Enterococcus faecalis, and Bifidobacterium genus were detected in colorectal carcinoma tissue in 140 (54%), 94 (36%), 193 (75%), and 89 (35%) of 256 cases, respectively. Compared with Bifidobacterium genus-negative cases, Bifidobacterium genus-high cases were associated with an increased risk of anastomotic leakage (multivariable odds ratio, 3.96; 95% confidence interval, 1.50 to 10.51; Ptrend = 0.004). The association of Fusobacterium nucleatum, pks-positive Escherichia coli, or Enterococcus faecalis with anastomotic leakage was not statistically significant. ConclusionsThe amount of Bifidobacterium genus in colorectal tissue is associated with an increased risk of anastomotic leakage after resection for colorectal cancer. These findings need to be validated to target gastrointestinal microflora for the prevention of anastomotic leakage after colorectal resection.

A polyketide synthase gene cluster associated with the sexual reproductive cycle of the banana pathogen, Pseudocercospora fijiensis.

Disease spread of Pseudocercospora fijiensis, causal agent of the black Sigatoka disease of banana, depends on ascospores produced through the sexual reproductive cycle. We used phylogenetic analysis to identify P. fijiensis homologs (PKS8-4 and Hybrid8-3) to the PKS4 polyketide synthases (PKS) from Neurospora crassa and Sordaria macrospora involved in sexual reproduction. These sequences also formed a clade with lovastatin, compactin, and betaenone-producing PKS sequences. Transcriptome analysis showed that both the P. fijiensis Hybrid8-3 and PKS8-4 genes have higher expression in infected leaf tissue compared to in culture. Domain analysis showed that PKS8-4 is more similar than Hybrid8-3 to PKS4. pPKS8-4:GFP transcriptional fusion transformants showed expression of GFP in flask-shaped structures in mycelial cultures as well as in crosses between compatible and incompatible mating types. Confocal microscopy confirmed expression in spermagonia in leaf substomatal cavities, consistent with a role in sexual reproduction. A disruption mutant of pks8-4 retained normal pathogenicity on banana, and no differences were observed in growth, conidial production, and spermagonia production. GC-MS profiling of the mutant and wild type did not identify differences in polyketide metabolites, but did identify changes in saturated fatty acid methyl esters and alkene and alkane derivatives. To our knowledge, this is the first report of a polyketide synthase pathway associated with spermagonia.

Heterologous expression of intact biosynthetic gene clusters in Fusarium graminearum

Filamentous fungi such as species from the genus Fusarium are capable of producing a wide palette of interesting metabolites relevant to health, agriculture and biotechnology. Secondary metabolites are formed from large synthase/synthetase enzymes often encoded in gene clusters containing additional enzymes cooperating in the metabolite’s biosynthesis. The true potential of fungal metabolomes remain untapped as the majority of secondary metabolite gene clusters are silent under standard laboratory growth conditions. One way to achieve expression of biosynthetic pathways is to clone the responsible genes and express them in a well-suited heterologous host, which poses a challenge since Fusarium polyketide synthase and non-ribosomal peptide synthetase gene clusters can be large (e.g. as large as 80 kb) and comprise several genes necessary for product formation. The major challenge associated with heterologous expression of fungal biosynthesis pathways is thus handling and cloning large DNA sequences. In this paper we present the successful workflow for cloning, reconstruction and heterologous production of two previously characterized Fusarium pseudograminearum natural product pathways in Fusarium graminearum. In vivo yeast recombination enabled rapid assembly of the W493 (NRPS32-PKS40) and the Fusarium Cytokinin gene clusters. F. graminearum transformants were obtained through protoplast-mediated and Agrobacterium tumefaciens-mediated transformation. Whole genome sequencing revealed isolation of transformants carrying intact copies the gene clusters was possible. Known Fusarium cytokinin metabolites; fusatin, 8-oxo-fusatin, 8-oxo-isopentenyladenine, fusatinic acid together with cis– and trans-zeatin were detected by liquid chromatography and mass spectrometry, which confirmed gene functionality in F. graminearum. In addition the non-ribosomal lipopeptide products W493 A and B was heterologously produced in similar amounts to that observed in the F. pseudograminearum doner. The Fusarium pan-genome comprises more than 60 uncharacterized putative secondary metabolite gene clusters. We nominate the well-characterized F. graminearum as a heterologous expression platform for Fusarium secondary metabolite gene clusters, and present our experience cloning and introducing gene clusters into this species. We expect the presented methods will inspire future endevours in heterologous production of Fusarium metabolites and potentially aid the production and characterization of novel natural products.

Heterologous Expression of a Cryptic Giant Type I PKS Gene Cluster Leads to the Production of Ansaseomycin.

Genome mining of the marine Streptomyces seoulensis A01 enabled the identification of a giant type I polyketide synthase gene cluster ( asm). Heterologous expression of the cryptic asm cluster using a bacterial artificial chromosome vector in heterologous host led to the production of ansaseomycins A (1) and B (2). A plausible biosynthetic pathway was also proposed. Additionally, compounds 1 and 2 are active against K562 cell lines with IC50 values of 13.3 and 18.1 μM, respectively.