Black yeasts are efficient heterologous hosts of a wide range of fungal polyketides

Black yeasts are efficient heterologous hosts of a wide range of fungal polyketides | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Black yeasts are efficient heterologous hosts of a wide range of fungal polyketides Pablo Cruz-Morales, Adrian Gadar-Lopez, Ana Calheiros de Carvalho, and 19 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6001933/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Fungal natural products, including polyketides, are a rich source of bioactive molecules. Their biosynthetic enzymes are encoded within biosynthetic gene clusters, which are often activated by specific environmental conditions. As a result, many natural products are not produced under standard laboratory conditions. Heterologous expression bypasses native regulation, enabling a systematic approach for polyketide discovery. The most widely used fungal hosts for natural product production are Saccharomycetales yeasts, and filamentous Eurotiomycetes. Yeasts are highly tractable but have a narrow product scope due to their limited secondary metabolism, while filamentous Eurotiomycetes have a richer secondary metabolism but are more difficult to engineer. In this work, we established two yeasts of the genera Exophiala and Knufia as novel heterologous hosts for a broad range of polyketides. These hosts combine the genetic tractability of yeast with the metabolic robustness of filamentous fungi. We developed genetic engineering tools for precise gene integration and genome editing, allowing us to heterologously express five fungal polyketide synthases with different domain architectures, including one involved in the biosynthesis of a previously undescribed 2-pyridone. Our findings demonstrate that these novel yeast hosts can efficiently produce complex polyketides, paving the way for systematic polyketide synthase expression and engineering. Biological sciences/Chemical biology/Natural products Biological sciences/Chemical biology/Biosynthesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Fungal natural products (NP) metabolism is an important source of molecules and enzymes that could enable the sustainable manufacturing of foods, bulk chemicals, polymers, pesticides, and fuels 1 . Among natural product enzymes, polyketide synthases (PKSs) stand out due to the diversity of their products and their biosynthetic mechanisms which can be engineered to create novel carbon-based compounds 2 , 3 . PKSs are multimodular enzyme complexes that can include acyl transferase (AT), ketosynthase (KS), C-methylation (M), ketoreductase (KR), dehydratase (DH), enoyl reductase (ER), acyl carrier protein (ACP), and thioesterase (TE) domains. A key aspect of the function of PKSs is the post-translational attachment of a phosphopantetheinyl group to a conserved serine in the ACP domain, a reaction catalyzed by a phosphopantetheinyl transferase (PPTase). This prosthetic group acts as an “arm”, shuttling the substrates and intermediates across the PKS domains 4 . Fungal iterative PKSs (iPKSs) produce polyketides using a single set of domains. In each iteration it is possible that the cycle skips some of the domains, creating structurally diverse products 4 . Given their biosynthetic capabilities, iPKSs are excellent systems for the development of new bioproducts 1 – 3 .Although many fungal iPKSs have been described, we are still far from exploiting their full chemical repertoire. One significant limitation in the discovery, production, and engineering of fungal polyketides is that many of them are not produced when fungi are cultivated under laboratory conditions. Several cultivation-based strategies have been developed to overcome this issue, but their efficacy is limited 5 , 6 . An alternative method that bypasses the native regulation of the PKSs is heterologous expression, in which known or putative biosynthetic genes are transferred into an amenable host under the control of orthogonal expression systems 7 . To date, few yeast species have been used for heterologous expression of fungal polyketides that produce phenolic and polycyclic products. Most efforts have focused on Saccharomyces cerevisiae 8 – 10 , Komagataella phaffii 11 – 13 , Kluyveromyces marxianus 14 , and Yarrowia lipolytica 15 ; all of them Saccharomycetales yeast that evolved a simplified metabolism by genome erosion 16 . Although their simpler genomes make them excellent for genetic engineering, genome erosion also reduced their ability to grow on complex substrates and to produce complex NPs, (Fig. 1 A, Supplementary table 2). This trait loss is underscored by the lack of PKSs and their cognate PPTase 17 , 18 . On the other hand, many filamentous fungi have large NP repertoires 19 , 20 (Supplementary tables 3–5) and can grow on complex substrates such as lignocellulosic biomass 21 and pre-treated plastic waste 22 . Among filamentous species, members of the genus Aspergillus are considered efficient hosts for the heterologous expression of complex NPs, and an increasing number of genetic engineering tools are available for them 23 , 24 . However, their filamentous morphology and genetic complexity implies longer design-build-test-learn cycles and greater challenges during process scale-up compared to Saccharomycetales yeast. Additionally, their complex metabolism and large repertoire of endogenous enzymes can modify the target products into other derivatives, complicating the detection and purification of the products 25 – 28 . Therefore, more efficient heterologous hosts with simplified genomes and robust-yet-simple metabolic backgrounds, unicellular growth, and the ability to use complex substrates are needed for the systematic discovery and sustainable manufacturing of polyketide-based bioproducts. In this work, we established two species of black yeast, Exophiala viscosa 29 and Knufia petricola 30 , 31 as heterologous hosts for the production of fungal polyketides. Black yeast is a common name for a polyphyletic group of unicellular, extremophilic fungi that can grow on complex substrates. Their dark phenotype is derived from melanin, a fungal polyketide that they copiously produce 29 , 31 . We reasoned that these black yeast species evolved a robust metabolism to thrive in harsh environments, and that their native metabolism likely includes a PKS-active PPTase and abundant acetyl-CoA and malonyl-CoA pools, which are essential precursors for melanin biosynthesis. We constructed non-homologous end-joining (NHEJ) deficient strains and developed a synthetic biology toolbox for efficient and marker-free genomic integrations. To test their potential as polyketide production hosts, we heterologously expressed and obtained the products of a set of PKSs representing the most frequent domain architectures found in the fungal kingdom. We achieved production of 6-methylsalicylic acid (6-MSA), YWA1, monocillin II, and farinosones, whereas the production was limited in S. cerevisiae . Finally, we demonstrated that our black yeast hosts can be used for rapid characterization of unknown biosynthetic pathways. Our work showcases the potential of black yeast for systematic expression and engineering of PKSs. Results Kingdom-wide polyketide genome mining reveals black yeast’s potential as heterologous hosts To select new yeast candidates to be engineered as heterologous hosts for PKS, we assembled and annotated a genome database with 1,979 strains covering 15 classes within the fungal kingdom (Supplementary file 1). We annotated these genomes and calculated the average number of polyketide synthases per class across the kingdom (Fig. 1 A, Supplementary table 1 and Supplementary file 2). The Eurotiomycetes class displayed the second highest number of PKSs. This group includes filamentous workhorses of the genus Aspergillus , and the genera Exophiala and Knufia . In contrast, PKSs are very rare among members of the Saccharomycetes class. We thus focused on the Eurotiomycetes K. petricola CBS123872 30,31 and E. viscosa strains JF 03-3F and JF 03-4F (CBS148801 and CBS148802) 29 which we acquired from the WI-KNAW collection. A desirable trait in a heterologous host is the ability to grow on second-generation feedstocks, such as lignocellulosic biomass. Thus, we tested E. viscosa and K. petricola for growth on beechwood hydrolysates and xylose media. We cultivated E. viscosa and K. petricola YPD ( 2% glucose), YPX (2% xylose), or MMBW, a minimal media supplemented with beechwood hydrolysate 32 . No significant differences were observed in the growth of E. viscosa , which grew as single cells (Fig. 1 B and 1 C) and reached an optical density (600 nm) of OD 600 40 in both YPD or YPX, OD 600 33, 39 in MMBW for strain JF 03-3F, and strain JF 03-4F respectively (Fig. 1 F and 1 G). K. petricola formed cell agglomerations during cultivation (Fig. 1 D). Therefore, we quantified its growth using biomass dry weight after 7 days of cultivation. The final average biomass obtained in these cultures was 298 mg +/-176 mg (YPD), 147 mg +/-14 mg (YPX), and 188 mg +/-13 mg (MMBW) ( Fig. 1 H ) . Overall, these results show that these black yeasts can grow on second generation feedstocks, even on beechwood hydrolysates which are toxic to many bacterial and fungal species 33 . The genomes of these species (Supplementary table 9) are larger than S. cerevisiae , but smaller than the frequently used Aspergillus spp. . Inspection of their genomes showed that these species harbor the potential to produce complex PKS, NRPS and terpenes but their repertoires are smaller when compared with Aspergillus spp . (Supplementary tables 2–8) . We reasoned that this may be advantageous, since a reduced NP repertoire may facilitate the redirection of precursors towards the heterologous pathway, could prevent unexpected modification done by endogenous enzymes and simplify the identification and purification of the products 25 – 28 . To assess the native metabolic complexity of these black yeasts, we compared the chemical profiles of E. viscosa , K. petricola , S. cerevisiae , and Aspergillus spp. . Our analysis showed that black yeasts have simpler chemical profiles than Aspergillus spp. (Fig. 1 E). Considering the growth capabilities and biosynthetic profile of our black yeasts we reasoned these species are excellent candidates to be developed into polyketide heterologous hosts. Development of synthetic biology tools for heterologous expression in black yeasts There are genetic engineering tools already available for K. petricola 34 , 35 . Thus, we constructed tools for E. viscosa . First, we determined that the E. viscosa strains are sensitive to Hygromycin B at 50 µg mL − 1 (Supplementary Fig. 1). We then transformed them with AMA1-based plasmids, which are episomal and can be cured in filamentous Eurotiomycetes 36 – 39 and in K. petricola 34 . For transformation, we adapted a protoplastation protocol used for filamentous fungi 40 – 42 . We observed colonies in the transformation plates after 21 days at 25°C (Fig. 2 A). We tested the functionality and efficiency of the CRISPR-Cas9 43,44 system in E. viscosa JF 03-3F and JF 03-4F. For this, we targeted the polyketide synthase gene pks1 involved in the biosynthesis of melanin, a dark colored product, and the phytoene synthase gene phs1 for carotenoid biosynthesis, an orange colored product 29 . Introduction of Cas9 and the cognate sgRNAs leads to a DNA double strand break (DSB) at the targeted site which may be repaired in an error-prone manner via NHEJ 45 often leading to deleterious insertions or deletions in the targeted genes. Therefore, successful introduction of DSBs in pks1 and phs1 is expected to cause a phenotypic change from black (wild-type genotype) to orange (mutated pks1) , or from black to white (mutated pks1 and phs1 ) (Fig. 2 B). We transformed E. viscosa JF 03-3F and JF 03-4F with plasmids for Cas9 expression and the Aspergillus fumigatus U3 promoter and terminator driving the expression of sgRNAs delivered via tRNA release mechanism targeting pks1 or both pks1 and phs1. This experiment resulted in the expected genotypic and phenotypic changes (Fig. 2 C), confirming that Cas9-sgRNA CRISPR nucleases are active in our black yeast host candidates. Gene-editing efficiencies are higher in NHEJ-deficient black yeast strains In many fungal species, inactivation of NHEJ DNA repair pathway allows controlled and precise integration of DNA, although with some fitness tradeoffs 46 . NHEJ deficient strains are usually obtained by knockout of the gene encoding Ku70 47 . After a DNA DSB has been made in NHEJ-deficient strains, the predominant DNA repairs mechanism to avoid death is homologous recombination (HR). This allows for efficient integration of exogenous DNA flanked with homologous sequences up-and downstream of the cut site 48 – 53 . To generate NHEJ deficient black yeast strains, we first identified ku70 orthologs in K. petricola and E. viscosa and targeted the first 30 nucleotides of these genes using Cas9-sgRNA plasmids. We provided a linearized DNA repair template harboring an XbaI restriction site and 500 bp upstream and downstream homology sequences to ku70 . We expected to obtain either full gene deletions by HR or gene disruption by NHEJ (Fig. 2 D). For K. petricola , a strain with full gene deletion was obtained and validated (Fig. 2 E). For E. viscosa JF 03-3F and JF 03-4F, full gene deletions affected the fitness of the strains (data not shown), but strains with ku70 frameshifts were viable and used for the following experiments (Fig. 2 F). Oligo-mediated engineering is a fast, precise and cheap gene-editing method for strain engineering in filamentous fungi 51 and non-conventional yeasts 52 , 53 , including K. petricola 34 . We tested its efficiency in both wild-type (WT) and NHEJ deficient strains of E. viscosa , and K. petricola . For this purpose, we transformed E. viscosa JF 03-3F and JF 03-4F and K. petricola with Cas9-sgRNA plasmids targeting pks1 and phs1 . We used 90 nucleotides single stranded DNA (ssDNA) oligos as repair templates for single or double gene deletions (Fig. 2 G). Up to ten colonies from each experiment with the expected phenotype were analyzed by diagnostic PCR. In E. viscosa , the number of transformants with full gene deletions was higher in NHEJ deficient strains for both single and double gene deletions when compared to the WT strains (Fig. 2 H and supplementary Fig. 2). In K. petricola , the number of transformants with full gene deletions was equal in NHEJ deficient strains for single gene deletions when compared to the WT. In contrast, the number of NHEJ deficient K. petricola transformants with double gene deletions was higher compared to the WT (supplementary Fig. 3). These experiments confirmed that inactivation of the NHEJ pathway improves the efficiency of gene edits in our black yeast (Supplementary Figs. 2 and 3), allowing us to demonstrate ssDNA-mediated gene editing in E. viscosa and establish the molecular biology pipeline for heterologous expression via genomic integration. Black yeast hosts enable efficient heterologous production of polyketides To test the efficacy of our black yeasts hosts for heterologous expression of polyketides, we chose a set of PKSs covering the most common domain architectures and degrees of complexity (Table 1 ) and (Fig. 3 A and 3 B). From the least to the most complex: 6-methylsalicylic acid synthase ( atX6MSA ) from Aspergillus terreus 54 , YWA1 ( pks12 ) synthase from Fusarium acasiae-mearnsii CBS10255, monocillin II ( pchI & pchE ) synthases from Pochonia chlamydosporia MUCL9880, and farinosone B ( adnE1-4 ) from Cordyceps sp. D1, a cryptic highly reducing PKS-NRPS predicted to direct the synthesis of a 2-pyridone product 55 – 57 (Fig. 3 B and 3 C). Our selection is representative of the most abundant PKSs in the fungal kingdom by covering 73% of the known architectures (Fig. 3 A). Table 1 PKSs selection for heterologous expression Product (gene) Domain architecture Origin 6MSA ( atX6MSA ) KS-AT-DH-KR-ACP A. terreus YWA1 ( pks12 ) SAT-KS-AT-PT-ACP-TE F. acaciae-mearnsii monocillin II ( pchI ) KS-AT-DH-ER-KR-ACP P. chamydosporia monocillin II ( pchE ) SAT-KS-AT-PT-ACP-TE P. chamydosporia YNP1 ( adnE1 ) KS-AT-DH-MT-ER-KR-ACP-C-A-ACP-Red Cordyceps sp. D1 6-MSA has been produced in S. cerevisiae 8 , 58 , 59 thus, we use it to benchmark our black yeast hosts. Given that black yeast are naturally proficient producers of melanins 29 – 31 , we reasoned that they must have a functional PPtase and abundant acetyl- and malonyl-CoA precursors to support polyketide production without further metabolic engineering 60 , 61 . In contrast, S. cerevisiae requires the incorporation of an active PPtase and increase of the acyl-coA pool to produce polyketides 17 , 18 , 59 . For the heterologous expression of 6-MSA, we transformed our black yeast hosts with Cas9-sgRNA plasmids targeting the melanin biosynthesis gene pks1 together with a linearized atX6MSA expression cassette codon-optimized for A. oryzae driven by the A. nidulans TEF1 promoter and the trpC terminator. The atX6MSA expression cassette flanked with 1.5 kilobases (Kb) homology arms was designed to replace pks1 upon Cas9 induced DSB. (Fig. 3 D). Our benchmarking strains were derived from S. cerevisiae IMX581 Y40593 62 : strain NPE9 carries a PPTase ( npgA ) from A. nidulans , and strain POP4 derives from NPE9 plus modifications to increase the acyl-coA, NADH and S-adenosyl methionine pools required for polyketide production (Supplementary table 10). These strains were transformed with a Cas9-sgRNA plasmid targeting the integration site ISX-2 63 together with a PCR fragment harboring a S. cerevisiae codon-optimized version of atX6MSA flanked by 60bp DNA homologous arms for the ISX-2 integration site (Fig. 3 D ) . Whole genome sequencing was carried out to confirm the integrity of the constructs and verify that only one copy of atX6MSA was integrated into their genomes. Validated transformants were propagated in liquid YPD and 6-MSA production was confirmed by liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) (Supplementary Fig. 4). We quantified 6-MSA titer in terms of absolute concentrations, and concentrations relative to dry biomass for three biological replicates with independent integration events (Supplementary Fig. 5) and (Fig. 3 E). From this experiment we concluded that S. cerevisiae POP4 produced the highest absolute amount of 6-MSA (296 mg L − 1 ± 93 mg L − 1 ) but required additional genetic modifications prior to its expression. In contrast, K. petricola with only one modification to achieve NHEJ-deficiency yielded comparable amounts of 6-MSA (234 mg L − 1 ± 47 mg L − 1 ), while Exophiala viscosa produced modest amounts of the product (67 mg L − 1 ± 6 mg L − 1 in JF 03-4F and 15 mg L − 1 ± 0.6 mg L − 1 in JF 03-3F). Overall, this experiment showed that our black yeast hosts are readily capable of producing a simple polyketide heterologously, confirming their endogenous PPTase is active on heterologous PKSs. The next polyketide that we produced was YWA1, an intermediate product in the biosynthesis of rubrofusarin in F. graminearum which is encoded in the pks12 gene 64 . For heterologous expression in S. cerevisiae , we identified an ortholog of pks12 from F. acaciae-mearnsii CBS10255, synthesized its predicted coding sequence and cloned it in a pESC-URA (Genscript®) shuttle vector. This plasmid was then used to transform the S. cerevisiae strain POP4 (Supplementary Fig. 6A). For the heterologous expression of pks12 in black yeasts, we transformed our strains with Cas9-sgRNA plasmids targeting pks1 . In this case we used 60 bp homology arms in PCR fragments designed to integrate pks12 under the control of the native pks1 promoter and terminator (Supplementary Fig. 6B and 6C). For all strains, yellow-dark transformants were obtained (Supplementary Fig. 6D, 6E, and 6F) and validated by diagnostic PCR prior propagation in liquid YPD. The strains were cultivated and the organic extracts of their supernatants was analyzed using LC-HRMS. YWA1 was detected in all transformants (Fig. 3 F and Supplementary Fig. 7). These results show that we can take advantage of the native pks1 promoters and terminators to integrate a PKS gene lacking promoters and terminators elements. At this point, we had obtained products from non-reducing PKSs, the simplest domain architecture among fungal PKSs, and which heterologous expression is viable in S. cerevisiae 64 . To test if our black yeast offer any advantage over S. cerevisiae as heterologous host, we tested the expression of more complex PKSs. These synthetases are typically produced in filamentous fungal hosts 65 – 72 , with a few exceptions that have been produced in Saccharomyces 73 – 76 . For this, we selected monocillin II, a resorcinol-lactone with nematocidal activity 77 . Monocillin II is biosynthesized by two PKSs, PchI a highly reducing PKS and PchE a non-reducing PKS 78 . For heterologous expression in S. cerevisiae POP4, we used a Cas9-sgRNA plasmid targeting the ISX-2 and ISXII-1 integration sites 63 , together with two PCR products harboring intron-less sequences of pchI and pchE from P. chlamydosporia MUCL9880 flanked by 60 base pairs (bp) homology arms (Supplementary Fig. 8). For heterologous production in black yeast, we used Cas9-sgRNA plasmids simultaneously targeting the pks1 and phs1 genes together with repair templates harboring the intron-less sequences of pchI and pchE ; pchI was flanked with 1 Kb homology arms to the promoter and terminators of pks1 (Supplementary Fig. 9), and pchE placed under the A. nidulans TEF1 promoter and the trpC terminator with 1.5 Kb homology arms for phs1 (Supplementary Fig. 10). Monocillin II was found in the supernatants of the cultures of our black yeast constructs (Fig. 3 F) and (Supplementary Fig. 11). While S. cerevisiae POP4 yielded traces of the product (Fig. 3 F) and (Supplementary Fig. 12). Overall, the production of 6-MSA, YWA1, and monocillin II demonstrated that our black yeasts are proficient in heterologous PKS production from distantly related species. Additionally, they are compatible with Aspergillus -derived promoters and terminators, allow for efficient simultaneous gene integration, and support the use of short homology arms. More importantly, these experiments highlight the potential of E. viscosa JF 03-3F, JF 03-4F, and K. petricola to produce complex PKSs using standardized methods, minimizing time-consuming troubleshooting steps. Heterologous expression in black yeasts lead to the discovery and pathway elucidation of a new 2-pyridone natural product After developing and benchmarking our black yeast hosts, we showcased their usefulness in the discovery and elucidation of new biosynthetic pathways. For this proof-of-concept experiment, we chose to express a previously undescribed BGC from Cordyceps sp. D1. We predicted that this BGC would produce a 2-pyridone natural product, but we could not detect it using cultivation-based approaches when applied to Cordyceps sp. D1 (unpublished results). To define the minimum set of genes for heterologous expression we used a phylogenomics approach 79 (Fig. 4 A). We selected three genes: adnE1 a hybrid, highly reducing PKS-NRPS with a domain organization that is characteristic of pyridine producing pathways 65 – 72 , adnE2 a cytochrome p450 (CYP450), and adnE3 a trans-enoyl reductase (trans-ER) fused to a CYP450. AdnE1 , a dnE2 and a dnE3 were integrated into the genome of all strains (Supplementary Figs. 13 and 14). The LC-HRMS analysis of extracellular fractions of all black yeast transformants revealed a peak with high intensity and m/z 438.1916, which corresponds to the calculated formula: [C 25 H 28 NO 6 ] + Δ + 1.141 ppm, while S. cerevisiae POP4 did not yield any products (Fig. 4 B). In addition, E. viscosa JF 03-3F and JF 03-4F, and K. petricola transformants displayed a visible yellow phenotype (Fig. 4 C), whereas S. cerevisiae POP4 did not. Mass to formula calculation and the mass fragmentation analysis of the ion was consistent with a PKS-derived 2-pyridone product (Supplementary Fig. 15). To elucidate the structure of the product, we cultivated E. viscosa JF 03-4F expressing adnE1 , adnE2 and adnE3 in YPD, YPX, MMX, and MMBW media for 7 days. We then extracted the resulting supernatants with ethyl acetate, and after solvent evaporation, we obtained approximately 88 mg L − 1 (YPD), 80 mg L − 1 (YPX), and 12 mg L − 1 (MMX) of an amorphous solid composed mostly of our product, whilst 204 mg L − 1 of an amorphous solid composed mostly of other products was obtained in MMBW (Supplementary Fig. 16). The 80 mg of product derived from YPX was further purified using semi preparative HPLC until we obtained 10 mg of material which was then analyzed with Nuclear Magnetic Resonance (NMR). Structural elucidation using 1 H NMR, COSY, HSQC, HMBC confirmed that the product is 17-Hydroxyfarinosone, a new member of the farinosones 80 which we named YNP1 (Fig. 4 D) (Supplementary Figs. 17–21). YNP1 did not show any fully saturated C-C bonds in the acyl chain as expected in PKS-derived pyridones with an active enoyl reductase domain 66 – 73 . We hypothesized that the gene fusion between the trans-ER and CYP450 in adnE3 might have inactivated the trans-ER. To test our hypothesis, we revisited the gene-calling of adnE3 and inspected its coding sequence. After a BLASTP search and comparison against other trans-ERs sequences, we identified misannotations at the C-terminal region of the trans-ER domain and the N-terminal of the CYP450 domain (Supplementary Fig. 22). These findings suggested that the trans-ER and CYP450 in adnE3 are in fact two individual genes artifactually fused. To validate the revisited annotation, we transformed the E. viscosa JF 03-4F YNP1 producer strain with a Cas9-sgRNA plasmid targeting the connecting DNA sequence between the trans-ER and CYP450 together with PCR products containing the A. nidulans trpC terminator, AO0583, an strong constitutive promoter 24 , and the missing DNA sequence of one, non, or both trans-ER and CYP450 flanked with 500 bp homology to the adnE3 coding sequence (Supplementary Fig. 23). Phenotypic changes from yellow to intense orange were observed in transformants where the missing trans-ER sequence was provided. On the other hand, complementation with the missing CYP450 sequence did not show obvious phenotypic changes. This suggested that the complementation with the missing trans-ER sequence restored the function of the trans-ER (Supplementary Fig. 24). After strain’s validation by diagnostic PCR and LC-HRMS analysis, peaks with the highest abundance m/z 422.1963 and m/z 406.2015 were observed in the metabolic profiles of the strains where the missing trans-ER sequence was introduced. The peak corresponding to m/z 422.1963 matched the formula and fragmentation pattern of farinosone B 80 , a polyketide-derived 2-pyridone bearing a fully saturated C-C bond in the acyl chain. This result confirmed the functional restoration of the trans-ER activity and the presence of a fourth gene adnE4 (Fig. 5 A) and (Supplementary Fig. 24). Based on our data, we concluded that farinosone B is the final product of the pathway encoded in adnE1-2-3-4 together with other minor products including YNP1 and farinosone A ( m/z 406.2015, calculated formula [C 25 H 28 NO 4 ] + Δ + 0.73 ppm) (Fig. 5 B) and (Supplementary Fig. 24). Thus, we propose a common biosynthetic pathway for YNP1, farinosone A and B (Fig. 5 C). Overall, this work demonstrates the potential of black yeast as heterologous hosts for the discovery of new polyketide natural products and highlights their potential for their sustainable production. Discussion E. viscosa and K. petricola are convenient heterologous hosts of fungal PKSs Yeast species are convenient hosts for heterologous expression of PKSs, as they are easier to manipulate and have simple metabolic backgrounds compared to filamentous fungi. In this work, we demonstrated that E. viscosa JF 03-3F, E. viscosa JF 03-4F , and K. petricola grow as yeasts, although K. petricola tends to form more clumps during cultivation. We also showed that these black yeastshave cleaner background metabolic profiles than Aspergillus hosts. This is advantageous because the heterologous products are easier to purify, and they are less likely to be modified by the host's metabolism. We also showed that E. viscosa and K. petricola can grow and make polyketides on xylose and in beechwood hydrolysates (Supplementary figure 16). In contrast S. cerevisiae, requires edible sugars as substrates. This opens the possibility to use sustainable feedstocks as substrates for the sustainable production of polyketide products. Black yeast hosts overcome S. cerevisiae limitations in polyketide production Our results show that black yeast hosts are more competent for PKS production than S. cerevisiae . Throughout our heterologous expression experiments we benchmarked the performance of our new yeast hosts by comparing them with an engineered strain of S. cerevisiae POP4 capable of high titer production of 6-MSA and robust YWA1 production. However, the same S. cerevisiae strain was not able to produce detectable amounts of monocillin II and farinosone A, B or YNP1. In both cases, we sequenced the whole genome of S. cerevisiae strains and confirmed the expected genotypes , expression of the PKSsby transcriptome analysis and the presence of the corresponding proteins by proteomics (Supplementary Figure 25-26). Production of monocillin II and lovastatin has already been reported in S. cerevisiae using 2μ high copy number plasmids 74,75,81,82 . In contrast, our strains for polyketide production in S. cerevisiae and black yeast were single-copy integrations which are stable and comparable. Nevertheless, since single-copy integration of PKS genes may limit the ability of S. cerevisiae to produce polyketides, we attempted expression of the PKS-NRPS encoded in a dnE1 in S. cerevisiae POP4 in a high copy number plasmid. However, the expected product, a five-membered ring intermediate 66 was not obtained (Supplementary figure 27). S. cerevisiae BJ-5464, a strain with low endoproteolytic activity, is commonly used for PKS expression 83 , as it is expected that this feature enhances PKS expression 84,85 . However, when we introduced a high copy number plasmid containing adnE1 into S. cerevisiae BJ-5464-NpgA we could not observe the expected product (Supplementary figure 28). Our troubleshooting efforts suggest that the bottleneck for production of farinosone A, B, YNP1 and monocillin II in S. cerevisiae is probably a post-translational event, e. g. misfolding, aggregation or toxicity of the proteins and/or intermediates in the pathway. Furthermore, farinosone B production in S. cerevisiae may require additional CYP450 reductases and/or cytochrome B5 proteins for proper function of CYP450 enzymes not encoded on its genome 86 . The fact that such modifications were not required in E. viscosa JF 03-3F and JF 03-4F , and K. petricola for farinosone A, B and YNP1 showcases their metabolic robustness. Overall, our work establishes black yeasts as heterologous hosts for the systematic expression of complex fungal PKSs, providing a streamlined workflow that can be applied to the systematic discovery of fungal polyketides and their enzymes, which we aim to fully exploit in sustainable biomanufacturing. Methods Strains and media The complete list of strains can be found in (Supplementary table 10). All strainswere propagated in liquid or solid (additional 2% (w/v) agar) YPD media (1% (w/v) yeast extract, 2% (w/v) Bacto™ peptone, 2% (w/v) glucose). When required, E. viscosa and K. petricola were propagated on YPX media (1% (w/v) yeast extract, 2% (w/v) Bacto™ peptone, 2% (w/v) xylose), or ME media 35 (2% malt extract (w/v), 0.1% (w/v) Bacto™ peptone, 2% (w/v) glucose) or minimal media with beechwood hydrolysate (MMBW) (1x nitrate salt solution 87 , 0.001% thiamine w/v, 1x trace metal solution 88 ) supplemented with beechwood hydrolysate (1% (w/v)) as carbon source 32 , or minimal media with xylose (MMX) supplemented with beechwood hydrolysate (1% (w/v)) as carbon source when required. For determination of Hygromycin B lethal concentrations, first-time transformation and construction of NHEJ-deficient strains of E. viscosa and K. petricola , transformants were selected in Transformation Media (TM) (1x nitrate salt solution 87 , 0.001% Thiamine w/v, 1x trace metal solution 88 ) supplemented with 1M sucrose and 50 µg mL -1 of Hygromycin B (Gibco™) for E. viscosa , or 25 µg mL -1 for K. petricola , and grown for 21 days. For heterologous expression experiments, transformants were selected in malt extract sucrose media 35 (MEAS) (2% (w/v) malt extract, 0.1% (w/v) peptone, 11% (w/v) sucrose) and Hygromycin B, and grown for 14 days. For S. cerevisiae, selection of transformants or plasmid maintenance was done using liquid or solid (media with additional 2% (w/v) agar) synthetic drop-out media (SD-Ura) (0.67% (w/v) yeast nitrogen base (Sigma-Aldrich®) without amino acids, 0.14% (w/v) yeast synthetic dropout medium supplements lacking uracil, 2% (w/v) glucose). Uracil was supplemented when required (0.0076% (w/v)). Counter-selection of plasmids in S. cerevisiae was carried out using SD media supplemented with uracil and 0.074% (w/v) 5-fluoroorotic acid (5-FOA) (Sigma®). S. cerevisiae was propagated at 30°C, E. viscosa and K. petricola at 25°C. E. coli DH5α was used for cloning and plasmid propagation. Liquid or solid (2% (w/v) agar) Lysogeny broth (LB) supplemented with 100 µg mL -1 of ampicillin was used for E. coli cultivation at 37°C. PCRs and plasmids construction For USER cloning 89 , all PCR reactions were performed using Phusion U Hot Start DNA Polymerase (Thermo Scientific™) according to the manufacturer's instructions. For in-vivo assemblies in S. cerevisiae and construction of DNA repair templates provided during transformations, PCR reactions were performed using Phusion™ Hot Start II DNA Polymerase (Thermo Scientific™) according to the manufacturer's instructions. All PCR fragments were gel or column-purified using NucleoSpin™ Gel and PCR Clean-up kit (Macherey-Nagel®) prior to USER cloning, or in-vivo assemblies, or transformations. All constructed plasmids and PCR amplifications were sequenced using long-read sequencing by Oxford nanopore technologies™ (Plasmidsaurs®) when required. For PCR reactions derived from gDNA, fresh biomass from plates was harvested using a 10uL loop and gDNA was extracted according to 42 . For diagnostic PCR, cell lysis was carried out using Platinum™ Direct PCR Universal Master Mix (Thermo Scientific™) cell lysis protocol, and PCR was performed using Phire™ Hot Start II DNA Polymerase (Thermo Scientific™) according to the manufacturer's instructions. All primers and ssDNA oligos used in this work are listed in (Supplementary table 11) and were obtained from Integrated DNA Technologies® (IDT). All PCR, cloning reactions, and constructed plasmids are listed in (Supplementary Tables 12-14). Cas9-sgRNA plasmids were constructed as per 51 , and protospacer sequences used in this work can be seen in (Supplementary table 15). All plasmids were purified using a NucleoSpin™ plasmid DNA purification kit (Macherey-Nagel®). When purifying plasmids from S. cerevisiae , the Zymoprep™ Yeast Plasmid Miniprep I (ZYMO RESEARCH®) was used. Plasmids containing DNA repair templates were linearized by enzymatic digestion with NotI or SmiI (Thermo Scientific™), and gel-purified prior to use in transformations.For heterologous expression of 6-MSA, the amino acid sequence of A. terreus 6MSA synthase 54 was retrieved from the minimum information about a biosynthetic gene cluster database 90 (MIBiG) accession number BGC0001276. The amino acid sequence was used for the DNA synthesis of S. cerevisiae and A. oryzae codon-optimized versions of an intron-less atX6MSA cloned into a pESC-URA 2μ plasmids (GenScript®). For S. cerevisiae , atX6MSA was under the control of TEF1 promoter and ADH1 terminator, and for E. viscosa and K. petricola , under the control of A. nidulans TEF1 promoter and trpC terminator. The S. cerevisiae codon-optimized version of atX6MSA was PCR-amplified with primers PR_YNP171 and PR_YNP172 (PCR62) flanked with 60bp DNA homologous sequences to the integration site ISX-2 63 and column-purified prior its transformation into S. cerevisiae strains NPE9 and POP4 (Supplementary figure 29). The A. oryzae codon-optimized version of atX6MSA was PCR-amplified with primers PR_YNP204 and PR_YNP205 (PCR77) including PacI/Nt.BbvcI tails to re-constitute them once the PCR product was USER cloned into plasmid pAC125 91 . The resultant plasmid pYNP59 was prepared for another round of USER cloning to be inserted with approx. 1.5 Kb of the upstream and downstream DNA sequences homologous to the pks1 locus from E. viscosa JF 03-3F and JF 03-4F, and K. petricola which were PCR-amplified using primer pairs PR_YNP125-PR_YNP126 (PCR46), PR_YNP127-PR_YNP128 (PCR47), PR_YNP121-PR_YNP122 (PCR44), PR_YNP123-PR_YNP124 (PCR45), PR_YNP129-PR_YNP130 (PCR48), PR_YNP131-PR_YNP132 (PCR49). The resultant plasmids pYNP70 (DONR_EVJ4_ATX6MSAORYOPT_PKS1), pYNP71 (DONR_EVJ3_ATX6MSAORYOPT_PKS1), and pYNP72 (DONR_KP_ATX6MSAORYOPT_PKS1) were linearized with NotI and gel-purified prior to transformation into their respective hosts (Supplementary figure 30). For heterologous expression of YWA1 64 , the intron-less coding sequence of pks12 was retrieved from an in-house isolate of F. acasiae-mearnsii strain CBS10255 and synthesized and cloned into a pESC-URA 2μ high copy plasmid (GenScript®) under the control of the TEF1 promoter and CYC1 terminator. The resultant plasmid pYNP100 was used to transform S. cerevisiae POP4. For black yeasts , primer pairs PR_YNP471-PR_YNP452 (PCR196 for E. viscosa ) and PR_YNP472-PR_YNP456 (PCR197 for K. petricola ) flanked with 60bp DNA sequences homologous to the pks1 promoter and terminator were used to amplify pks12 from plasmid pYNP100. The obtained PCR products were column-purified prior to transformation into their respective hosts (Supplementary figure 6). For the heterologous expression of the uncharacterized BGC from Cordyceps sp. D1, the intron-less coding sequences of the PKS-NRPS synthetase ( adnE1 )were PCR-amplified from Cordyceps sp. D1 . gDNA with primer pairs flanked with 50bp complementary homology PR_YNP284-PR_YNP285 (PCR117), PR_YNP286-PR_YNP289 (PCR114), PR_YNP290-PR_YNP293 (PCR115), and assembled into a 2μ high copy plasmid backbone PCR-amplified with primer pair PR_YNP294-PR_YNP295 (PCR122). PCR fragments were in-vivo assembled in S. cerevisiae POP4. The resultant plasmid pYNP78 expresses adnE1 synthetase under the control of TEF1 promoter and CYC1 terminator (Supplementary figure 13). The intron-less coding sequences of the CP450 ( adnE2) and TransER-CP450 ( adnE3) coding sequences were retrieved from Cordyceps sp. D1 and synthesized and cloned into pUC57 plasmid backbones (GenScript®). For E. viscosa and K. petricola , adnE1 was PCR-amplified from plasmid pYNP78 using primer pair PR_YNP317-PR_YNP318 (PCR129) which also reconstitutes PacI/Nt.BbvcI USER cloning cassettes after cloning. Subsequently, the PCR fragment was USER cloned into plasmid pAC125, resulting in plasmid pYNP83 (Supplementary figure 14A). Thereafter, approx. 1 Kb of the upstream and downstream DNA sequences flanking pks1 from E. viscosa JF 03-3F andJF 03-4F,and K. petricola were PCR-amplified from gDNA with primer pairs PR_YNP319-PR_YNP320 (PCR130), PR_YNP321-PR_YNP322 (PCR131), PR_YNP323-PR_YNP324 (PCR132), PR_YNP325-PR_YNP326 (PCR133), PR_YNP327-PR_YNP328 (PCR134), PR_YNP329-PR_YNP330 (PCR135), and USER cloned into pYNP83, resulting in plasmids pYNP84 (DONR_EVJ4_adnE1_PKS1), pYNP85 (DONR_EVJ3_adnE1_PKS1), and pYNP86 (DONR_KP_adnE1_PKS1) expressing adnE1 under the control of pks1 promoter and terminator. Plasmids pYNP84, pYNP85, and pYNP86 were linearized with SmiI (Thermo Scientific™), and gel-purified prior to use for transformations into their respective hosts (Supplementary figure 14C and 14E). adnE2 and adnE3 were PCR-amplified from pUC57 plasmids with primer pairs PR_YNP307-PR_YNP308 (PCR116) and PR_YNP281-PR_YNP282 (PCR109), respectively. A. nidulans promoters gpdA and TEF1 were PCR-amplified from plasmid pDIV652 with primer pair PR_YNP306-PR_YNP296 (PCR110). All PCR products were cloned into plasmid pAC125, which also reconstituted PacI/Nt.BbvcI cassettes after cloning, resulting in plasmid pYNP79 which expresses the adnE2 and adnE3 under A. nidulans TEF1 and gpdA promoters, and argB and pkiA terminators, respectively (Supplementary figure 14B). Thereafter, approx. 1.5 Kb of the upstream and downstream DNA sequences flanking phs1 from E. viscosa and K. petricola were PCR-amplified from gDNA with primer pairs PR_YNP253-PR_YNP254 (PCR103), PR_YNP255-PR_YNP256 (PCR104), PR_YNP259-PR_YNP260 (PCR105), PR_YNP261-PR_YNP262 (PCR106), PR_YNP263-PR_YNP264 (PCR107), PR_YNP265-PR_YNP266 (PCR108). PCR products were cloned into plasmid pYNP79, resulting in plasmids pYNP80 (DONR_EVJ3_adnE_2_3_PHS1), pYNP81 (DONR_EVJ4_adnE_2_3_PHS1), and pYNP82 (DONR_KP_adnE_2_3_PHS1). Plasmids pYNP80, pYNP81, and pYNP82 were linearized with NotI (Thermo Scientific™), and gel-purified prior to transformation into their respective hosts (Supplementary figure 14D and 14E). For S. cerevisiae POP4, adnE2 and adnE3 were PCR-amplified from plasmid pYNP79 with primer pairs PR_YNP346-PR_YNP347 (PCR144) and PR_YNP344-PR_YNP345 (PCR143), respectively. S. cerevisiae promoters TDH3 and PGK1 were PCR-amplified from plasmid pCFB2909-BIK with primer pair PR_YNP348-PR_YNP349 (PCR145). All PCR products were cloned into plasmid backbone pCFB2909-BIK PCR-amplified with primer pair PR_YNP342-PR_YNP343 (PCR142). The resultant plasmid pYNP88 expresses adnE2 under the control of PGK1 promoter and ADH1 terminator, and adnE3 under control of TDH3 and TEF1 terminator. adnE1 was PCR-amplified from pYNP78 using primer pair PR_YNP171-PR_YNP297 (PCR146) with 60bp homology to integration site ISX-2 63 , and adnE2 and adnE3 were PCR-amplified from pYNP88 using primer pair PR_YNP358-PR_YNP359 (PCR147) with 60bp homology to integration site ISXII-1 63 . The PCR products were column-purified prior to transformation into S. cerevisiae POP4 (Supplementary figure 13). For heterologous expression of monocillin II, the intron-less coding sequences of monocillin II pchI and pchE were PCR-amplified from P. chlamydosporia strain MUCL9880 using primers PR_YNP394-PR_YNP395 (PCR168), PR_YNP396-PR_YNP397 (PCR169), and PR_YNP398-PR_YNP399 (PCR170) flanked with 50bp complementary homology. Thereafter, the backbone of plasmid pYNP78 was PCR-amplified using PR_YNP400-PR_YNP401 (PCR171) and PR_YNP400-PR_YNP402 (PCR183) flanked with 60bp homology to pchI or pchE . Thereafter,PCR products were assembled in-vivo in S. cerevisiae POP4, resulting in plasmids pYNP98 and pYNP99 expressing pchI and pchE , under the control of S. cerevisiae TEF1 promoter and CYC1 terminator. PchI was PCR-amplified from plasmid pYNP98 using primer pair PR_YNP480-PR_YNP481 (PCR184) with 60bp homology to integration site ISX-2 63 , and pchE was PCR-amplified from pYNP99 using primer pair PR_YNP482-PR_YNP483 (PCR185) with 60bp homology to integration site ISXII-1 63 . The PCR products were column-purified prior to transformation into S. cerevisiae POP4 (Supplementary figure 8). For E. viscosa and K. petricola , pchI and pchE were PCR-amplified from plasmids pYNP98 and pYNP99 with primers PR_YNP447-PR_YNP448 (PCR194) and PR_YNP467-PR_YNP468 (PCR203), respectively. A. nidulans TEF1 promoter and trpC terminator were PCR-amplified from plasmid pYNP59 with primer pairs PR_YNP465-PR_YNP466 (PCR202) and PR_YNP469-PR_YNP470 (PCR204), respectively. Backbones from plasmids pYNP80, pYNP81, pYNP82, pYNP84, pYNP85, and pYNP86, were PCR-amplified with primer pairs PR_YNP437-PR_YNP438 (PCR189), PR_YNP435-PR_YNP436 (PCR188), PR_YNP441-PR_YNP442 (PCR191), PR_YNP433-PR_YNP434 (PCR186 and PCR187), and PR_YNP439-PR_YNP440 (PCR190). PchI PCR product was cloned into pYNP84-85-86 open plasmid backbones. Then, pchE PCR product, A. nidulans TEF1 promoter, and trpC terminator were cloned into pYNP80-81-82 open plasmid backbones. The resultant plasmids pYNP102 (DONR_EVJ4_pchI_PKS1), pYNP103 (DONR_EVJ3_pchI_PKS1), and pYNP106 (DONR_KP_pchI_PKS1) express pchI under the control of pks1 promoter and terminator (Supplementary figure 9), while the resultant plasmids pYNP104 (DONR_EVJ4_pchE_PHS1), pYNP105 (DONR_EVJ3_pchE_PHS1), and pYNP107 (DONR_KP_pchE_PHS1) express pchE under the control of A. nidulans TEF1 promoter and trpC terminator (Supplementary figure 10). Plasmids pYNP102, pYNP103, pYNP104, pYNP105, pYNP106, and pYNP107 were linearized with NotI (Thermo Scientific™), and gel-purified prior use in transformations. Transformation and strain construction Black yeastswere inoculated in 50 mL of YPD ( E. viscosa ) or MEB 35 ( K. petricola ) in 250 mL shake flasks and incubated for 5 days at 200 RPM and 25°C. E. viscosa cultures were diluted to OD 600 0.1 in 100 mL of YPD in 500 mL shake flasks and continued incubation for 3 days. For K. petricola , 1 mL of the seed semi-dispersed culture was transferred to 100 mL of ME in a 500 mL shake flask and continued incubation for 4 days. Strains were protoplast according to 40–42 with a few modifications. Briefly, the cultures were transferred to 50 mL Falcon tubes by filtering them through a sterile funnel with miracloth paper to remove non-dispersed cells. The filtered cultures were centrifuged at 3500 G for five minutes and the supernatants were discarded. The cell pellets were washed two times in 25 mL APB solution and centrifuged at 3500 G. The washed cell pellets were resuspended in a 0.45 μm sterile-filtered APB solution containing either Vinotaste-pro™ (Novonesis™) or Extralyse™ (Laffort™) cell wall digestive enzymes in a concentration of 100 mg mL -1 in a final volume of 40 mL. The Falcon tubes containing the cell resuspensions were horizontally placed into a shaking incubator and incubated at 30°C and 150 RPM shaking for 3-4 h. After incubations, cell resuspensions were transferred to new 50 mL Falcon tubes by filtering them through a sterile funnel with a miracloth paper to remove cell debri, APB was added to the filtered resuspensions to a final volume of 40 mL. The cell resuspensions were overlaid with 5 mL of ATB and MQ water solution in 1::1 ratio. The Falcon tubes were centrifuged at 3000 G (acc. 9, deacc. 5) for eleven minutes. The obtained phase of protoplasts was transferred to new 50 mL Falcon tubes and ATB was added to a final volume of 40 mL, gently mixed, and centrifuged at 3000 G (acc. 9, deacc. 9) for eleven minutes. The supernatant was discarded, and the obtained protoplasts were gently resuspended in 1 mL of ATB prior dilutions to desired concentrations and kept on ice. For transformations, 50 μl ( E. viscosa ) or 100 μl ( K. petricola ) of protoplasts in a concentration of 10 7 mL -1 ( E. viscosa ) 10 8 mL -1 ( K. petricola ) were mix with 1 μg of Cas9-sgRNA plasmid and 1 μg of linearized DNA repair templates or column-purified PCRs, or 4 μg of ssDNA oligonucleotides. 150 μl of PCT solution was added to the transformation mix and gently mixed by pipetting. The transformation mix was incubated on ice for 30 minutes. ATB solution was added to the transformation mix up to a final volume of 450 μL and gently mixed before plating on TM or MEAS 35 supplemented with Hygromycin B. Transformation plates were incubated at 25°C at least 10 days before further analysis. S. cerevisiae was transformed according to 92 . For S. cerevisiae transformations, 300 ng of Cas9-sgRNA plasmid and 1 µg of column-purified PCRs were used when required. For in-vivo assemblies in S. cerevisiae ,DNA fragments were mixed in equi-molar amounts.All transformants were streaked-purified prior to validation by diagnostic PCR. For diagnostic PCRs, a primer pair binding upstream and downstream of the genomic integration site or a primer binding outside the integration site and another binding within the integrated DNA sequence were used for all strains validations. Chemical extractions For chemical detection of 6-MSA (or quantification), monocillin II, and farinosone A, B, and YNP1, E. viscosa and K. petricola strains were inoculated in 50 mL of YPD and incubated at 25°C for 5 daysand 200 RPM shaking, cultures were diluted to OD 600 0.1 ( E. viscosa ) or 1 mL was diluted in 50 mL ( K. petricola ) in fresh media, and incubated for seven days prior to culture's processing, except for YWA1 producer strains, which incubation was changed for five days instead. For scale-up of E. viscosa JF 03-4F farinosone A, B, and YNP1 producer strain, 5 days seed cultures were diluted to OD 600 0.1 in 500 mL of fresh media in 2 L shake flasks and incubated for seven days prior to culture's processing. For A. nidulans, A. oryzae, and A. niger , 10 6 spores were inoculated in 50 mL of YPD and incubated at 25°C for 3 days 200 RPM shaking prior extractions. For S. cerevisiae , strains were inoculated in 50 mL of YPD (or SD dropout media with or without uracil when necessary) and incubated at 30°C for three daysand 200 RPM shaking. For S. cerevisiae , chemical detection of 6-MSA (or quantification), YWA1, monocillin II, and YNP1, 24 h seed cultures were diluted to OD 600 0.1 in 50 mL of fresh media and incubated for three days prior to culture's processing. Prior chemical extractions of PKS products, 50 mL cultures were transferred to 50 mL Falcon tubes and centrifuged at 3,500G for five minutes. The liquid phase was then transferred to new Falcon tubes and 25 mL of the liquid phase was extracted in a glass funnel with 2:1 ethyl acetate. The organic phase was recovered and transferred to 50 mL glass tubes, dried in a rotovapor, resuspended in 1 mL of methanol, and diluted 15 times prior LCMS or HPLC analysis. Farinosone A, B, and YNP1 produced during the scale-up culture of E. viscosa JF 03-4F, both the liquid and biomass phases were extracted in a glass funnel with 2:1 ethyl acetate. The organic phase was recovered and transferred to 50 mL glass tubes, dried in a rotovapor, resuspended in 1 mL of methanol, and diluted 300 times prior LCMS or HPLC analysis. For 6-MSA yield quantification purposes, the obtained biomass from cultures was dried for 3 days at 60°C prior weighting. For S. cerevisiae intracellular extractions, the biomass from 50 mL Falcon tubes was resuspended in 10 mL of methanol and transferred to 50 mL glass tubes. The glass tubes were shaken vigorously at 1,800 RPM for 30 minutes, and thereafter the biomass-methanol mix was transferred again to 50 mL Falcon tubes. Falcon tubes were centrifuged at 3,500G for five minutes, and the methanol phase transferred to new 50 mL glass tubes. The samples were dried concentrated in a rotovapor, resuspended in 1 mL of methanol, and diluted 15 times prior to LCMS analysis. Prior chemical extractions of E. viscosa JF 03-4F strain producing farinosone A, B, and YNP1 was grown in 1L YPD, YPX, and 50 mL MMBW, the whole cultures were extracted with 2:1 ethyl acetate. The organic phase was partitioned with 1:1 MQ. The recovered organic phase was dried in a rotovapor, resuspended in 1 mL of methanol, and diluted 300 times for YPD and YPX, and 15 times for MMBW cultures, prior to LCMS analysis. The resuspended extractions were transferred to 1 mL Eppendorf tubes and dried using a rotary evaporator at 45°C prior to weighing. LC-HRMS and LC-UV analyses LC-HRMS analyses were carried out using a Vanquish Duo UHPLC binary system (Thermo Fisher Scientific®, Waltham, MA, USA) coupled to an Orbitrap IDX Tribrid Mass Spectrometer (Thermo Fisher Scientific®). The chromatographic separation was achieved in reverse phase conditions as previously described 93 . The MS measurements were performed in positive- and negative-heated electrospray ionization (+HESI) mode with a voltage of 3500 V and 2500 V, respectively, acquiring in full MS/MS spectra (Data dependent Acquisition-driven MS/MS) in the mass range of 70-1000 Da. The DDA settings were the following: automatic gain control (AGC) target value was set at 4 5 for the full MS and 5 4 for the MS/MS spectral acquisition, the mass resolution was set to 120,000 for full scan MS and 30,000 for MS/MS events. Precursor ions were fragmented by stepped High-energy collision dissociation (HCD) using collision energies of 20, 40, and 60. For 6-MSA compound detection and quantification by HPLC-UV, 10 points serial dilutions of a 6-MSA standard (CAS No.567-61-3, 98%, Thermo Scientific Chemicals®) were used for calibration curve and quantification. The supernatant was analyzed using a Agilent 1100 HPLC system (Agilent Technologies®, Santa Clara, CA, USA) with a diode array detector. The chromatographic separation was achieved using a Poroshell 120 Phenyl-Hexyl column (2.1 mm × 100 mm, 2.7 μm; Agilent Technologies®) with H 2 O + 0.1% formic acid as eluent A and with HPLC grade acetonitrile as eluent B. Gradient elution was performed at a flow rate of 0.5 mL min -1 according to the following: 0-0.5 min 15% B, 0.5-6 min 15% to 65% B, 6-7 min 65% to 100% B, 7-8.5 min 100% B. The column was then re-equilibrated at 15% B. An injection volume of 1 μL was used, and the column oven was maintained at 50°C throughout the analyses. The analytes were detected at a wavelength of 240 nm at a width of 4 nm. Proteomics For proteomics analysis, S. cerevisiae cell pelletswereobtainedfrom cultures grown for 6 h in 25 mL of YPD media incubated at 30 °C and shaken at 200 RPM. These cultures were inoculated at 0.1 OD 600 with cells from 4 mL pre cultures incubated for 24 h. Proteins were extracted using a iST sample preparation kit (PreOmics®) according to manufacturer's instructions. The samples were analyzed by online nano-scale liquid chromatography tandem mass spectrometry (LC-MS/MS) in turn. Peptides were separated on a 15 cm C18-column (Thermo EasySpray™ ES804A) using an EASY-nLC 1200 system (Thermo Scientific®). The column temperature was maintained at 30°C. Buffer A consisted of 0.1% Formic acid in water, and buffer B of 80% ACN, 0.1% Formic acid. The flow rate of the gradient was kept at 250 nL min -1 , and started at 6% Buffer B, going to 23% buffer B in 43 minutes. This was followed by a 12-minute step going to 38% buffer B, increasing to 60% Buffer B in 5 minutes, and finally ramping up to 95% buffer B in 3 minutes, holding it for 7 minutes to wash the column. The Q Exactive Classic instrument (Thermo Scientific®) was run in data dependent acquisition mode using a top 10 Higher-energy Collisional Dissociation (HCD)-MS/MS method with the following settings. Scan range was limited to m/z 350-1750. Full scan resolution was set to m/z 70,000, with an AGC target of 3 6 and a maximum injection time (IT) value of 20 ms. Peptides were fragmented with a normalized collision energy of 25, having a dynamic exclusion of 30 s, excluding unassigned ions and those with a charge state of 1. MS/MS resolution was set at m/z 17,500, with an AGC target of 3 6 and a maximum IT of 60 ms. The spectral data was analysed with Maxquant 94 using the protein sequences of PchI and PchE (monocillin II) and AdnE1-3(YNP1)plus NpgA. Bioinformatic methods For whole genome sequencing, S. cerevisiae cell pellets were obtained from 4 mL YPD cultures grown overnight at 30°C and shaken at 200 RPM. E. viscosa and K. petricola cell pellets were obtained from cultures grown in 25 mL of YPD media in 250 mL flasks for seven days at 25 °C and shaken at 200 RPM, in all cases the cell pellets were washed once with deionized water. For transcriptome analyses, overnight cultures of S. cerevisiae were diluted to OD 600 0.1and grown for 6 h in 25 mL of YPD media incubated at 30 °C and shaken at 200 RPM. 2mL of culture was pelleted and washed once with deionized water and used transcriptome sequencing GENEWIZ (Azenta Life Science technologies) using the Illumina NovaSeq platform in the 150 bases pair-end format. For genome assembly the reads were trimmed using TrimmomaticPE v0.39 95 and assembled using the SPAdes assembler v3.13.1 96 . The gene calling on the assembled scaffolds was performed using Augustus v3.4.0 97 with genes model trained with the annotated genome of E. viscosa JF 03-3F (RefSeq accession GCF_022695815) or S. cerevisiae S288C (RefSeq accession GCF_000146045). The assemblies and gene-calling files were used for genome annotation with antiSMASH v7.0.0 98 . To assess the expression of the monocillin II and YNP1 in S. cerevisiae we used bowtie v. 2.4.4 99 to map the RNA reads from strains sYNP19213 and sYNP19220 against the coding sequences of npgA , p chI and p chE (monocillin II) and a dnE1 - 3 (YNP1) plus the coding sequence of act1 from S. cerevisiae S288C (RefSeq accession GCF_000146045). The resulting Sequence Alignment/Map files were then parsed to count the number of reads mapped for each gene; the expression level was recorded as the number of reads mapped for each coding sequence divided by their length in kilobases (RPK) and then normalized to the RPK value for the housekeeping gene act1 . Genome sequencing, mining and Phylogenomics To estimate the distribution of PKSs across the fungal kingdom, We compiled a collection of 1979 fungal genomes obtained from the GenBank (supplementary file1), these genomes were gene-called using Augustus v3.4.0 97 and their natural polyketide synthase repertoire was annotated with antiSMASH v7.0.0 98 . The PKSs found in these genomes were identified and their domain organization extracted using a Perl script (https://github.com/WeMakeMolecules/Megasynthase_string_miner/blob/main/antismash_domain_parser.pl) the extracted PKSs were then parsed to classify and count them , the complete list of analyzed PKSs is available on (supplementary file 2). To compare known 2-pyridone biosynthetic gene cluster families to the YNP1 BGC (Figure 4A) we used FUNGISON, a implementation of the CORASON pipeline 79 for fungal genomes available at https://github.com/WeMakeMolecules/fungison. The reference BGCs were obtained from the MiBig database 90 . Declarations Data availability: All the genomes in the dataset used for this project are available in GenBank, the complete list of accession numbers is available in Supplementary file 2. All the sequences of PKSs analyzed are available in Supplementary file 2. The genomics and transcriptomics data for S. cerevisiae strains will be available at the NCBI sequence read archive before publication. Acknowledgements We want to thank Assoc. Prof. Jakob Blæsbjerg Hoof for providing Aspergillus spp.. strains from the IBT fungal strain collection used in this work, and to Andreas Møllerhøj Vestergaard for sharing S. cerevisiae strain BJ-5464-NpgA and plasmid pCFB2909-BIK1. We also thank Asst. Prof. Vayu Hill-Maini, and all the members of the Yeast Natural Products laboratory for reviewing this manuscript and providing insightful comments. We are deeply grateful to the IT team and the staff of the Novo Nordisk Center for Biosustainability for their invaluable support. This research was funded by the Novo Nordisk Foundation NNF20CC0035580 Author contributions: AEGL, XL, CCP, LB, DA, LC, SJ, JSB: performed molecular biology experiments; AEGL, LB, ACC, PCM: Performed metabolite extractions and purifications; MVL: Performed proteomic analyses; DR, LH, ACC: developed LC-MS methods and collected spectral data; CHG, ACC: performed NMR analysis; AEGL, ACC, PCM analyzed mass spectrometry data; AEGL, MVB and PCM performed bioinformatic analyses; AF and SIJ, prepared beechwood extracts and designed cultivations; EC, SH, UHM, TS, helped in the design of the experiments, data analysis and discussion; JDK and PCM supervised the research, AEGL and PCM designed the experiments; AEGL, JDK, UM and PCM prepared the manuscript. PCM conceived the idea. Competing interests: JDK has financial interests in Amyris, Ansa Biotechnologies, Apertor Pharma, Berkeley Yeast, Cyklos Materials, Demetrix, Lygos, Napigen, ResVita Bio, and Zero Acre Farms. 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Bargen","suffix":""},{"id":417081021,"identity":"a58457cd-d913-4f57-8533-594d9e6c4c46","order_by":13,"name":"Laasya Bhagavan","email":"","orcid":"","institution":"Yeast Natural Products Laboratory, Novo Nordisk Foundation Center for Biosustainability","correspondingAuthor":false,"prefix":"","firstName":"Laasya","middleName":"","lastName":"Bhagavan","suffix":""},{"id":417081022,"identity":"edbfca82-5e55-44d8-9d44-e340097abd13","order_by":14,"name":"Tomas Strucko","email":"","orcid":"","institution":"Tecnical University of Denmark","correspondingAuthor":false,"prefix":"","firstName":"Tomas","middleName":"","lastName":"Strucko","suffix":""},{"id":417081023,"identity":"b485b139-024a-4990-ac61-beb4ceb07379","order_by":15,"name":"Adrian Frey","email":"","orcid":"https://orcid.org/0009-0008-7716-4042","institution":"Novo Nordisk Foundation Center for Biosustainability","correspondingAuthor":false,"prefix":"","firstName":"Adrian","middleName":"","lastName":"Frey","suffix":""},{"id":417081024,"identity":"f3beeb9f-ce32-4d98-815b-e04a4d3d1b3b","order_by":16,"name":"Dushica Arsovska","email":"","orcid":"","institution":"Technical University of Denmark","correspondingAuthor":false,"prefix":"","firstName":"Dushica","middleName":"","lastName":"Arsovska","suffix":""},{"id":417081025,"identity":"ef009d0d-98ad-4f26-9f51-e7be1e1db9f1","order_by":17,"name":"Erin Carr","email":"","orcid":"https://orcid.org/0000-0001-6551-9207","institution":"University of Nebraska Lincoln","correspondingAuthor":false,"prefix":"","firstName":"Erin","middleName":"","lastName":"Carr","suffix":""},{"id":417081026,"identity":"e42a651c-cb66-4fc2-99ab-ab04dabf69dc","order_by":18,"name":"Sheila Jensen","email":"","orcid":"","institution":"Novo Nordisk Foundation Center for Biosustainability","correspondingAuthor":false,"prefix":"","firstName":"Sheila","middleName":"","lastName":"Jensen","suffix":""},{"id":417081027,"identity":"a1e7afcb-2e4d-4d52-b037-1467e4e0c326","order_by":19,"name":"Steven Harris","email":"","orcid":"","institution":"Iowa State University","correspondingAuthor":false,"prefix":"","firstName":"Steven","middleName":"","lastName":"Harris","suffix":""},{"id":417081028,"identity":"e0639140-72fd-492b-bd6d-fa4ed8ab8b78","order_by":20,"name":"Uffe Mortensen","email":"","orcid":"https://orcid.org/0000-0002-7794-7273","institution":"Department of Biotechnology and Bioengineering, Technical University of Denmark","correspondingAuthor":false,"prefix":"","firstName":"Uffe","middleName":"","lastName":"Mortensen","suffix":""},{"id":417081029,"identity":"1412e9c4-8c8e-438a-ae09-8a1594c61979","order_by":21,"name":"Jay Kiesling","email":"","orcid":"https://orcid.org/0000-0003-4170-6088","institution":"5\tDepartment of Chemical \u0026 Biomolecular Engineering, University of California, Berkeley; California Institute of Quantitative Biosciences (QB3), University of California, Berkeley; Joint BioEnergy I","correspondingAuthor":false,"prefix":"","firstName":"Jay","middleName":"","lastName":"Kiesling","suffix":""}],"badges":[],"createdAt":"2025-02-10 20:20:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6001933/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6001933/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":76766189,"identity":"6ff53435-bcb8-4629-a058-78ef1d481e3c","added_by":"auto","created_at":"2025-02-20 13:25:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":249722,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBioprospection of new yeast hosts for heterologous polyketide production.\u003c/strong\u003e \u003cstrong\u003eA)\u003c/strong\u003eDistribution of PKSs across the fungal kingdom. Lecanoromycetes and Eurotiomycetes contain the highest average number of PKS, whilst Saccharomycetes yeast exhibit a significant reduction in their ability to produce polyktides; \u003cstrong\u003eB)\u003c/strong\u003e40x microscopy showing the yeast morphology of \u003cem\u003eE. viscosa\u003c/em\u003e JF 03-3F; \u003cstrong\u003eC)\u003c/strong\u003e40x microscopy showing the yeast morphology of \u003cem\u003eE. viscosa\u003c/em\u003e JF 03-4F; \u003cstrong\u003eD)\u003c/strong\u003e40x microscopy showing the yeast morphology of \u003cem\u003eK. petricola\u003c/em\u003e. \u003cstrong\u003eE) \u003c/strong\u003eTotal ion chromatograms of \u003cem\u003eAspergillus spp..\u003c/em\u003e, \u003cem\u003eE. viscosa, K. petricola, \u003c/em\u003eand \u003cem\u003eS. cerevisiae\u003c/em\u003e. \u003cstrong\u003eF)\u003c/strong\u003e Growth of \u003cem\u003eE. viscosa \u003c/em\u003eJF 03-3F cultivated in YPD, YPX, and MMBW; \u003cstrong\u003eG)\u003c/strong\u003eGrowth of \u003cem\u003eE. viscosa \u003c/em\u003eJF 03-4F\u003cem\u003e \u003c/em\u003ecultivated in YPD, YPX, and MMBW; \u003cstrong\u003eH)\u003c/strong\u003e Initial and final dry biomass measurements of \u003cem\u003eK. petricola \u003c/em\u003eduring cultures in YPD, YPX, and MMBW. Panels F, G and H show the average value and standard deviation of three biological replicates.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6001933/v1/c43dadfa4b86079145f7d590.png"},{"id":76766190,"identity":"c35902f0-1e6a-494d-bcc9-c562a51b4d9a","added_by":"auto","created_at":"2025-02-20 13:25:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":365103,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDevelopment of genetic engineering tools for black yeast engineering\u003c/strong\u003e \u003cstrong\u003eA)\u003c/strong\u003eTransformation plates of \u003cem\u003eE. viscosa \u003c/em\u003eand \u003cem\u003eK. petricola \u003c/em\u003eafter 21 days at 25°C in TM media; \u003cstrong\u003eB)\u003c/strong\u003e Visual Screening of \u003cem\u003eE. viscosa \u003c/em\u003etransformats with Cas9-sgRNA plasmids targeting \u003cem\u003epks1 \u003c/em\u003eand \u003cem\u003epks1 \u003c/em\u003egenes. A visual screening is carried out to identify phenotypic changes, indicating a DSB was made and might be repaired in an error prone manner by NHEJ \u003cstrong\u003eC)\u003c/strong\u003e On top, pictures of \u003cem\u003eE. viscosa \u003c/em\u003estrains transformed with plasmid pYNP64, arrows indicate the phenotype of Δ\u003cem\u003epks1 \u003c/em\u003eand\u003cem\u003e \u003c/em\u003eΔ\u003cem\u003epks1 \u003c/em\u003eΔ\u003cem\u003ephs1 \u003c/em\u003egenes. On the bottom, sanger sequencing results within Cas9 cutting site; three Δ\u003cem\u003epks1 \u003c/em\u003eΔ\u003cem\u003ephs1 \u003c/em\u003ecolonies displaying a white phenotype were analyzed and all nucleotide changes resulted in \u003cem\u003epks1\u003c/em\u003e,\u003cem\u003e \u003c/em\u003eand \u003cem\u003ephs1 \u003c/em\u003eframeshift mutations; \u003cstrong\u003eD)\u003c/strong\u003e Construction of NHEJ deficient strains by \u003cem\u003eku70\u003c/em\u003e gene deletions or by \u003cem\u003eku70\u003c/em\u003e frameshift mutations; \u003cstrong\u003eE)\u003c/strong\u003e Validation of NHEJ deficient \u003cem\u003eK. petricola\u003c/em\u003e. Gel from left to right: 1 Kb ladder, amplification of \u003cem\u003eK. petricola ku70\u003c/em\u003egene deletion (2.5 Kb), amplification of \u003cem\u003eK. petricola ku70\u003c/em\u003e gene deletion digested with XbaI (1.25 Kb); \u003cstrong\u003eF)\u003c/strong\u003e \u003cem\u003eE. viscosa \u003c/em\u003eNHEJ deficient strains sanger sequencing results. Mutations in \u003cem\u003eku70\u003c/em\u003e resulted in frameshifts; \u003cstrong\u003eG)\u003c/strong\u003e ssDNA-mediated gene deletions in \u003cem\u003eE. viscosa\u003c/em\u003e. A DSB is made by Cas9 in one or both \u003cem\u003epks1 \u003c/em\u003eand \u003cem\u003ephs1 \u003c/em\u003egenes, thereafter DNA should be repaired via HR using a 90nt ssDNA as repair template; \u003cstrong\u003eH)\u003c/strong\u003e Frequencies of gene deletions in WT and NHEJ deficient strains of \u003cem\u003eE. viscosa\u003c/em\u003e using 90nt ssDNA as repair template. Higher gene deletion frequencies were observed in NHEJ deficient strains compared to WT strains.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6001933/v1/b15522b3725d064cb7cf680c.png"},{"id":76767877,"identity":"15541ece-9504-4d28-9a56-4325a6051d63","added_by":"auto","created_at":"2025-02-20 13:49:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":278907,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeterologous production of polyketides. A)\u003c/strong\u003eDistribution of PKSs domain architectures in the fungal kingdom; \u003cstrong\u003eB) \u003c/strong\u003eSet of PKSs selected for heterologous expression. \u003cstrong\u003eC) \u003c/strong\u003eChemical structures, formulas and theoretical masses for the products of the PKSs selected for heterologous production; \u003cstrong\u003eD)\u003c/strong\u003e Genomic integration of \u003cem\u003eatX6MSA\u003c/em\u003ein black yeast and \u003cem\u003eS. cerevisiae \u003c/em\u003ePOP4. A construct \u003cem\u003eatX6MSA\u003c/em\u003e driven by the \u003cem\u003eA. nidulans\u003c/em\u003e TEF1 promoter and trpC terminator flanked with 1.5 Kb sequences homologous to \u003cem\u003epks1 \u003c/em\u003ewas used for black yeasts. For \u003cem\u003eS. cerevisiae \u003c/em\u003ePOP4, \u003cem\u003eatX6MSA\u003c/em\u003edriven by \u003cem\u003eS. cerevisiae \u003c/em\u003eTEF1 promoter and CYC1 terminator, flanked with 60 bp sequences homologous to the ISX-2 was used; \u003cstrong\u003eE)\u003c/strong\u003e Absolute concentrations of 6-MSA made by the heterologous strains. The graph shows the average value and standard deviation of three biological replicates \u003cstrong\u003eF) \u003c/strong\u003eTotal ion chromatograms obtained from the organic extracts of the supernatants of YWA1 and monocillin II heterologous producers.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6001933/v1/6f6fa08f8e213e689a3525cf.png"},{"id":76766192,"identity":"3af33fac-63d9-4d6f-834e-22387721783c","added_by":"auto","created_at":"2025-02-20 13:25:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":261719,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDiscovery of YNP1. A)\u003c/strong\u003e Genome mining for a highly reducing methylating hybrid PKS-NRPSs. The BGC from \u003cem\u003eCordyces sp. \u003c/em\u003eD1 is closely related to BGCs encoding the biosynthetic pathways of 2-pyridones. The core biosynthetic genes from the BGCs are represented in colors red (PKS-NRPS synthetase), yellow (CYP450), and light blue and dark blue (trans-ER and CYP450). In \u003cem\u003eC. sp. \u003c/em\u003eD1 the trans-ER was annotated as a fusion to the CYP450; \u003cstrong\u003eB) \u003c/strong\u003eMetabolic profiles\u003cstrong\u003e \u003c/strong\u003e(LC-HRMS) of the strains with the 2-pyridone BGC from \u003cem\u003eCordyceps sp.\u003c/em\u003e D1\u003cem\u003e.\u003c/em\u003e Peaks for a mass corresponding to [C\u003csub\u003e25\u003c/sub\u003eH\u003csub\u003e26\u003c/sub\u003eNO\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e+\u003c/sup\u003e were found in \u003cem\u003eE. viscosa \u003c/em\u003eJF 03-3F, JF 03-4F\u003cem\u003e, \u003c/em\u003eand \u003cem\u003eK. petricola\u003c/em\u003e, but not in \u003cem\u003eS. cerevisiae\u003c/em\u003e POP4; \u003cstrong\u003eC)\u003c/strong\u003e Phenotype of the strains with the 2-pyridone BGC from \u003cem\u003eCordyceps sp.\u003c/em\u003e D1. \u003cstrong\u003eD)\u003c/strong\u003e Structure of YNP1. Displayed are \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e1\u003c/sup\u003eH COSY and \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e13\u003c/sup\u003eC HMBC main correlations of YNP1 (17-Hydroxylfarinosone).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6001933/v1/fc4ad610e40a4ae44cf39e49.png"},{"id":76766210,"identity":"1ccafeb0-6f4d-4215-b7f0-c105b475bcf6","added_by":"auto","created_at":"2025-02-20 13:25:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":134380,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTrans-ER restoration and proposed pathway for \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCordyceps sp. \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eD1 farinosone B biosynthesis. A) \u003c/strong\u003eExtracted ion chromatograms of the strains where corrected ER and CYP450 sequences were introduced. Farinosone B was found to be the final product of \u003cem\u003eadnE1-2-3-4\u003c/em\u003e. Parental strains without corrected ER sequences did not produce farinosone B; \u003cstrong\u003eB) \u003c/strong\u003eExtracted ion chromatograms of the strains where corrected ER and CYP450 sequences were introduced, YNP1 was still produced in these strains; \u003cstrong\u003eC)\u003c/strong\u003eProposed biosynthetic pathway of farinosone B\u003cem\u003e. AdnE1 \u003c/em\u003eproduces a tyrosylated intermediate which is cyclized upon reductive release to form a five-membered ring,\u003cem\u003e adnE2 \u003c/em\u003ecatalyzes the ring expansion, \u003cem\u003eadnE3\u003c/em\u003e reduces a double bond in the acyl chain, and \u003cem\u003eadnE4\u003c/em\u003ehydroxylates the amide group.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6001933/v1/053bc23e939c37175cb4cf8b.png"},{"id":76769050,"identity":"00404ad1-284a-4e9b-9b5e-e26c7ccef16b","added_by":"auto","created_at":"2025-02-20 13:57:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2772013,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6001933/v1/2d7f5c04-acc0-41f4-8609-b14f89c8affc.pdf"},{"id":76766227,"identity":"69f30f6e-495d-4c37-87cb-4c63f241790a","added_by":"auto","created_at":"2025-02-20 13:25:20","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":86181,"visible":true,"origin":"","legend":"Supplementary Data Set 1","description":"","filename":"Supplementaryfile1Genomesdatabase.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6001933/v1/d91d3f36885bd9319e3e11d5.xlsx"},{"id":76766201,"identity":"16cebfaa-1e8b-49f3-8521-282f10f20f7c","added_by":"auto","created_at":"2025-02-20 13:25:18","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17198351,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Data Set 2\u003c/p\u003e","description":"","filename":"Supplementaryfile2PKSDatabase.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6001933/v1/619b40642825a2d9a7b163bd.xlsx"},{"id":76766203,"identity":"7b8ecf70-28c7-4c87-9038-153c55e2f76c","added_by":"auto","created_at":"2025-02-20 13:25:18","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":13061098,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary_figures_and tables\u003c/p\u003e","description":"","filename":"Supplementaryfiguresandtables10022025forsubmission.docx","url":"https://assets-eu.researchsquare.com/files/rs-6001933/v1/d31b2502f8288249ff0dc77c.docx"},{"id":76766197,"identity":"9f05e95c-a688-41c0-82fa-da0e04e3d837","added_by":"auto","created_at":"2025-02-20 13:25:18","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1471313,"visible":true,"origin":"","legend":"editorial checklist","description":"","filename":"Editorialpolicychecklist.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6001933/v1/f40a8f78853265505eb96f84.pdf"},{"id":76766207,"identity":"88ae7755-0d36-4c22-9c1f-726344eb9ee5","added_by":"auto","created_at":"2025-02-20 13:25:18","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1790397,"visible":true,"origin":"","legend":"reporting summary","description":"","filename":"nrreportingsummary.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6001933/v1/db5bd0befc8aa83358ebb89b.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nJay D. Keasling has financial interests in Amyris, Ansa Biotechnologies, Apertor Pharma, Berkeley Yeast, Cyklos Materials, Demetrix, Lygos, Napigen, ResVita Bio, and Zero Acre Farms. The other authors declare no competing interests.","formattedTitle":"Black yeasts are efficient heterologous hosts of a wide range of fungal polyketides","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFungal natural products (NP) metabolism is an important source of molecules and enzymes that could enable the sustainable manufacturing of foods, bulk chemicals, polymers, pesticides, and fuels \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Among natural product enzymes, polyketide synthases (PKSs) stand out due to the diversity of their products and their biosynthetic mechanisms which can be engineered to create novel carbon-based compounds \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. PKSs are multimodular enzyme complexes that can include acyl transferase (AT), ketosynthase (KS), C-methylation (M), ketoreductase (KR), dehydratase (DH), enoyl reductase (ER), acyl carrier protein (ACP), and thioesterase (TE) domains. A key aspect of the function of PKSs is the post-translational attachment of a phosphopantetheinyl group to a conserved serine in the ACP domain, a reaction catalyzed by a phosphopantetheinyl transferase (PPTase). This prosthetic group acts as an \u0026ldquo;arm\u0026rdquo;, shuttling the substrates and intermediates across the PKS domains \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFungal iterative PKSs (iPKSs) produce polyketides using a single set of domains. In each iteration it is possible that the cycle skips some of the domains, creating structurally diverse products \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Given their biosynthetic capabilities, iPKSs are excellent systems for the development of new bioproducts \u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.Although many fungal iPKSs have been described, we are still far from exploiting their full chemical repertoire. One significant limitation in the discovery, production, and engineering of fungal polyketides is that many of them are not produced when fungi are cultivated under laboratory conditions. Several cultivation-based strategies have been developed to overcome this issue, but their efficacy is limited \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. An alternative method that bypasses the native regulation of the PKSs is heterologous expression, in which known or putative biosynthetic genes are transferred into an amenable host under the control of orthogonal expression systems \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo date, few yeast species have been used for heterologous expression of fungal polyketides that produce phenolic and polycyclic products. Most efforts have focused on \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e \u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eKomagataella phaffii\u003c/em\u003e \u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eKluyveromyces marxianus\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, and \u003cem\u003eYarrowia lipolytica\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e; all of them Saccharomycetales yeast that evolved a simplified metabolism by genome erosion \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Although their simpler genomes make them excellent for genetic engineering, genome erosion also reduced their ability to grow on complex substrates and to produce complex NPs, (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Supplementary table 2). This trait loss is underscored by the lack of PKSs and their cognate PPTase \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. On the other hand, many filamentous fungi have large NP repertoires \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e (Supplementary tables 3\u0026ndash;5) and can grow on complex substrates such as lignocellulosic biomass \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e and pre-treated plastic waste \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Among filamentous species, members of the genus \u003cem\u003eAspergillus\u003c/em\u003e are considered efficient hosts for the heterologous expression of complex NPs, and an increasing number of genetic engineering tools are available for them \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. However, their filamentous morphology and genetic complexity implies longer design-build-test-learn cycles and greater challenges during process scale-up compared to Saccharomycetales yeast. Additionally, their complex metabolism and large repertoire of endogenous enzymes can modify the target products into other derivatives, complicating the detection and purification of the products \u003csup\u003e\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Therefore, more efficient heterologous hosts with simplified genomes and robust-yet-simple metabolic backgrounds, unicellular growth, and the ability to use complex substrates are needed for the systematic discovery and sustainable manufacturing of polyketide-based bioproducts.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn this work, we established two species of black yeast, \u003cem\u003eExophiala viscosa\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e and \u003cem\u003eKnufia petricola\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e as heterologous hosts for the production of fungal polyketides. Black yeast is a common name for a polyphyletic group of unicellular, extremophilic fungi that can grow on complex substrates. Their dark phenotype is derived from melanin, a fungal polyketide that they copiously produce \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. We reasoned that these black yeast species evolved a robust metabolism to thrive in harsh environments, and that their native metabolism likely includes a PKS-active PPTase and abundant acetyl-CoA and malonyl-CoA pools, which are essential precursors for melanin biosynthesis. We constructed non-homologous end-joining (NHEJ) deficient strains and developed a synthetic biology toolbox for efficient and marker-free genomic integrations. To test their potential as polyketide production hosts, we heterologously expressed and obtained the products of a set of PKSs representing the most frequent domain architectures found in the fungal kingdom. We achieved production of 6-methylsalicylic acid (6-MSA), YWA1, monocillin II, and farinosones, whereas the production was limited in \u003cem\u003eS. cerevisiae\u003c/em\u003e. Finally, we demonstrated that our black yeast hosts can be used for rapid characterization of unknown biosynthetic pathways. Our work showcases the potential of black yeast for systematic expression and engineering of PKSs.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eKingdom-wide polyketide genome mining reveals black yeast\u0026rsquo;s potential as heterologous hosts\u003c/h2\u003e \u003cp\u003eTo select new yeast candidates to be engineered as heterologous hosts for PKS, we assembled and annotated a genome database with 1,979 strains covering 15 classes within the fungal kingdom (Supplementary file 1). We annotated these genomes and calculated the average number of polyketide synthases per class across the kingdom (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Supplementary file 2). The Eurotiomycetes class displayed the second highest number of PKSs. This group includes filamentous workhorses of the genus \u003cem\u003eAspergillus\u003c/em\u003e, and the genera \u003cem\u003eExophiala\u003c/em\u003e and \u003cem\u003eKnufia\u003c/em\u003e. In contrast, PKSs are very rare among members of the Saccharomycetes class. We thus focused on the Eurotiomycetes \u003cem\u003eK. petricola\u003c/em\u003e CBS123872 \u003csup\u003e30,31\u003c/sup\u003e and \u003cem\u003eE. viscosa\u003c/em\u003e strains JF 03-3F and JF 03-4F (CBS148801 and CBS148802) \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e which we acquired from the WI-KNAW collection.\u003c/p\u003e \u003cp\u003eA desirable trait in a heterologous host is the ability to grow on second-generation feedstocks, such as lignocellulosic biomass. Thus, we tested \u003cem\u003eE. viscosa\u003c/em\u003e and \u003cem\u003eK. petricola\u003c/em\u003e for growth on beechwood hydrolysates and xylose media. We cultivated \u003cem\u003eE. viscosa\u003c/em\u003e and \u003cem\u003eK. petricola\u003c/em\u003e YPD ( 2% glucose), YPX (2% xylose), or MMBW, a minimal media supplemented with beechwood hydrolysate \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. No significant differences were observed in the growth of \u003cem\u003eE. viscosa\u003c/em\u003e, which grew as single cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) and reached an optical density (600 nm) of OD\u003csub\u003e600\u003c/sub\u003e 40 in both YPD or YPX, OD\u003csub\u003e600\u003c/sub\u003e 33, 39 in MMBW for strain JF 03-3F, and strain JF 03-4F respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). \u003cem\u003eK. petricola\u003c/em\u003e formed cell agglomerations during cultivation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Therefore, we quantified its growth using biomass dry weight after 7 days of cultivation. The final average biomass obtained in these cultures was 298 mg +/-176 mg (YPD), 147 mg +/-14 mg (YPX), and 188 mg +/-13 mg (MMBW) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH\u003cb\u003e)\u003c/b\u003e. Overall, these results show that these black yeasts can grow on second generation feedstocks, even on beechwood hydrolysates which are toxic to many bacterial and fungal species \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. The genomes of these species \u003cb\u003e(Supplementary table 9)\u003c/b\u003e are larger than \u003cem\u003eS. cerevisiae\u003c/em\u003e, but smaller than the frequently used \u003cem\u003eAspergillus spp.\u003c/em\u003e. Inspection of their genomes showed that these species harbor the potential to produce complex PKS, NRPS and terpenes but their repertoires are smaller when compared with \u003cem\u003eAspergillus spp\u003c/em\u003e. \u003cb\u003e(Supplementary tables 2\u0026ndash;8)\u003c/b\u003e. We reasoned that this may be advantageous, since a reduced NP repertoire may facilitate the redirection of precursors towards the heterologous pathway, could prevent unexpected modification done by endogenous enzymes and simplify the identification and purification of the products \u003csup\u003e\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. To assess the native metabolic complexity of these black yeasts, we compared the chemical profiles of \u003cem\u003eE. viscosa\u003c/em\u003e, \u003cem\u003eK. petricola\u003c/em\u003e, \u003cem\u003eS. cerevisiae\u003c/em\u003e, and \u003cem\u003eAspergillus spp.\u003c/em\u003e. Our analysis showed that black yeasts have simpler chemical profiles than \u003cem\u003eAspergillus spp.\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Considering the growth capabilities and biosynthetic profile of our black yeasts we reasoned these species are excellent candidates to be developed into polyketide heterologous hosts.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDevelopment of synthetic biology tools for heterologous expression in black yeasts\u003c/h3\u003e\n\u003cp\u003eThere are genetic engineering tools already available for \u003cem\u003eK. petricola\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Thus, we constructed tools for \u003cem\u003eE. viscosa\u003c/em\u003e. First, we determined that the \u003cem\u003eE. viscosa\u003c/em\u003e strains are sensitive to Hygromycin B at 50 \u0026micro;g mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Supplementary Fig.\u0026nbsp;1). We then transformed them with AMA1-based plasmids, which are episomal and can be cured in filamentous Eurotiomycetes \u003csup\u003e\u003cspan additionalcitationids=\"CR37 CR38\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e and in \u003cem\u003eK. petricola\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. For transformation, we adapted a protoplastation protocol used for filamentous fungi \u003csup\u003e\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. We observed colonies in the transformation plates after 21 days at 25\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe tested the functionality and efficiency of the CRISPR-Cas9 \u003csup\u003e43,44\u003c/sup\u003e system in \u003cem\u003eE. viscosa\u003c/em\u003e JF 03-3F and JF 03-4F. For this, we targeted the polyketide synthase gene \u003cem\u003epks1\u003c/em\u003e involved in the biosynthesis of melanin, a dark colored product, and the phytoene synthase gene \u003cem\u003ephs1\u003c/em\u003e for carotenoid biosynthesis, an orange colored product \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. Introduction of Cas9 and the cognate sgRNAs leads to a DNA double strand break (DSB) at the targeted site which may be repaired in an error-prone manner via NHEJ \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e often leading to deleterious insertions or deletions in the targeted genes. Therefore, successful introduction of DSBs in \u003cem\u003epks1\u003c/em\u003e and \u003cem\u003ephs1\u003c/em\u003e is expected to cause a phenotypic change from black (wild-type genotype) to orange (mutated \u003cem\u003epks1)\u003c/em\u003e, or from black to white (mutated \u003cem\u003epks1\u003c/em\u003e and \u003cem\u003ephs1\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). We transformed \u003cem\u003eE. viscosa\u003c/em\u003e JF 03-3F and JF 03-4F with plasmids for Cas9 expression and the \u003cem\u003eAspergillus fumigatus\u003c/em\u003e U3 promoter and terminator driving the expression of sgRNAs delivered via tRNA release mechanism targeting \u003cem\u003epks1\u003c/em\u003e or both \u003cem\u003epks1\u003c/em\u003e and \u003cem\u003ephs1.\u003c/em\u003e This experiment resulted in the expected genotypic and phenotypic changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), confirming that Cas9-sgRNA CRISPR nucleases are active in our black yeast host candidates.\u003c/p\u003e\n\u003ch3\u003eGene-editing efficiencies are higher in NHEJ-deficient black yeast strains\u003c/h3\u003e\n\u003cp\u003eIn many fungal species, inactivation of NHEJ DNA repair pathway allows controlled and precise integration of DNA, although with some fitness tradeoffs \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. NHEJ deficient strains are usually obtained by knockout of the gene encoding Ku70 \u003csup\u003e47\u003c/sup\u003e. After a DNA DSB has been made in NHEJ-deficient strains, the predominant DNA repairs mechanism to avoid death is homologous recombination (HR). This allows for efficient integration of exogenous DNA flanked with homologous sequences up-and downstream of the cut site \u003csup\u003e\u003cspan additionalcitationids=\"CR49 CR50 CR51 CR52\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo generate NHEJ deficient black yeast strains, we first identified \u003cem\u003eku70\u003c/em\u003e orthologs in \u003cem\u003eK. petricola\u003c/em\u003e and \u003cem\u003eE. viscosa\u003c/em\u003e and targeted the first 30 nucleotides of these genes using Cas9-sgRNA plasmids. We provided a linearized DNA repair template harboring an XbaI restriction site and 500 bp upstream and downstream homology sequences to \u003cem\u003eku70\u003c/em\u003e. We expected to obtain either full gene deletions by HR or gene disruption by NHEJ (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). For \u003cem\u003eK. petricola\u003c/em\u003e, a strain with full gene deletion was obtained and validated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). For \u003cem\u003eE. viscosa\u003c/em\u003e JF 03-3F and JF 03-4F, full gene deletions affected the fitness of the strains (data not shown), but strains with \u003cem\u003eku70\u003c/em\u003e frameshifts were viable and used for the following experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eOligo-mediated engineering is a fast, precise and cheap gene-editing method for strain engineering in filamentous fungi \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e and non-conventional yeasts \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, including \u003cem\u003eK. petricola\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. We tested its efficiency in both wild-type (WT) and NHEJ deficient strains of \u003cem\u003eE. viscosa\u003c/em\u003e, and \u003cem\u003eK. petricola\u003c/em\u003e. For this purpose, we transformed \u003cem\u003eE. viscosa\u003c/em\u003e JF 03-3F and JF 03-4F and \u003cem\u003eK. petricola\u003c/em\u003e with Cas9-sgRNA plasmids targeting \u003cem\u003epks1\u003c/em\u003e and \u003cem\u003ephs1\u003c/em\u003e. We used 90 nucleotides single stranded DNA (ssDNA) oligos as repair templates for single or double gene deletions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Up to ten colonies from each experiment with the expected phenotype were analyzed by diagnostic PCR. In \u003cem\u003eE. viscosa\u003c/em\u003e, the number of transformants with full gene deletions was higher in NHEJ deficient strains for both single and double gene deletions when compared to the WT strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH and supplementary Fig.\u0026nbsp;2). In \u003cem\u003eK. petricola\u003c/em\u003e, the number of transformants with full gene deletions was equal in NHEJ deficient strains for single gene deletions when compared to the WT. In contrast, the number of NHEJ deficient \u003cem\u003eK. petricola\u003c/em\u003e transformants with double gene deletions was higher compared to the WT (supplementary Fig.\u0026nbsp;3). These experiments confirmed that inactivation of the NHEJ pathway improves the efficiency of gene edits in our black yeast (Supplementary Figs.\u0026nbsp;2 and 3), allowing us to demonstrate ssDNA-mediated gene editing in \u003cem\u003eE. viscosa\u003c/em\u003e and establish the molecular biology pipeline for heterologous expression via genomic integration.\u003c/p\u003e\n\u003ch3\u003eBlack yeast hosts enable efficient heterologous production of polyketides\u003c/h3\u003e\n\u003cp\u003eTo test the efficacy of our black yeasts hosts for heterologous expression of polyketides, we chose a set of PKSs covering the most common domain architectures and degrees of complexity (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). From the least to the most complex: 6-methylsalicylic acid synthase (\u003cem\u003eatX6MSA\u003c/em\u003e) from \u003cem\u003eAspergillus terreus\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e, YWA1 (\u003cem\u003epks12\u003c/em\u003e) synthase from \u003cem\u003eFusarium acasiae-mearnsii\u003c/em\u003e CBS10255, monocillin II (\u003cem\u003epchI \u0026amp; pchE\u003c/em\u003e) synthases from \u003cem\u003ePochonia chlamydosporia\u003c/em\u003e MUCL9880, and farinosone B (\u003cem\u003eadnE1-4\u003c/em\u003e) from \u003cem\u003eCordyceps sp.\u003c/em\u003e D1, a cryptic highly reducing PKS-NRPS predicted to direct the synthesis of a 2-pyridone product \u003csup\u003e\u003cspan additionalcitationids=\"CR56\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Our selection is representative of the most abundant PKSs in the fungal kingdom by covering 73% of the known architectures (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePKSs selection for heterologous expression\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProduct (gene)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDomain architecture\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOrigin\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6MSA (\u003cem\u003eatX6MSA\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKS-AT-DH-KR-ACP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eA. terreus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYWA1 (\u003cem\u003epks12\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSAT-KS-AT-PT-ACP-TE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eF. acaciae-mearnsii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emonocillin II (\u003cem\u003epchI\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKS-AT-DH-ER-KR-ACP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eP. chamydosporia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003emonocillin II (\u003cem\u003epchE\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSAT-KS-AT-PT-ACP-TE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eP. chamydosporia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYNP1 (\u003cem\u003eadnE1\u003c/em\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKS-AT-DH-MT-ER-KR-ACP-C-A-ACP-Red\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eCordyceps sp.\u003c/em\u003e D1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e6-MSA has been produced in \u003cem\u003eS. cerevisiae\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e thus, we use it to benchmark our black yeast hosts. Given that black yeast are naturally proficient producers of melanins \u003csup\u003e\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, we reasoned that they must have a functional PPtase and abundant acetyl- and malonyl-CoA precursors to support polyketide production without further metabolic engineering \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. In contrast, \u003cem\u003eS. cerevisiae\u003c/em\u003e requires the incorporation of an active PPtase and increase of the acyl-coA pool to produce polyketides \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFor the heterologous expression of 6-MSA, we transformed our black yeast hosts with Cas9-sgRNA plasmids targeting the melanin biosynthesis gene \u003cem\u003epks1\u003c/em\u003e together with a linearized \u003cem\u003eatX6MSA\u003c/em\u003e expression cassette codon-optimized for \u003cem\u003eA. oryzae\u003c/em\u003e driven by the \u003cem\u003eA. nidulans\u003c/em\u003e TEF1 promoter and the trpC terminator. The \u003cem\u003eatX6MSA\u003c/em\u003e expression cassette flanked with 1.5 kilobases (Kb) homology arms was designed to replace \u003cem\u003epks1\u003c/em\u003e upon Cas9 induced DSB. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Our benchmarking strains were derived from \u003cem\u003eS. cerevisiae\u003c/em\u003e IMX581 Y40593 \u003csup\u003e62\u003c/sup\u003e: strain NPE9 carries a PPTase (\u003cem\u003enpgA\u003c/em\u003e) from \u003cem\u003eA. nidulans\u003c/em\u003e, and strain POP4 derives from NPE9 plus modifications to increase the acyl-coA, NADH and S-adenosyl methionine pools required for polyketide production (Supplementary table 10). These strains were transformed with a Cas9-sgRNA plasmid targeting the integration site ISX-2 \u003csup\u003e63\u003c/sup\u003e together with a PCR fragment harboring a \u003cem\u003eS. cerevisiae\u003c/em\u003e codon-optimized version of \u003cem\u003eatX6MSA\u003c/em\u003e flanked by 60bp DNA homologous arms for the ISX-2 integration site (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. Whole genome sequencing was carried out to confirm the integrity of the constructs and verify that only one copy of \u003cem\u003eatX6MSA\u003c/em\u003e was integrated into their genomes. Validated transformants were propagated in liquid YPD and 6-MSA production was confirmed by liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) (Supplementary Fig.\u0026nbsp;4). We quantified 6-MSA titer in terms of absolute concentrations, and concentrations relative to dry biomass for three biological replicates with independent integration events (Supplementary Fig.\u0026nbsp;5) and (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). From this experiment we concluded that \u003cem\u003eS. cerevisiae\u003c/em\u003e POP4 produced the highest absolute amount of 6-MSA (296 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u0026plusmn; 93 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) but required additional genetic modifications prior to its expression. In contrast, \u003cem\u003eK. petricola\u003c/em\u003e with only one modification to achieve NHEJ-deficiency yielded comparable amounts of 6-MSA (234 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u0026plusmn; 47 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), while \u003cem\u003eExophiala viscosa\u003c/em\u003e produced modest amounts of the product (67 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u0026plusmn; 6 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in JF 03-4F and 15 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e \u0026plusmn; 0.6 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in JF 03-3F). Overall, this experiment showed that our black yeast hosts are readily capable of producing a simple polyketide heterologously, confirming their endogenous PPTase is active on heterologous PKSs.\u003c/p\u003e \u003cp\u003eThe next polyketide that we produced was YWA1, an intermediate product in the biosynthesis of rubrofusarin in \u003cem\u003eF. graminearum\u003c/em\u003e which is encoded in the \u003cem\u003epks12\u003c/em\u003e gene \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. For heterologous expression in \u003cem\u003eS. cerevisiae\u003c/em\u003e, we identified an ortholog of \u003cem\u003epks12\u003c/em\u003e from \u003cem\u003eF. acaciae-mearnsii\u003c/em\u003e CBS10255, synthesized its predicted coding sequence and cloned it in a pESC-URA (Genscript\u0026reg;) shuttle vector. This plasmid was then used to transform the \u003cem\u003eS. cerevisiae\u003c/em\u003e strain POP4 (Supplementary Fig.\u0026nbsp;6A). For the heterologous expression of \u003cem\u003epks12\u003c/em\u003e in black yeasts, we transformed our strains with Cas9-sgRNA plasmids targeting \u003cem\u003epks1\u003c/em\u003e. In this case we used 60 bp homology arms in PCR fragments designed to integrate \u003cem\u003epks12\u003c/em\u003e under the control of the native \u003cem\u003epks1\u003c/em\u003e promoter and terminator (Supplementary Fig.\u0026nbsp;6B and 6C). For all strains, yellow-dark transformants were obtained (Supplementary Fig.\u0026nbsp;6D, 6E, and 6F) and validated by diagnostic PCR prior propagation in liquid YPD. The strains were cultivated and the organic extracts of their supernatants was analyzed using LC-HRMS. YWA1 was detected in all transformants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF and Supplementary Fig.\u0026nbsp;7). These results show that we can take advantage of the native \u003cem\u003epks1\u003c/em\u003e promoters and terminators to integrate a PKS gene lacking promoters and terminators elements.\u003c/p\u003e \u003cp\u003eAt this point, we had obtained products from non-reducing PKSs, the simplest domain architecture among fungal PKSs, and which heterologous expression is viable in \u003cem\u003eS. cerevisiae\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. To test if our black yeast offer any advantage over \u003cem\u003eS. cerevisiae\u003c/em\u003e as heterologous host, we tested the expression of more complex PKSs. These synthetases are typically produced in filamentous fungal hosts \u003csup\u003e\u003cspan additionalcitationids=\"CR66 CR67 CR68 CR69 CR70 CR71\" citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e, with a few exceptions that have been produced in \u003cem\u003eSaccharomyces\u003c/em\u003e \u003csup\u003e\u003cspan additionalcitationids=\"CR74 CR75\" citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. For this, we selected monocillin II, a resorcinol-lactone with nematocidal activity \u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. Monocillin II is biosynthesized by two PKSs, PchI a highly reducing PKS and PchE a non-reducing PKS \u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. For heterologous expression in \u003cem\u003eS. cerevisiae\u003c/em\u003e POP4, we used a Cas9-sgRNA plasmid targeting the ISX-2 and ISXII-1 integration sites \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e, together with two PCR products harboring intron-less sequences of \u003cem\u003epchI\u003c/em\u003e and \u003cem\u003epchE\u003c/em\u003e from \u003cem\u003eP. chlamydosporia\u003c/em\u003e MUCL9880 flanked by 60 base pairs (bp) homology arms (Supplementary Fig.\u0026nbsp;8). For heterologous production in black yeast, we used Cas9-sgRNA plasmids simultaneously targeting the \u003cem\u003epks1\u003c/em\u003e and \u003cem\u003ephs1\u003c/em\u003e genes together with repair templates harboring the intron-less sequences of \u003cem\u003epchI\u003c/em\u003e and \u003cem\u003epchE\u003c/em\u003e; \u003cem\u003epchI\u003c/em\u003e was flanked with 1 Kb homology arms to the promoter and terminators of \u003cem\u003epks1\u003c/em\u003e (Supplementary Fig.\u0026nbsp;9), and \u003cem\u003epchE\u003c/em\u003e placed under the \u003cem\u003eA. nidulans\u003c/em\u003e TEF1 promoter and the trpC terminator with 1.5 Kb homology arms for \u003cem\u003ephs1\u003c/em\u003e (Supplementary Fig.\u0026nbsp;10). Monocillin II was found in the supernatants of the cultures of our black yeast constructs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) and (Supplementary Fig.\u0026nbsp;11). While \u003cem\u003eS. cerevisiae\u003c/em\u003e POP4 yielded traces of the product (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) and (Supplementary Fig.\u0026nbsp;12).\u003c/p\u003e \u003cp\u003eOverall, the production of 6-MSA, YWA1, and monocillin II demonstrated that our black yeasts are proficient in heterologous PKS production from distantly related species. Additionally, they are compatible with \u003cem\u003eAspergillus\u003c/em\u003e-derived promoters and terminators, allow for efficient simultaneous gene integration, and support the use of short homology arms. More importantly, these experiments highlight the potential of \u003cem\u003eE. viscosa\u003c/em\u003e JF 03-3F, JF 03-4F, and \u003cem\u003eK. petricola\u003c/em\u003e to produce complex PKSs using standardized methods, minimizing time-consuming troubleshooting steps.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHeterologous expression in black yeasts lead to the discovery and pathway elucidation of a new 2-pyridone natural product\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAfter developing and benchmarking our black yeast hosts, we showcased their usefulness in the discovery and elucidation of new biosynthetic pathways. For this proof-of-concept experiment, we chose to express a previously undescribed BGC from \u003cem\u003eCordyceps sp.\u003c/em\u003e D1. We predicted that this BGC would produce a 2-pyridone natural product, but we could not detect it using cultivation-based approaches when applied to \u003cem\u003eCordyceps sp.\u003c/em\u003e D1 (unpublished results). To define the minimum set of genes for heterologous expression we used a phylogenomics approach \u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). We selected three genes: \u003cem\u003eadnE1\u003c/em\u003e a hybrid, highly reducing PKS-NRPS with a domain organization that is characteristic of pyridine producing pathways \u003csup\u003e\u003cspan additionalcitationids=\"CR66 CR67 CR68 CR69 CR70 CR71\" citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e, \u003cem\u003eadnE2\u003c/em\u003e a cytochrome p450 (CYP450), and \u003cem\u003eadnE3\u003c/em\u003e a trans-enoyl reductase (trans-ER) fused to a CYP450. \u003cem\u003eAdnE1\u003c/em\u003e, a\u003cem\u003ednE2\u003c/em\u003e and a\u003cem\u003ednE3\u003c/em\u003e were integrated into the genome of all strains (Supplementary Figs.\u0026nbsp;13 and 14). The LC-HRMS analysis of extracellular fractions of all black yeast transformants revealed a peak with high intensity and m/z 438.1916, which corresponds to the calculated formula: [C\u003csub\u003e25\u003c/sub\u003eH\u003csub\u003e28\u003c/sub\u003eNO\u003csub\u003e6\u003c/sub\u003e]\u003csup\u003e+\u003c/sup\u003e Δ\u0026thinsp;+\u0026thinsp;1.141 ppm, while \u003cem\u003eS. cerevisiae\u003c/em\u003e POP4 did not yield any products (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In addition, \u003cem\u003eE. viscosa\u003c/em\u003e JF 03-3F and JF 03-4F, and \u003cem\u003eK. petricola\u003c/em\u003e transformants displayed a visible yellow phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), whereas \u003cem\u003eS. cerevisiae\u003c/em\u003e POP4 did not. Mass to formula calculation and the mass fragmentation analysis of the ion was consistent with a PKS-derived 2-pyridone product (Supplementary Fig.\u0026nbsp;15). To elucidate the structure of the product, we cultivated \u003cem\u003eE. viscosa\u003c/em\u003e JF 03-4F expressing \u003cem\u003eadnE1\u003c/em\u003e, \u003cem\u003eadnE2\u003c/em\u003e and \u003cem\u003eadnE3\u003c/em\u003e in YPD, YPX, MMX, and MMBW media for 7 days. We then extracted the resulting supernatants with ethyl acetate, and after solvent evaporation, we obtained approximately 88 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (YPD), 80 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (YPX), and 12 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (MMX) of an amorphous solid composed mostly of our product, whilst 204 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of an amorphous solid composed mostly of other products was obtained in MMBW (Supplementary Fig.\u0026nbsp;16). The 80 mg of product derived from YPX was further purified using semi preparative HPLC until we obtained 10 mg of material which was then analyzed with Nuclear Magnetic Resonance (NMR). Structural elucidation using \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003eH NMR, COSY, HSQC, HMBC confirmed that the product is 17-Hydroxyfarinosone, a new member of the farinosones \u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e which we named YNP1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) (Supplementary Figs.\u0026nbsp;17\u0026ndash;21).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eYNP1 did not show any fully saturated C-C bonds in the acyl chain as expected in PKS-derived pyridones with an active enoyl reductase domain \u003csup\u003e\u003cspan additionalcitationids=\"CR67 CR68 CR69 CR70 CR71 CR72\" citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. We hypothesized that the gene fusion between the trans-ER and CYP450 in \u003cem\u003eadnE3\u003c/em\u003e might have inactivated the trans-ER. To test our hypothesis, we revisited the gene-calling of \u003cem\u003eadnE3\u003c/em\u003e and inspected its coding sequence. After a BLASTP search and comparison against other trans-ERs sequences, we identified misannotations at the C-terminal region of the trans-ER domain and the N-terminal of the CYP450 domain (Supplementary Fig.\u0026nbsp;22). These findings suggested that the trans-ER and CYP450 in \u003cem\u003eadnE3\u003c/em\u003e are in fact two individual genes artifactually fused. To validate the revisited annotation, we transformed the \u003cem\u003eE. viscosa\u003c/em\u003e JF 03-4F YNP1 producer strain with a Cas9-sgRNA plasmid targeting the connecting DNA sequence between the trans-ER and CYP450 together with PCR products containing the \u003cem\u003eA. nidulans\u003c/em\u003e trpC terminator, AO0583, an strong constitutive promoter \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, and the missing DNA sequence of one, non, or both trans-ER and CYP450 flanked with 500 bp homology to the \u003cem\u003eadnE3\u003c/em\u003e coding sequence (Supplementary Fig.\u0026nbsp;23). Phenotypic changes from yellow to intense orange were observed in transformants where the missing trans-ER sequence was provided. On the other hand, complementation with the missing CYP450 sequence did not show obvious phenotypic changes. This suggested that the complementation with the missing trans-ER sequence restored the function of the trans-ER (Supplementary Fig.\u0026nbsp;24). After strain\u0026rsquo;s validation by diagnostic PCR and LC-HRMS analysis, peaks with the highest abundance \u003cem\u003em/z\u003c/em\u003e 422.1963 and \u003cem\u003em/z\u003c/em\u003e 406.2015 were observed in the metabolic profiles of the strains where the missing trans-ER sequence was introduced. The peak corresponding to \u003cem\u003em/z\u003c/em\u003e 422.1963 matched the formula and fragmentation pattern of farinosone B \u003csup\u003e\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e\u003c/sup\u003e, a polyketide-derived 2-pyridone bearing a fully saturated C-C bond in the acyl chain. This result confirmed the functional restoration of the trans-ER activity and the presence of a fourth gene \u003cem\u003eadnE4\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) and (Supplementary Fig.\u0026nbsp;24). Based on our data, we concluded that farinosone B is the final product of the pathway encoded in \u003cem\u003eadnE1-2-3-4\u003c/em\u003e together with other minor products including YNP1 and farinosone A (\u003cem\u003em/z\u003c/em\u003e 406.2015, calculated formula [C\u003csub\u003e25\u003c/sub\u003eH\u003csub\u003e28\u003c/sub\u003eNO\u003csub\u003e4\u003c/sub\u003e]\u003csup\u003e+\u003c/sup\u003e Δ\u0026thinsp;+\u0026thinsp;0.73 ppm) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) and (Supplementary Fig.\u0026nbsp;24). Thus, we propose a common biosynthetic pathway for YNP1, farinosone A and B (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Overall, this work demonstrates the potential of black yeast as heterologous hosts for the discovery of new polyketide natural products and highlights their potential for their sustainable production.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eE. viscosa\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eand\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;K. petricola\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eare convenient heterologous hosts of fungal PKSs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYeast species are convenient hosts for heterologous expression of PKSs, as they are easier to manipulate and have simple metabolic backgrounds compared to filamentous fungi. In this work, we demonstrated that \u003cem\u003eE. viscosa\u003c/em\u003e JF 03-3F, \u003cem\u003eE. viscosa\u003c/em\u003e JF 03-4F\u003cem\u003e,\u0026nbsp;\u003c/em\u003eand \u003cem\u003eK. petricola\u003c/em\u003e grow as yeasts, although \u003cem\u003eK. petricola\u003c/em\u003e tends to form more clumps during cultivation. We also showed that these black yeastshave cleaner background metabolic profiles than \u003cem\u003eAspergillus\u003c/em\u003e hosts. This is advantageous because the heterologous products are easier to purify, and they are less likely to be modified by the host's metabolism. We also showed that \u003cem\u003eE. viscosa\u003c/em\u003e and \u003cem\u003eK. petricola\u003c/em\u003e can grow and make polyketides on xylose and in beechwood hydrolysates (Supplementary figure 16). In contrast \u003cem\u003eS. cerevisiae,\u0026nbsp;\u003c/em\u003erequires edible sugars as substrates. This opens the possibility to use sustainable feedstocks as substrates for the sustainable production of polyketide products.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlack yeast hosts overcome\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eS. cerevisiae\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;limitations in polyketide production\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur results show that black yeast hosts are more competent for PKS production than \u003cem\u003eS. cerevisiae\u003c/em\u003e. Throughout our heterologous expression experiments we benchmarked the performance of our new yeast hosts by comparing them with an engineered strain of \u003cem\u003eS. cerevisiae\u0026nbsp;\u003c/em\u003ePOP4 capable of high titer production of 6-MSA and robust YWA1 production. However, the same \u003cem\u003eS. cerevisiae\u003c/em\u003e strain was not able to produce detectable amounts of monocillin II and farinosone A, B or YNP1. In both cases, we sequenced the whole genome of \u003cem\u003eS. cerevisiae\u0026nbsp;\u003c/em\u003estrains and confirmed the expected genotypes\u003cem\u003e,\u0026nbsp;\u003c/em\u003eexpression of the PKSsby transcriptome analysis and the presence of the corresponding proteins by proteomics (Supplementary Figure 25-26).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProduction of monocillin II and lovastatin has already been reported in \u003cem\u003eS. cerevisiae\u003c/em\u003e using 2μ high copy number plasmids\u003csup\u003e74,75,81,82\u003c/sup\u003e. In contrast, our strains for polyketide production in \u003cem\u003eS. cerevisiae\u003c/em\u003e and black yeast were single-copy integrations which are stable and comparable. Nevertheless, since single-copy integration of PKS genes may limit the ability of \u003cem\u003eS. cerevisiae\u0026nbsp;\u003c/em\u003eto produce polyketides, we attempted expression of the PKS-NRPS encoded in \u003cem\u003ea\u003c/em\u003e\u003cem\u003ednE1\u003c/em\u003e in \u003cem\u003eS. cerevisiae\u0026nbsp;\u003c/em\u003ePOP4 in a high copy number plasmid. However, the expected product, a five-membered ring intermediate\u0026nbsp;\u003csup\u003e66\u003c/sup\u003e was not obtained (Supplementary figure 27). \u003cem\u003eS. cerevisiae\u003c/em\u003e BJ-5464, a strain with low endoproteolytic activity, is commonly used for PKS expression\u0026nbsp;\u003csup\u003e83\u003c/sup\u003e, as it is expected that this feature enhances PKS expression\u0026nbsp;\u003csup\u003e84,85\u003c/sup\u003e. However, when we introduced a high copy number plasmid containing \u003cem\u003eadnE1\u003c/em\u003e into \u003cem\u003eS. cerevisiae\u0026nbsp;\u003c/em\u003eBJ-5464-NpgA we could not observe the expected product (Supplementary figure 28). Our troubleshooting efforts suggest that the bottleneck for production of farinosone A, B, YNP1 and monocillin II in \u003cem\u003eS. cerevisiae\u003c/em\u003e is probably a post-translational event, \u003cem\u003ee. g.\u003c/em\u003e\u0026nbsp; misfolding, aggregation or toxicity of the proteins and/or intermediates in the pathway. Furthermore, farinosone B production in \u003cem\u003eS. cerevisiae\u003c/em\u003e may require additional CYP450 reductases and/or cytochrome B5 proteins for proper function of CYP450 enzymes not encoded on its genome\u0026nbsp;\u003csup\u003e86\u003c/sup\u003e. The fact that such modifications were not required in \u003cem\u003eE. viscosa\u003c/em\u003e JF 03-3F and JF 03-4F\u003cem\u003e,\u0026nbsp;\u003c/em\u003eand \u003cem\u003eK. petricola\u003c/em\u003e for farinosone A, B and YNP1 showcases their metabolic robustness.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOverall, our work establishes black yeasts as heterologous hosts for the systematic expression of complex fungal PKSs, providing a streamlined workflow that can be applied to the systematic discovery of fungal polyketides and their enzymes, which we aim to fully exploit in sustainable biomanufacturing.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eStrains and media\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe complete list of strains can be found in (Supplementary table 10). All strainswere propagated in liquid or solid (additional 2% (w/v) agar) YPD media (1% (w/v) yeast extract, 2% (w/v) Bacto™ peptone, 2% (w/v) glucose). When required, \u003cem\u003eE. viscosa \u003c/em\u003eand \u003cem\u003eK. petricola \u003c/em\u003ewere propagated on YPX media (1% (w/v) yeast extract, 2% (w/v) Bacto™ peptone, 2% (w/v) xylose), or ME media \u003csup\u003e35\u003c/sup\u003e (2% malt extract (w/v), 0.1% (w/v) Bacto™ peptone, 2% (w/v) glucose) or minimal media with beechwood hydrolysate (MMBW) (1x nitrate salt solution \u003csup\u003e87\u003c/sup\u003e, 0.001% thiamine w/v, 1x trace metal solution \u003csup\u003e88\u003c/sup\u003e) supplemented with beechwood hydrolysate (1% (w/v)) as carbon source \u003csup\u003e32\u003c/sup\u003e, or minimal media with xylose (MMX) supplemented with beechwood hydrolysate (1% (w/v)) as carbon source when required. For determination of Hygromycin B lethal concentrations, first-time transformation and construction of NHEJ-deficient strains of \u003cem\u003eE. viscosa \u003c/em\u003eand \u003cem\u003eK. petricola\u003c/em\u003e, transformants were selected in Transformation Media (TM) (1x nitrate salt solution \u003csup\u003e87\u003c/sup\u003e, 0.001% Thiamine w/v, 1x trace metal solution \u003csup\u003e88\u003c/sup\u003e) supplemented with 1M sucrose and 50 µg mL\u003csup\u003e-1\u003c/sup\u003e of Hygromycin B (Gibco™) for \u003cem\u003eE. viscosa\u003c/em\u003e, or 25 µg mL\u003csup\u003e-1\u003c/sup\u003e for \u003cem\u003eK. petricola\u003c/em\u003e, and grown for 21 days. For heterologous expression experiments, transformants were selected in malt extract sucrose media \u003csup\u003e35\u003c/sup\u003e (MEAS) (2% (w/v) malt extract, 0.1% (w/v) peptone, 11% (w/v) sucrose) and Hygromycin B, and grown for 14 days. For \u003cem\u003eS. cerevisiae,\u003c/em\u003e selection of transformants or plasmid maintenance was done using liquid or solid (media with additional 2% (w/v) agar) synthetic drop-out media (SD-Ura) (0.67% (w/v) yeast nitrogen base (Sigma-Aldrich®) without amino acids, 0.14% (w/v) yeast synthetic dropout medium supplements lacking uracil, 2% (w/v) glucose). Uracil was supplemented when required (0.0076% (w/v)). Counter-selection of plasmids in \u003cem\u003eS. cerevisiae \u003c/em\u003ewas carried out using SD media supplemented with uracil and 0.074% (w/v) 5-fluoroorotic acid (5-FOA) (Sigma®). \u003cem\u003eS. cerevisiae \u003c/em\u003ewas propagated at 30°C, \u003cem\u003eE. viscosa \u003c/em\u003eand \u003cem\u003eK. petricola \u003c/em\u003eat 25°C. \u003cem\u003eE. coli\u003c/em\u003e DH5α was used for cloning and plasmid propagation. Liquid or solid (2% (w/v) agar) Lysogeny broth (LB) supplemented with 100 µg mL\u003csup\u003e-1\u003c/sup\u003e of ampicillin was used for \u003cem\u003eE. coli \u003c/em\u003ecultivation at 37°C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCRs and plasmids construction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor USER cloning \u003csup\u003e89\u003c/sup\u003e, all PCR reactions were performed using Phusion U Hot Start DNA Polymerase (Thermo Scientific™) according to the manufacturer's instructions. For \u003cem\u003ein-vivo \u003c/em\u003eassemblies in \u003cem\u003eS. cerevisiae \u003c/em\u003eand construction of DNA repair templates provided during transformations, PCR reactions were performed using Phusion™ Hot Start II DNA Polymerase (Thermo Scientific™) according to the manufacturer's instructions. All PCR fragments were gel or column-purified using NucleoSpin™ Gel and PCR Clean-up kit (Macherey-Nagel®) prior to USER cloning, or \u003cem\u003ein-vivo \u003c/em\u003eassemblies, or transformations. All constructed plasmids and PCR amplifications were sequenced using long-read sequencing by Oxford nanopore technologies™ (Plasmidsaurs®) when required. For PCR reactions derived from gDNA, fresh biomass from plates was harvested using a 10uL loop and gDNA was extracted according to \u003csup\u003e42\u003c/sup\u003e. For diagnostic PCR, cell lysis was carried out using Platinum™ Direct PCR Universal Master Mix (Thermo Scientific™) cell lysis protocol, and PCR was performed using Phire™ Hot Start II DNA Polymerase (Thermo Scientific™) according to the manufacturer's instructions. All primers and ssDNA oligos used in this work are listed in (Supplementary table 11) and were obtained from Integrated DNA Technologies® (IDT). All PCR, cloning reactions, and constructed plasmids are listed in (Supplementary Tables 12-14). Cas9-sgRNA plasmids were constructed as per \u003csup\u003e51\u003c/sup\u003e, and protospacer sequences used in this work can be seen in (Supplementary table 15). \u003c/p\u003e\n\u003cp\u003eAll plasmids were purified using a NucleoSpin™ plasmid DNA purification kit (Macherey-Nagel®). When purifying plasmids from \u003cem\u003eS. cerevisiae\u003c/em\u003e, the Zymoprep™ Yeast Plasmid Miniprep I (ZYMO RESEARCH®) was used. Plasmids containing DNA repair templates were linearized by enzymatic digestion with NotI or SmiI (Thermo Scientific™), and gel-purified prior to use in transformations.For heterologous expression of 6-MSA, the amino acid sequence of \u003cem\u003eA. terreus\u003c/em\u003e 6MSA synthase \u003csup\u003e54\u003c/sup\u003e was retrieved from the minimum information about a biosynthetic gene cluster database \u003csup\u003e90\u003c/sup\u003e (MIBiG) accession number BGC0001276. The amino acid sequence was used for the DNA synthesis of \u003cem\u003eS. cerevisiae \u003c/em\u003eand \u003cem\u003eA. oryzae \u003c/em\u003ecodon-optimized versions of an intron-less \u003cem\u003eatX6MSA\u003c/em\u003e cloned into a pESC-URA 2μ plasmids (GenScript®). For \u003cem\u003eS. cerevisiae\u003c/em\u003e,\u003cem\u003e atX6MSA\u003c/em\u003e was under the control of TEF1 promoter and ADH1 terminator, and for \u003cem\u003eE. viscosa \u003c/em\u003eand \u003cem\u003eK. petricola\u003c/em\u003e, under the control of \u003cem\u003eA. nidulans \u003c/em\u003eTEF1 promoter and trpC terminator. The \u003cem\u003eS. cerevisiae \u003c/em\u003ecodon-optimized version of \u003cem\u003eatX6MSA\u003c/em\u003e was PCR-amplified with primers PR_YNP171 and PR_YNP172 (PCR62) flanked with 60bp DNA homologous sequences to the integration site ISX-2 \u003csup\u003e63\u003c/sup\u003e and column-purified prior its transformation into \u003cem\u003eS. cerevisiae \u003c/em\u003estrains NPE9 and POP4 (Supplementary figure 29). \u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eA. oryzae \u003c/em\u003ecodon-optimized version of \u003cem\u003eatX6MSA\u003c/em\u003e was PCR-amplified with primers PR_YNP204 and PR_YNP205 (PCR77) including PacI/Nt.BbvcI tails to re-constitute them once the PCR product was USER cloned into plasmid pAC125 \u003csup\u003e91\u003c/sup\u003e. The resultant plasmid pYNP59 was prepared for another round of USER cloning to be inserted with approx. 1.5 Kb of the upstream and downstream DNA sequences homologous to the \u003cem\u003epks1 \u003c/em\u003elocus from \u003cem\u003eE. viscosa\u003c/em\u003e JF 03-3F and JF 03-4F, and \u003cem\u003eK. petricola \u003c/em\u003ewhich were PCR-amplified using primer pairs PR_YNP125-PR_YNP126 (PCR46), PR_YNP127-PR_YNP128 (PCR47), PR_YNP121-PR_YNP122 (PCR44), PR_YNP123-PR_YNP124 (PCR45), PR_YNP129-PR_YNP130 (PCR48), PR_YNP131-PR_YNP132 (PCR49). The resultant plasmids pYNP70 (DONR_EVJ4_ATX6MSAORYOPT_PKS1), pYNP71 (DONR_EVJ3_ATX6MSAORYOPT_PKS1), and pYNP72 (DONR_KP_ATX6MSAORYOPT_PKS1) were linearized with NotI and gel-purified prior to transformation into their respective hosts (Supplementary figure 30). \u003c/p\u003e\n\u003cp\u003eFor heterologous expression of YWA1 \u003csup\u003e64\u003c/sup\u003e, the intron-less coding sequence of \u003cem\u003epks12\u003c/em\u003e was retrieved from an in-house isolate of \u003cem\u003eF. acasiae-mearnsii \u003c/em\u003estrain CBS10255 and synthesized and cloned into a pESC-URA 2μ high copy plasmid (GenScript®) under the control of the TEF1 promoter and CYC1 terminator. The resultant plasmid pYNP100 was used to transform \u003cem\u003eS. cerevisiae \u003c/em\u003ePOP4. For black yeasts\u003cem\u003e,\u003c/em\u003e primer pairs PR_YNP471-PR_YNP452 (PCR196 for \u003cem\u003eE. viscosa\u003c/em\u003e) and PR_YNP472-PR_YNP456 (PCR197 for \u003cem\u003eK. petricola\u003c/em\u003e) flanked with 60bp DNA sequences homologous to the \u003cem\u003epks1 \u003c/em\u003epromoter and terminator were used to amplify \u003cem\u003epks12\u003c/em\u003e from plasmid pYNP100. The obtained PCR products were column-purified prior to transformation into their respective hosts (Supplementary figure 6). \u003c/p\u003e\n\u003cp\u003eFor the heterologous expression of the uncharacterized BGC from \u003cem\u003eCordyceps sp. \u003c/em\u003eD1, the intron-less coding sequences of the PKS-NRPS synthetase (\u003cem\u003eadnE1\u003c/em\u003e)were PCR-amplified from \u003cem\u003eCordyceps sp. \u003c/em\u003eD1\u003cem\u003e.\u003c/em\u003e gDNA with primer pairs flanked with 50bp complementary homology PR_YNP284-PR_YNP285 (PCR117), PR_YNP286-PR_YNP289 (PCR114), PR_YNP290-PR_YNP293 (PCR115), and assembled into a 2μ high copy plasmid backbone PCR-amplified with primer pair PR_YNP294-PR_YNP295 (PCR122). PCR fragments were \u003cem\u003ein-vivo\u003c/em\u003e assembled in \u003cem\u003eS. cerevisiae \u003c/em\u003ePOP4. The resultant plasmid pYNP78 expresses \u003cem\u003eadnE1\u003c/em\u003e synthetase under the control of TEF1 promoter and CYC1 terminator (Supplementary figure 13). The intron-less coding sequences of the CP450 (\u003cem\u003eadnE2)\u003c/em\u003e and TransER-CP450 (\u003cem\u003eadnE3)\u003c/em\u003e coding sequences were retrieved from \u003cem\u003eCordyceps sp. \u003c/em\u003eD1 and synthesized and cloned into pUC57 plasmid backbones (GenScript®). For \u003cem\u003eE. viscosa \u003c/em\u003eand \u003cem\u003eK. petricola\u003c/em\u003e, \u003cem\u003eadnE1\u003c/em\u003e was PCR-amplified from plasmid pYNP78 using primer pair PR_YNP317-PR_YNP318 (PCR129) which also reconstitutes PacI/Nt.BbvcI USER cloning cassettes after cloning. Subsequently, the PCR fragment was USER cloned into plasmid pAC125, resulting in plasmid pYNP83 (Supplementary figure 14A). Thereafter, approx. 1 Kb of the upstream and downstream DNA sequences flanking \u003cem\u003epks1\u003c/em\u003e from \u003cem\u003eE. viscosa \u003c/em\u003eJF 03-3F andJF 03-4F,and \u003cem\u003eK. petricola \u003c/em\u003ewere PCR-amplified from gDNA with primer pairs PR_YNP319-PR_YNP320 (PCR130), PR_YNP321-PR_YNP322 (PCR131), PR_YNP323-PR_YNP324 (PCR132), PR_YNP325-PR_YNP326 (PCR133), PR_YNP327-PR_YNP328 (PCR134), PR_YNP329-PR_YNP330 (PCR135), and USER cloned into pYNP83, resulting in plasmids pYNP84 (DONR_EVJ4_adnE1_PKS1), pYNP85 (DONR_EVJ3_adnE1_PKS1), and pYNP86 (DONR_KP_adnE1_PKS1) expressing \u003cem\u003eadnE1 \u003c/em\u003eunder the control of pks1 promoter and terminator. Plasmids pYNP84, pYNP85, and pYNP86 were linearized with SmiI (Thermo Scientific™), and gel-purified prior to use for transformations into their respective hosts (Supplementary figure 14C and 14E). \u003c/p\u003e\n\u003cp\u003e\u003cem\u003eadnE2\u003c/em\u003e and \u003cem\u003eadnE3\u003c/em\u003e were PCR-amplified from pUC57 plasmids with primer pairs PR_YNP307-PR_YNP308 (PCR116) and PR_YNP281-PR_YNP282 (PCR109), respectively. \u003cem\u003eA. nidulans \u003c/em\u003epromoters gpdA and TEF1 were PCR-amplified from plasmid pDIV652 with primer pair PR_YNP306-PR_YNP296 (PCR110). All PCR products were cloned into plasmid pAC125, which also reconstituted PacI/Nt.BbvcI cassettes after cloning, resulting in plasmid pYNP79 which expresses the \u003cem\u003eadnE2\u003c/em\u003e and \u003cem\u003eadnE3 \u003c/em\u003eunder \u003cem\u003eA. nidulans \u003c/em\u003eTEF1 and gpdA promoters, and argB and pkiA terminators, respectively (Supplementary figure 14B). Thereafter, approx. 1.5 Kb of the upstream and downstream DNA sequences flanking \u003cem\u003ephs1\u003c/em\u003e from \u003cem\u003eE. viscosa \u003c/em\u003eand \u003cem\u003eK. petricola \u003c/em\u003ewere PCR-amplified from gDNA with primer pairs PR_YNP253-PR_YNP254 (PCR103), PR_YNP255-PR_YNP256 (PCR104), PR_YNP259-PR_YNP260 (PCR105), PR_YNP261-PR_YNP262 (PCR106), PR_YNP263-PR_YNP264 (PCR107), PR_YNP265-PR_YNP266 (PCR108). PCR products were cloned into plasmid pYNP79, resulting in plasmids pYNP80 (DONR_EVJ3_adnE_2_3_PHS1), pYNP81 (DONR_EVJ4_adnE_2_3_PHS1), and pYNP82 (DONR_KP_adnE_2_3_PHS1). Plasmids pYNP80, pYNP81, and pYNP82 were linearized with NotI (Thermo Scientific™), and gel-purified prior to transformation into their respective hosts (Supplementary figure 14D and 14E). \u003c/p\u003e\n\u003cp\u003eFor \u003cem\u003eS. cerevisiae \u003c/em\u003ePOP4, \u003cem\u003eadnE2\u003c/em\u003e and \u003cem\u003eadnE3\u003c/em\u003e were PCR-amplified from plasmid pYNP79 with primer pairs PR_YNP346-PR_YNP347 (PCR144) and PR_YNP344-PR_YNP345 (PCR143), respectively. \u003cem\u003eS. cerevisiae \u003c/em\u003epromoters TDH3 and PGK1 were PCR-amplified from plasmid pCFB2909-BIK with primer pair PR_YNP348-PR_YNP349 (PCR145). All PCR products were cloned into plasmid backbone pCFB2909-BIK PCR-amplified with primer pair PR_YNP342-PR_YNP343 (PCR142). The resultant plasmid pYNP88 expresses \u003cem\u003eadnE2\u003c/em\u003e under the control of PGK1 promoter and ADH1 terminator, and \u003cem\u003eadnE3\u003c/em\u003e under control of TDH3 and TEF1 terminator. \u003cem\u003eadnE1\u003c/em\u003e was PCR-amplified from pYNP78 using primer pair PR_YNP171-PR_YNP297 (PCR146) with 60bp homology to integration site ISX-2 \u003csup\u003e63\u003c/sup\u003e, and \u003cem\u003eadnE2\u003c/em\u003e and \u003cem\u003eadnE3\u003c/em\u003e were PCR-amplified from pYNP88 using primer pair PR_YNP358-PR_YNP359 (PCR147) with 60bp homology to integration site ISXII-1 \u003csup\u003e63\u003c/sup\u003e. The PCR products were column-purified prior to transformation into \u003cem\u003eS. cerevisiae \u003c/em\u003ePOP4 (Supplementary figure 13).\u003c/p\u003e\n\u003cp\u003eFor heterologous expression of monocillin II, the intron-less coding sequences of monocillin II \u003cem\u003epchI \u003c/em\u003eand \u003cem\u003epchE\u003c/em\u003e were PCR-amplified from \u003cem\u003eP. chlamydosporia \u003c/em\u003estrain MUCL9880 using primers PR_YNP394-PR_YNP395 (PCR168), PR_YNP396-PR_YNP397 (PCR169), and PR_YNP398-PR_YNP399 (PCR170) flanked with 50bp complementary homology. Thereafter, the backbone of plasmid pYNP78 was PCR-amplified using PR_YNP400-PR_YNP401 (PCR171) and PR_YNP400-PR_YNP402 (PCR183) flanked with 60bp homology to \u003cem\u003epchI \u003c/em\u003eor \u003cem\u003epchE\u003c/em\u003e. Thereafter,PCR products were assembled \u003cem\u003ein-vivo\u003c/em\u003e in \u003cem\u003eS. cerevisiae \u003c/em\u003ePOP4, resulting in plasmids pYNP98 and pYNP99 expressing \u003cem\u003epchI \u003c/em\u003eand \u003cem\u003epchE\u003c/em\u003e, under the control of \u003cem\u003eS. cerevisiae \u003c/em\u003eTEF1 promoter and CYC1 terminator. \u003cem\u003ePchI \u003c/em\u003ewas PCR-amplified from plasmid pYNP98 using primer pair PR_YNP480-PR_YNP481 (PCR184) with 60bp homology to integration site ISX-2 \u003csup\u003e63\u003c/sup\u003e, and \u003cem\u003epchE \u003c/em\u003ewas PCR-amplified from pYNP99 using primer pair PR_YNP482-PR_YNP483 (PCR185) with 60bp homology to integration site ISXII-1 \u003csup\u003e63\u003c/sup\u003e. The PCR products were column-purified prior to transformation into \u003cem\u003eS. cerevisiae \u003c/em\u003ePOP4 (Supplementary figure 8). \u003c/p\u003e\n\u003cp\u003eFor \u003cem\u003eE. viscosa \u003c/em\u003eand \u003cem\u003eK. petricola\u003c/em\u003e, \u003cem\u003epchI \u003c/em\u003eand \u003cem\u003epchE \u003c/em\u003ewere PCR-amplified from plasmids pYNP98 and pYNP99 with primers PR_YNP447-PR_YNP448 (PCR194) and PR_YNP467-PR_YNP468 (PCR203), respectively. \u003cem\u003eA. nidulans \u003c/em\u003eTEF1 promoter and trpC terminator were PCR-amplified from plasmid pYNP59 with primer pairs PR_YNP465-PR_YNP466 (PCR202) and PR_YNP469-PR_YNP470 (PCR204), respectively. Backbones from plasmids pYNP80, pYNP81, pYNP82, pYNP84, pYNP85, and pYNP86, were PCR-amplified with primer pairs PR_YNP437-PR_YNP438 (PCR189), PR_YNP435-PR_YNP436 (PCR188), PR_YNP441-PR_YNP442 (PCR191), PR_YNP433-PR_YNP434 (PCR186 and PCR187), and PR_YNP439-PR_YNP440 (PCR190). \u003cem\u003ePchI \u003c/em\u003ePCR product was cloned into pYNP84-85-86 open plasmid backbones. Then, \u003cem\u003epchE \u003c/em\u003ePCR product, \u003cem\u003eA. nidulans \u003c/em\u003eTEF1 promoter, and trpC terminator were cloned into pYNP80-81-82 open plasmid backbones. The resultant plasmids pYNP102 (DONR_EVJ4_pchI_PKS1), pYNP103 (DONR_EVJ3_pchI_PKS1), and pYNP106 (DONR_KP_pchI_PKS1) express \u003cem\u003epchI \u003c/em\u003e under the control of pks1 promoter and terminator (Supplementary figure 9), while the resultant plasmids pYNP104 (DONR_EVJ4_pchE_PHS1), pYNP105 (DONR_EVJ3_pchE_PHS1), and pYNP107 (DONR_KP_pchE_PHS1) express \u003cem\u003epchE \u003c/em\u003eunder the control of \u003cem\u003eA. nidulans\u003c/em\u003e TEF1 promoter and trpC terminator (Supplementary figure 10). Plasmids pYNP102, pYNP103, pYNP104, pYNP105, pYNP106, and pYNP107 were linearized with NotI (Thermo Scientific™), and gel-purified prior use in transformations. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransformation and strain construction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlack yeastswere inoculated in 50 mL of YPD (\u003cem\u003eE. viscosa\u003c/em\u003e) or MEB \u003csup\u003e35\u003c/sup\u003e (\u003cem\u003eK. petricola\u003c/em\u003e) in 250 mL shake flasks and incubated for 5 days at 200 RPM and 25°C.\u003cem\u003e E. viscosa \u003c/em\u003ecultures were diluted to OD\u003csub\u003e600\u003c/sub\u003e 0.1 in 100 mL of YPD in 500 mL shake flasks and continued incubation for 3 days. For \u003cem\u003eK. petricola\u003c/em\u003e, 1 mL of the seed semi-dispersed culture was transferred to 100 mL of ME in a 500 mL shake flask and continued incubation for 4 days. Strains were protoplast according to \u003csup\u003e40–42\u003c/sup\u003e with a few modifications. Briefly, the cultures were transferred to 50 mL Falcon tubes by filtering them through a sterile funnel with miracloth paper to remove non-dispersed cells. The filtered cultures were centrifuged at 3500 G for five minutes and the supernatants were discarded. The cell pellets were washed two times in 25 mL APB solution and centrifuged at 3500 G. The washed cell pellets were resuspended in a 0.45 μm sterile-filtered APB solution containing either Vinotaste-pro™ (Novonesis™) or Extralyse™ (Laffort™) cell wall digestive enzymes in a concentration of 100 mg mL\u003csup\u003e-1 \u003c/sup\u003ein a final volume of 40 mL. The Falcon tubes containing the cell resuspensions were horizontally placed into a shaking incubator and incubated at 30°C and 150 RPM shaking for 3-4 h. After incubations, cell resuspensions were transferred to new 50 mL Falcon tubes by filtering them through a sterile funnel with a miracloth paper to remove cell debri, APB was added to the filtered resuspensions to a final volume of 40 mL. The cell resuspensions were overlaid with 5 mL of ATB and MQ water solution in 1::1 ratio. The Falcon tubes were centrifuged at 3000 G (acc. 9, deacc. 5) for eleven minutes. The obtained phase of protoplasts was transferred to new 50 mL Falcon tubes and ATB was added to a final volume of 40 mL, gently mixed, and centrifuged at 3000 G (acc. 9, deacc. 9) for eleven minutes. The supernatant was discarded, and the obtained protoplasts were gently resuspended in 1 mL of ATB prior dilutions to desired concentrations and kept on ice. For transformations, 50 μl (\u003cem\u003eE. viscosa\u003c/em\u003e) or 100 μl (\u003cem\u003eK. petricola\u003c/em\u003e) of protoplasts in a concentration of 10\u003csup\u003e7\u003c/sup\u003e mL\u003csup\u003e-1\u003c/sup\u003e (\u003cem\u003eE. viscosa\u003c/em\u003e) 10\u003csup\u003e8\u003c/sup\u003e mL\u003csup\u003e-1\u003c/sup\u003e (\u003cem\u003eK. petricola\u003c/em\u003e) were mix with 1 μg of Cas9-sgRNA plasmid and 1 μg of linearized DNA repair templates or column-purified PCRs, or 4 μg of ssDNA oligonucleotides. 150 μl of PCT solution was added to the transformation mix and gently mixed by pipetting. The transformation mix was incubated on ice for 30 minutes. ATB solution was added to the transformation mix up to a final volume of 450 μL and gently mixed before plating on TM or MEAS \u003csup\u003e35\u003c/sup\u003e supplemented with Hygromycin B. Transformation plates were incubated at 25°C at least 10 days before further analysis. \u003cem\u003eS. cerevisiae \u003c/em\u003ewas transformed according to \u003csup\u003e92\u003c/sup\u003e. For \u003cem\u003eS. cerevisiae \u003c/em\u003etransformations, 300 ng of Cas9-sgRNA plasmid and 1 µg of column-purified PCRs were used when required. For \u003cem\u003ein-vivo \u003c/em\u003eassemblies in \u003cem\u003eS. cerevisiae\u003c/em\u003e,DNA fragments were mixed in equi-molar amounts.All transformants were streaked-purified prior to validation by diagnostic PCR. For diagnostic PCRs, a primer pair binding upstream and downstream of the genomic integration site or a primer binding outside the integration site and another binding within the integrated DNA sequence were used for all strains validations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChemical extractions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor chemical detection of 6-MSA (or quantification), monocillin II, and farinosone A, B, and YNP1, \u003cem\u003eE. viscosa \u003c/em\u003eand \u003cem\u003eK. petricola\u003c/em\u003e strains were inoculated in 50 mL of YPD and incubated at 25°C for 5 daysand 200 RPM shaking, cultures were diluted to OD\u003csub\u003e600\u003c/sub\u003e 0.1 (\u003cem\u003eE. viscosa\u003c/em\u003e) or 1 mL was diluted in 50 mL (\u003cem\u003eK. petricola\u003c/em\u003e) in fresh media, and incubated for seven days prior to culture's processing, except for YWA1 producer strains, which incubation was changed for five days instead. For scale-up of \u003cem\u003eE. viscosa \u003c/em\u003eJF 03-4F farinosone A, B, and YNP1 producer strain, 5 days seed cultures were diluted to OD\u003csub\u003e600\u003c/sub\u003e 0.1 in 500 mL of fresh media in 2 L shake flasks and incubated for seven days prior to culture's processing. For \u003cem\u003eA. nidulans, A. oryzae, \u003c/em\u003eand\u003cem\u003e A. niger\u003c/em\u003e, 10\u003csup\u003e6\u003c/sup\u003e spores were inoculated in 50 mL of YPD and incubated at 25°C for 3 days 200 RPM shaking prior extractions. For \u003cem\u003eS. cerevisiae\u003c/em\u003e, strains were inoculated in 50 mL of YPD (or SD dropout media with or without uracil when necessary) and incubated at 30°C for three daysand 200 RPM shaking. For \u003cem\u003eS. cerevisiae\u003c/em\u003e, chemical detection of 6-MSA (or quantification), YWA1, monocillin II, and YNP1, 24 h seed cultures were diluted to OD\u003csub\u003e600\u003c/sub\u003e 0.1 in 50 mL of fresh media and incubated for three days prior to culture's processing. \u003c/p\u003e\n\u003cp\u003ePrior chemical extractions of PKS products, 50 mL cultures were transferred to 50 mL Falcon tubes and centrifuged at 3,500G for five minutes. The liquid phase was then transferred to new Falcon tubes and 25 mL of the liquid phase was extracted in a glass funnel with 2:1 ethyl acetate. The organic phase was recovered and transferred to 50 mL glass tubes, dried in a rotovapor, resuspended in 1 mL of methanol, and diluted 15 times prior LCMS or HPLC analysis. Farinosone A, B, and YNP1 produced during the scale-up culture of \u003cem\u003eE. viscosa \u003c/em\u003eJF 03-4F, both the liquid and biomass phases were extracted in a glass funnel with 2:1 ethyl acetate. The organic phase was recovered and transferred to 50 mL glass tubes, dried in a rotovapor, resuspended in 1 mL of methanol, and diluted 300 times prior LCMS or HPLC analysis. For 6-MSA yield quantification purposes, the obtained biomass from cultures was dried for 3 days at 60°C prior weighting. For \u003cem\u003eS. cerevisiae \u003c/em\u003eintracellular extractions, the biomass from 50 mL Falcon tubes was resuspended in 10 mL of methanol and transferred to 50 mL glass tubes. The glass tubes were shaken vigorously at 1,800 RPM for 30 minutes, and thereafter the biomass-methanol mix was transferred again to 50 mL Falcon tubes. Falcon tubes were centrifuged at 3,500G for five minutes, and the methanol phase transferred to new 50 mL glass tubes. The samples were dried concentrated in a rotovapor, resuspended in 1 mL of methanol, and diluted 15 times prior to LCMS analysis. Prior chemical extractions of \u003cem\u003eE. viscosa \u003c/em\u003eJF 03-4F strain producing farinosone A, B, and YNP1 was grown in 1L YPD, YPX, and 50 mL MMBW, the whole cultures were extracted with 2:1 ethyl acetate. The organic phase was partitioned with 1:1 MQ. The recovered organic phase was dried in a rotovapor, resuspended in 1 mL of methanol, and diluted 300 times for YPD and YPX, and 15 times for MMBW cultures, prior to LCMS analysis. The resuspended extractions were transferred to 1 mL Eppendorf tubes and dried using a rotary evaporator at 45°C prior to weighing. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLC-HRMS and LC-UV analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLC-HRMS analyses were carried out using a Vanquish Duo UHPLC binary system (Thermo Fisher Scientific®, Waltham, MA, USA) coupled to an Orbitrap IDX Tribrid Mass Spectrometer (Thermo Fisher Scientific®). The chromatographic separation was achieved in reverse phase conditions as previously described \u003csup\u003e93\u003c/sup\u003e. The MS measurements were performed in positive- and negative-heated electrospray ionization (+HESI) mode with a voltage of 3500 V and 2500 V, respectively, acquiring in full MS/MS spectra (Data dependent Acquisition-driven MS/MS) in the mass range of 70-1000 Da. The DDA settings were the following: automatic gain control (AGC) target value was set at 4\u003csup\u003e5\u003c/sup\u003e for the full MS and 5\u003csup\u003e4\u003c/sup\u003e for the MS/MS spectral acquisition, the mass resolution was set to 120,000 for full scan MS and 30,000 for MS/MS events. Precursor ions were fragmented by stepped High-energy collision dissociation (HCD) using collision energies of 20, 40, and 60. For 6-MSA compound detection and quantification by HPLC-UV, 10 points serial dilutions of a 6-MSA standard (CAS No.567-61-3, 98%, Thermo Scientific Chemicals®) were used for calibration curve and quantification. The supernatant was analyzed using a Agilent 1100 HPLC system (Agilent Technologies®, Santa Clara, CA, USA) with a diode array detector. The chromatographic separation was achieved using a Poroshell 120 Phenyl-Hexyl column (2.1 mm × 100 mm, 2.7 μm; Agilent Technologies®) with H\u003csub\u003e2\u003c/sub\u003eO + 0.1% formic acid as eluent A and with HPLC grade acetonitrile as eluent B. Gradient elution was performed at a flow rate of 0.5 mL min\u003csup\u003e-1\u003c/sup\u003e according to the following: 0-0.5 min 15% B, 0.5-6 min 15% to 65% B, 6-7 min 65% to 100% B, 7-8.5 min 100% B. The column was then re-equilibrated at 15% B. An injection volume of 1 μL was used, and the column oven was maintained at 50°C throughout the analyses. The analytes were detected at a wavelength of 240 nm at a width of 4 nm. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProteomics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor proteomics analysis, \u003cem\u003eS. cerevisiae \u003c/em\u003ecell pelletswereobtainedfrom cultures grown for 6 h in 25 mL of YPD media incubated at 30 °C and shaken at 200 RPM. These cultures were inoculated at 0.1 OD\u003csub\u003e600 \u003c/sub\u003ewith cells from 4 mL pre cultures incubated for 24 h. Proteins were extracted using a iST sample preparation kit (PreOmics®) according to manufacturer's instructions. The samples were analyzed by online nano-scale liquid chromatography tandem mass spectrometry (LC-MS/MS) in turn. Peptides were separated on a 15 cm C18-column (Thermo EasySpray™ ES804A) using an EASY-nLC 1200 system (Thermo Scientific®). The column temperature was maintained at 30°C. Buffer A consisted of 0.1% Formic acid in water, and buffer B of 80% ACN, 0.1% Formic acid. The flow rate of the gradient was kept at 250 nL min\u003csup\u003e-1\u003c/sup\u003e, and started at 6% Buffer B, going to 23% buffer B in 43 minutes. This was followed by a 12-minute step going to 38% buffer B, increasing to 60% Buffer B in 5 minutes, and finally ramping up to 95% buffer B in 3 minutes, holding it for 7 minutes to wash the column. The Q Exactive Classic instrument (Thermo Scientific®) was run in data dependent acquisition mode using a top 10 Higher-energy Collisional Dissociation (HCD)-MS/MS method with the following settings. Scan range was limited to \u003cem\u003em/z\u003c/em\u003e 350-1750. Full scan resolution was set to \u003cem\u003em/z \u003c/em\u003e70,000, with an AGC target of 3\u003csup\u003e6\u003c/sup\u003e and a maximum injection time (IT) value of 20 ms. Peptides were fragmented with a normalized collision energy of 25, having a dynamic exclusion of 30 s, excluding unassigned ions and those with a charge state of 1. MS/MS resolution was set at \u003cem\u003em/z \u003c/em\u003e17,500, with an AGC target of 3\u003csup\u003e6\u003c/sup\u003e and a maximum IT of 60 ms. The spectral data was analysed with Maxquant \u003csup\u003e94\u003c/sup\u003e using the protein sequences of PchI and PchE (monocillin II) and AdnE1-3(YNP1)plus NpgA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioinformatic methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor whole genome sequencing, \u003cem\u003eS. cerevisiae \u003c/em\u003ecell pellets were obtained from 4 mL YPD cultures grown overnight at 30°C and shaken at 200 RPM. \u003cem\u003eE. viscosa \u003c/em\u003eand \u003cem\u003eK. petricola\u003c/em\u003e cell pellets were obtained from cultures grown in 25 mL of YPD media in 250 mL flasks for seven days at 25 °C and shaken at 200 RPM, in all cases the cell pellets were washed once with deionized water. For transcriptome analyses, overnight cultures of \u003cem\u003eS. cerevisiae \u003c/em\u003ewere diluted to OD\u003csub\u003e600 \u003c/sub\u003e0.1and grown for 6 h in 25 mL of YPD media incubated at 30 °C and shaken at 200 RPM. 2mL of culture was pelleted and washed once with deionized water and used transcriptome sequencing GENEWIZ (Azenta Life Science technologies) using the Illumina NovaSeq platform in the 150 bases pair-end format. For genome assembly the reads were trimmed using TrimmomaticPE v0.39 \u003csup\u003e95\u003c/sup\u003e and assembled using the SPAdes assembler v3.13.1 \u003csup\u003e96\u003c/sup\u003e. The gene calling on the assembled scaffolds was performed using Augustus v3.4.0 \u003csup\u003e97\u003c/sup\u003e with genes model trained with the annotated genome of \u003cem\u003eE. viscosa\u003c/em\u003e JF 03-3F (RefSeq accession GCF_022695815) or \u003cem\u003eS. cerevisiae \u003c/em\u003eS288C (RefSeq accession GCF_000146045). The assemblies and gene-calling files were used for genome annotation with antiSMASH v7.0.0 \u003csup\u003e98\u003c/sup\u003e. To assess the expression of the monocillin II and YNP1 in \u003cem\u003eS. cerevisiae\u003c/em\u003e we used bowtie v. 2.4.4 \u003csup\u003e99\u003c/sup\u003e to map the RNA reads from strains sYNP19213 and sYNP19220 against the coding sequences of \u003cem\u003enpgA\u003c/em\u003e, \u003cem\u003ep\u003c/em\u003e\u003cem\u003echI\u003c/em\u003e and \u003cem\u003ep\u003c/em\u003e\u003cem\u003echE\u003c/em\u003e (monocillin II) and \u003cem\u003ea\u003c/em\u003e\u003cem\u003ednE1\u003c/em\u003e-\u003cem\u003e3\u003c/em\u003e (YNP1) plus the coding sequence of \u003cem\u003eact1\u003c/em\u003e from\u003cem\u003e S. cerevisiae \u003c/em\u003eS288C (RefSeq accession GCF_000146045). The resulting Sequence Alignment/Map files were then parsed to count the number of reads mapped for each gene; the expression level was recorded as the number of reads mapped for each coding sequence divided by their length in kilobases (RPK) and then normalized to the RPK value for the housekeeping gene \u003cem\u003eact1\u003c/em\u003e. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e Genome sequencing, mining and Phylogenomics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo estimate the distribution of PKSs across the fungal kingdom, We compiled a collection of 1979 fungal genomes obtained from the GenBank (supplementary file1), these genomes were gene-called using Augustus v3.4.0 \u003csup\u003e97\u003c/sup\u003e and their natural polyketide synthase repertoire was annotated with antiSMASH v7.0.0 \u003csup\u003e98\u003c/sup\u003e. The PKSs found in these genomes were identified and their domain organization extracted using a Perl script (https://github.com/WeMakeMolecules/Megasynthase_string_miner/blob/main/antismash_domain_parser.pl) the extracted PKSs were then parsed to classify and count them , the complete list of analyzed PKSs is available on (supplementary file 2). To compare known 2-pyridone biosynthetic gene cluster families to the YNP1 BGC (Figure 4A) we used FUNGISON, a implementation of the CORASON pipeline \u003csup\u003e79\u003c/sup\u003e for fungal genomes available at https://github.com/WeMakeMolecules/fungison. The reference BGCs were obtained from the MiBig database \u003csup\u003e90\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the genomes in the dataset used for this project are available in GenBank, the complete list of accession numbers is available in Supplementary file 2. All the sequences of PKSs analyzed are available in Supplementary file 2. The genomics and transcriptomics data for \u003cem\u003eS. cerevisiae\u003c/em\u003e strains will be available at the NCBI sequence read archive before publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe want to thank Assoc. Prof. Jakob Blæsbjerg Hoof for providing \u003cem\u003eAspergillus spp..\u0026nbsp;\u003c/em\u003estrains from the IBT fungal strain collection used in this work, and to Andreas Møllerhøj Vestergaard for sharing \u003cem\u003eS. cerevisiae\u003c/em\u003e strain BJ-5464-NpgA and plasmid pCFB2909-BIK1. We also thank Asst. Prof. Vayu Hill-Maini, and all the members of the Yeast Natural Products laboratory for reviewing this manuscript and providing insightful comments. We are deeply grateful to the IT team and the staff of the Novo Nordisk Center for Biosustainability for their invaluable support.\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Novo Nordisk Foundation NNF20CC0035580\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAEGL, XL, CCP, LB, DA, LC, SJ, JSB: performed molecular biology experiments; AEGL, LB, ACC, PCM: Performed metabolite extractions and purifications; MVL: Performed proteomic analyses; DR, LH, ACC: developed LC-MS methods and collected spectral data; CHG, ACC: performed NMR analysis; AEGL, ACC, PCM analyzed mass spectrometry data; AEGL, MVB and PCM performed bioinformatic analyses; AF and SIJ, prepared beechwood extracts and designed cultivations; EC, SH, UHM, TS, helped in the design of the experiments, data analysis and discussion; JDK and PCM supervised the research, AEGL and PCM designed the experiments; AEGL, JDK, UM and PCM prepared the manuscript. PCM conceived the idea.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJDK has financial interests in Amyris, Ansa Biotechnologies, Apertor Pharma, Berkeley Yeast, Cyklos Materials, Demetrix, Lygos, Napigen, ResVita Bio, and Zero Acre Farms. The other authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eLi, X. \u003cem\u003eet al.\u003c/em\u003e Mining natural products for advanced biofuels and sustainable bioproducts. \u003cem\u003eCurr. Opin. Biotechnol.\u003c/em\u003e \u003cstrong\u003e84\u003c/strong\u003e, 103003 (2023).\u003c/li\u003e\n \u003cli\u003eYuzawa, S. \u003cem\u003eet al.\u003c/em\u003e Short-chain ketone production by engineered polyketide synthases in Streptomyces albus. \u003cem\u003eNat. 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Methods\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 357\u0026ndash;359 (2012).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6001933/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6001933/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFungal natural products, including polyketides, are a rich source of bioactive molecules. Their biosynthetic enzymes are encoded within biosynthetic gene clusters, which are often activated by specific environmental conditions. As a result, many natural products are not produced under standard laboratory conditions. Heterologous expression bypasses native regulation, enabling a systematic approach for polyketide discovery. The most widely used fungal hosts for natural product production are Saccharomycetales yeasts, and filamentous Eurotiomycetes. Yeasts are highly tractable but have a narrow product scope due to their limited secondary metabolism, while filamentous Eurotiomycetes have a richer secondary metabolism but are more difficult to engineer.\u003c/p\u003e\n\u003cp\u003eIn this work, we established two yeasts of the genera \u003cem\u003eExophiala \u003c/em\u003eand \u003cem\u003eKnufia \u003c/em\u003eas novel heterologous hosts for a broad range of polyketides. These hosts combine the genetic tractability of yeast with the metabolic robustness of filamentous fungi. We developed genetic engineering tools for precise gene integration and genome editing, allowing us to heterologously express five fungal polyketide synthases with different domain architectures, including one involved in the biosynthesis of a previously undescribed 2-pyridone. Our findings demonstrate that these novel yeast hosts can efficiently produce complex polyketides, paving the way for systematic polyketide synthase expression and engineering.\u003c/p\u003e","manuscriptTitle":"Black yeasts are efficient heterologous hosts of a wide range of fungal polyketides","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-20 13:25:13","doi":"10.21203/rs.3.rs-6001933/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-chemical-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nchembio","sideBox":"Learn more about [Nature Chemical Biology](http://www.nature.com/nchembio/)","snPcode":"","submissionUrl":"","title":"Nature Chemical Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9f32018d-2e3e-43ed-a5ab-945c4269f5ea","owner":[],"postedDate":"February 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":44467815,"name":"Biological sciences/Chemical biology/Natural products"},{"id":44467816,"name":"Biological sciences/Chemical biology/Biosynthesis"}],"tags":[],"updatedAt":"2026-02-06T16:56:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-20 13:25:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6001933","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6001933","identity":"rs-6001933","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}