Characterization of the unconventional meroterpenoid gene cluster from Stachybotrys sp. CPCC 401591 for phenylspirodrimanes biosynthesis

Characterization of the unconventional meroterpenoid gene cluster from Stachybotrys sp. CPCC 401591 for phenylspirodrimanes biosynthesis | 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 Research Article Characterization of the unconventional meroterpenoid gene cluster from Stachybotrys sp. CPCC 401591 for phenylspirodrimanes biosynthesis Hui Lv, Bailin Song, Lingjin Bu, Wenni He, Lu Wang, Liyan Yu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7289101/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Oct, 2025 Read the published version in Microbial Cell Factories → Version 1 posted 9 You are reading this latest preprint version Abstract Background: Phenylspirodrimanes (PSDs) are a unique class of meroterpenoids that have been extensively studied due to their diverse biological activities. These metabolites are supposedly biosynthesized through the farnesylation of orsellinate. However, the molecular basis has not yet been elucidated. Results: Herein, using a combination of genome mining, bioinformatic alignment, and gene deletion approach, we characterized the dedicated gene cluster psd responsible for biosynthesizing PSDs in the fungus Stachybotrys sp. CPCC 401591. RNA sequencing-based transcriptomic analyses broadened our understanding of the genetic basis, regulation, and mechanisms of PSDs biosynthesis. Furthermore, based on the chemical structures of PSD derivatives characterized and deduced gene functions of the cluster psd , a hypothetical metabolic pathway for biosynthesizing chartarlactam K was proposed. These results revealed the underlying mechanism of phenylspirodrimanes generation, and these findings might facilitate downstream metabolic engineering studies of PSDs or the production of new chemical entities. Conclusions: Genome discovery and gene deletion experiments unveiled a previously uncharacterized biosynthetic gene cluster psd involved in the biosynthesizing PSD derivatives in Stachybotrys sp. CPCC 401591. In addition, based on the gene cluster data and transcriptomic analyses, a hypothetical biosynthetic pathway of chartarlactam K was put forward. Phenylspirodrimanes genome mining gene deletion transcriptome biosynthesis Stachybotrys Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Phenylspirodrimanes (PSDs), belonging to meroterpenoids, represent a structurally unique family of fungal secondary metabolites [ 1 ]. Naturally occurring PSDs with structural diversity have attracted increasing interest from research communities due to their diverse bioactivities, e . g ., anti-tumor, antibacterial, anti-diabetic, antiviral, anti-hyperlipidemic, anti-inflammatory, and anti-osteoporosis properties [ 1 – 6 ]. Monomeric and dimeric PSDs comprise the largest group with over 120 compounds bearing a phenylpropylfuran skeleton, fused by spirocyclic drimane with a benzene ring via a spirofuran moiety [ 4 , 7 ]. The structural differences among PSD dimers are attributed to various dimerization patterns of two monomers to form different linkage units. The linkage units include alkyl diamine chain [ 6 , 8 ], nitrogen-containing heterocycle [ 3 , 9 ], 6/7 oxygen heterocycle [ 10 ], [ 5 , 6 ]-spiroketal aromatic skeleton [ 7 ], C − C coupled bridge with a cyclopentanone core [ 11 , 12 ], and C − C coupled connection through two furan rings [ 13 ]. Many PSDs, such as bistachybotrysins M, S, and U, selectively inhibited the proliferation of human tumor cell lines HCT116, BGC 823, Daoy, NCI-H460, and HepG2 with IC 50 values in the range of 1.8–3.5 µ mol/L, making them potential drug leads [ 7 ]. Phenylspirodrimanes were mainly isolated from the fungi of the Stachybotrys genus, and closely related strains exemplified as Memnoniella echinata and Hansfordia sinuosae [ 4 , 14 , 15 ]. Stachybotrys is a genus including approximately 60 species and is widely distributed, which has been isolated from plant substrates, soil, rhizomes, sponges, and corals [ 3 , 13 , 16 – 18 ]. Species from this genus are prolific producers of a variety of structurally diversified metabolites, including phenylspirodrimanes, atranones, trichothecenes, dolabellane diterpenes, isoindolinone, cyclosporins, cochlioquinones, xanthones, and alachalasins [ 2 , 19 – 24 ]. Recently, postgenomic analyses have accelerated the biosynthetic studies on prenylated aryl-aldehyde of SMs, including stachybisbin B and ascofuranone, and the formation of intermediate ilicicolin B (LL-Z1272β) is associated with a three-gene ensemble that encodes proteins including non-reducing polyketide synthase (NR-PKS), UbiA-family prenyltransferase, and nonribosomal peptide synthetase (NRPS)-like reductase [ 25 , 26 ]. Biosynthetically, PSDs are believed to be generated from orsellinic acid and farnesyl diphosphate via an on-pathway intermediate ilicicolin B (Fig. 1 ) [ 27 , 28 ]. However, no molecular basis for the biosynthesis of phenylspirodrimanes has been characterized. Previously, we isolated the compound chartarlactam K from the wetland fungus Stachybotrys sp. CPCC 401591, along with seven phenylspirodrimane derivatives ( Fig. S1 , supporting file) [ 23 ]. Herein, we investigated an unconventional biosynthetic cluster in the strain CPCC 401591. Intriguingly, using a combination of bioinformatic analyses, gene deletion, and RNA sequencing-based transcriptomic profiling, a plausible metabolic pathway of chartarlactam K was also proposed. Deciphering the biosynthesis and identification of the biocatalysts for the formation of PSDs could broaden our knowledge of fungal meroterpenoid biosynthesis. Our study could facilitate future applications for generation of bioactive molecules through metabolic engineering. MATERIALS AND METHODS Strains and culture conditions The fungus Stachybotrys sp. CPCC 401591 and the deletant used in this study were deposited in the China Pharmaceutical Culture Collection Center (CPCC) [24]. The fungi were routinely cultivated on potato dextrose agar (BD, USA) and preserved in 15% glycerol at − 80°C. For genomic DNA (gDNA) extraction, the fungus was cultured in liquid potato dextrose broth (PDB) for one week. For RNA sequencing, the wild-type fungus was cultivated in PDB broth and TBI broth for seven days, respectively [29]. Small-scale fermentation of the parental strain CPCC 401591 and the deletant ΔpsdA was performed using rice medium (100 g rice/100 mL of deionized water in a 500 mL Erlenmeyer flask) for thirty days [30, 31]. The cultivation temperature of the strains is 30°C. DNA cloning was conducted using Escherichia coli DH5α as the host. The derived engineered E . coli cells were incubated in Luria-Bertani (LB) medium supplemented with kanamycin (50 µ g/mL) at 37°C. Sequencing and bioinformatic tools The gDNA from the strain CPCC 401591 was extracted using E.Z.N.A.® Fungal DNA Mini Kit (Omega, USA) as per the manufacturer’s instructions [30]. Genome sequencing was performed at Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China) using the Illumina HiSeq 2500 system. Sequence assembly was performed with SOAPdenovo 1.05 [32]. The systematic bioinformatic analysis of biosynthetic gene clusters (BGCs) was conducted using the antiSMASH platform [33]. Gene annotations of the BGCs coding sequences were performed on FGENESH platform () and then verified manually for BLAST alignments in the NCBI database. RNA-sequencing and Real-time PCR analysis Total RNA extraction was performed using a PureLink™ RNA Mini Kit (Invitrogen, USA). gDNA was further removed using RNase-free DNase I (Takara, Japan), and the EasyPure® RNA purification kit (TransGen, China) was employed for RNA preparation. RNA concentration was determined using a Nanodrop 2000/2000c spectrometer (Thermo Fisher Scientific, USA). The cDNA synthesis was recovered from RNA (1,250 ng) using HiScript III RT SuperMix (Vazyme, China) as described by the manufacturer. Quantitative PCR was conducted using SYBR qPCR assay (Vazyme, China) according to the standard manual. The primers used in this study are listed in Table S2. The relative quantification of mRNAs was then normalized against the levels of the β-tubulin gene according to the 2 −ΔΔCT method [31, 34, 35]. Data was calculated with the average value from triplicate experiments. RNA-sequencing was accomplished at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China), and an Illumina NovaSeq 6000 sequencer was employed. General techniques for DNA manipulation To construct the disruption cassette of the gene psdA , the homologous arms of the gene psdA were amplified from gDNA of the parental strain using primer sets ( psdA -Up-F/R and psdA -Dn-F/R, respectively) listed in Table S1 (Supporting Information) . PCR amplifications were performed using Phanta Flash Super-Fidelity DNA Polymerase (Vazyme, China). The plasmid pAg1-ble was digested with the Kpn I/ Pac I enzyme sets. Then, the downstream amplicon psd -Dn was ligated into the linearized pAg1-ble vector, in which the Clonexpress Ultra One Step Cloning Kit was employed (Vazyme, China). In addition, the upstream amplicon psd -Up was ligated into Sbf I-digested linear fragment pAg1-ble-Dn to generate the deletion plasmid pAg1-ble- ΔpsdA , in which the in vitro recombination cloning strategy was employed (Vazyme, China). Fungal transformation and generation of the deletant ΔpsdA Fresh spores of Stachybotrys sp. were harvested from PDA plates, which were cultured for 7–8 days at 30°C. The deletant of the strain CPCC 401591 was created using the standard protoplast-polyethylene glycol (PEG) strategy previously reported [36–38]. The transformants were inoculated into selective PDA medium (200 µ g/mL zeocin) with stationary incubation for 7 days. Diagnostic PCRs were further utilized to confirm the genotype of transformants [37, 39]. The specific primers for the construction of deletion cassettes and deletants verification are listed in Table S1 . Fermentation, extraction, LC-MS, and GNPS analysis The mutant and the wild-type strain CPCC 401591 were initially cultivated on PDA plates for 7 days. Subsequently, 1×10 8 fungal spores were inoculated in 100 mL PDB broth and incubated on a rotary shaker at 200 rpm to afford a seed culture. Then, 10 mL seed culture was inoculated in rice fermentation medium and cultured at 30°C for thirty days, respectively. The fermented material was extracted three times with ethyl acetate, and the organic layer was concentrated to dryness under vacuum. The extract was redissolved in 1 mL acetonitrile, and the sample (5 µ L) was analyzed by high-performance liquid chromatography analysis (Agilent InfinityII 1260, Agilent Eclipse SB-C18 column, 5 µ m, 4.6 × 250 mm). LC-MS/MS measurements were performed using Thermo Fisher Scientific LTQ Orbitrap XL mass spectrometer equipped with a heated electrospray ion source (HESI). Chromatographic separation was acquired using a reversed-phase Agilent Eclipse SB-C18 column (5 µ m particle size, 4.6×250 mm). The elution was conducted with a linear gradient of 10 − 100% acetonitrile-water (v/v) at a flow rate of 1.0 mL/min − 1 for 25 min, followed by a 5 min wash with 100% acetonitrile for 5 min. MS/MS spectra were achieved using electrospray ionization in positive ion mode. The optimized detection parameters were set as follows: ion spray voltage, 4.5 kV; ion source temperature: 550°C; capillary temperature, 220°C; scan range of m / z : 150 − 2000 Da, profile mode; a high collision energy of 45 eV was employed for fragmentation. HPLC solvent gradients and MS scan functions were controlled using the Xcalibur data system (Thermo Fisher Scientific). RESULTS Mining and bioinformatic analysis of the gene cluster psd in Stachybotrys sp. To characterize the genetic locus responsible for phenylspirodrimanes biosynthesis, we initially performed the whole genome sequencing of the strain Stachybotrys sp. The Illumina HiSeq 2500 sequencing of the fungus generated 276 scaffolds, which concluded 38.44 million non-redundant bases. In the course of antiSMASH-based genome mining endeavors in the fungus, one unique meroterpenoid biosynthetic cluster was acquired, which encodes a conserved (> 74.69% identity) non-reducing polyketide synthase (NR-PKS, PsdA), adenylate-forming reductase (PsdB), and UbiA-family prenyltransferase (PsdC) ensemble. It has been reported that a 5-methyl orsellinic acid encoding NR-PKS, a prenyltransferase, and an NRPS-like reductase could function collaboratively to produce ilicicolin B, an on-pathway intermediate during the process of ascochlorin and stachybisbin B biosyntheses [ 1 , 15 ]. This cluster is designated as psd (Fig. 1 and Table S2 , Supplementary Information). Furthermore, investigation of the bilateral regions of the skeleton forming genes psdA , psd B, and psdC supported the discovery of other genes encoding post-modification enzymes. The enzymes included one 3-dehydroshikimate dehydratase, one short-chain dehydrogenase/reductase, one oxidoreductase, two methyltransferases, one O-acetyltransferase, one MFS-type transporter, and one pathway-specific transcriptional regulatory factor that correspond to the structural features of phenylspirodrimanes. Nevertheless, the gene cluster psd was inserted by many genes that encode proteins involved in primary metabolism or obviously unrelated to the biosynthesis of the PSDs, exemplified as gene 08153 (coding glycosyl hydrolase), gene 08166 (coding alpha-L-arabinofuranosidase), gene 08170 (coding neutral protease). Therefore, these redundant genes were easily eliminated. In the light of the PsdA, PsdB, and PsdC encoded by the cluster psd exhibited highly similar identities with cluster stb and asc , involved in biosynthesizing L-Z1272β and ascochlorin, respectively [ 1 , 15 ]. We hypothesize that the BGC psd might be responsible for phenylspirodrimanes biosynthesis. Characterization of the cluster psd responsible for PSDs biosynthesis To verify the participation of PsdA in the generation of phenylspirodrimanes, deletion of the NR-PKS coding gene psdA was performed. We established a protoplast transformation-based gene disruption experiment to support the proposal that psdA is functioning in PSDs biosynthesis [ 36 ]. The construction of the knockout plasmid was conducted using the scaffold of pAg1-ble by replacing the bilateral fragments of the BleR selective cassette with homologous arms of the target gene psdA . Next, after protoplast transformation experiment, of thirty-eight putative zeocin + Stachybotrys transformants analyzed, the mutant ΔpsdA -16 was verified by diagnostic PCRs characterization with four primer sets. With one Zeo R specific primer set, the mutant gave two amplicons of 1,300 bp and 1,392 bp in size, respectively, while the WT had no amplicons ( Figure S2 , Supporting Information). Further confirming using one primer set resided in the deletion region of the gene psdA , two amplicons with 4,553 bp and 4,533 bp in size were recovered from WT, while no amplicons were obtained from the mutant ( Figure S2 , Supporting Information). The thus-acquired crude extracts from ΔpsdA and the parental strain were analyzed using Liquid Chromatography-Mass Spectrometry (LC-MS) analysis (Fig. 3 ). Knock-out of the psdA gene completely abolished the generation of the compounds stachybotrylactam ( t R =15.15 min) and chartarlactam F ( t R =16.45 min), thereby setting up the linkage between the psd biosynthetic cluster and the yielding of the PSDs. At the same HPLC conditions, the peaks of chartarlactam F and stachybotrylactam detected in the parental strain (WT) that two target compounds were further verified along with the evidence of m/z = 386.3 [M + H] + and m/z = 771.2 [2M + H] + using LC-MS/MS analysis. However, as shown in Fig. 3 , the strain ΔpsdA lacked these characteristic molecular ion peaks, indicating the absence of the target two compounds in metabolic profiling of the mutant ( Figure S3 , Supporting Information). Collectively, the experimental evidence further corroborated with our prediction regarding the function of the gene cluster psd , confirming that the gene psd A is a key gene for PSDs biosynthesis in Stachybotrys sp. Preliminary investigation of the genetic basis for PSDs biosynthesis To reveal the expression level of the gene cluster psd during PSDs biosynthesis on TBI and PDB broth, transcriptome sequencing was performed to characterize the differentially expressed genes (DEGs) of intergroup comparison (TBI/PDB) (Fig. 4 A). There were 3,874 DEGs, in which the up-regulated and down-regulated genes were 2,053 and 1,821, respectively (Fig. 4 B). Gene Ontology (GO) enrichment analysis manifested that the up-regulated genes were approximately engaged in biological processes including lipid metabolism, organophosphate catabolism, and secondary metabolites biosynthesis (Fig. 4 C). The KEGG enrichment data demonstrated that the up-regulated genes were significantly enriched in primary metabolic pathways participating in the processes of fatty acid, amino acid, starch, sucrose, glycolysis and gluconeogenesis ( Figure S4 , Supporting Information). These pathways were related to primary metabolism, including carbon metabolic reactions for strain growth and secondary metabolism. In addition, significant enrichment was also observed in the biosynthetic pathways of terpenoids and mycotoxins. Next, when we further examined the cluster psd , one phenomenon observed is that there are many dispensable genes within the cluster. Therefore, transcriptomic analyses of the encoded genes might be helpful to distinguish genes directly involved in PSDs biosynthesis. The expression level of psdA (gene 08149) and psdN (gene 08175, coding methyltransferase) in TBI sample were significantly up-regulated than PDB condition (Fig. 4 B, Figure S5 , and Table S2 , Supporting Information). Further deciphering the genes 08145–08148 and 08176–08180, gene 08148 encoded a hypothetical protein and was significantly down-regulated, while genes 08145–08147, gene 08176, and gene 08180 exhibited no detectable expression level in both conditions. Moreover, the genes (08177–08179) were annotated as phosphatase, mannitol dehydrogenase and accessory effector CFEM5, respectively. These three genes are obviously not associated with secondary metabolism and could be excluded from further analysis. Therefore, we speculated that psdA and psdN should be located on the left and right boundaries of the cluster psd (Fig. 2 A). Similarly, statistical analyses revealed that seven genes ( psdA , psdC - F , psdJ and psdN ) were significantly up-regulated (FC > 2, p < 0.05) in TBI condition, demonstrating that corresponding coded proteins should participate in PSDs generation. Also, seven genes ( psdB , and psdG - I , and psdK - M ) from both culture conditions exhibited high expression level in TPM (transcripts per million) values and moderately up-regulated (1 < FC < 2) in TBI sample, indicating these genes might contribute in PSDs biosynthesis or metabolites transport. Based on the transcriptome data, it was supposed that psdI might serve as regulatory factor, which could trigger the catalysis of proteins encoded by the cluster psd . In contrast, eleven genes displayed no detectable expression level in both conditions, which are unlikely involved in biosynthesizing PSDs ( Figure S5 , Table S2 , Supporting Information). Real-time reverse-transcription PCR further demonstrated that transcriptional level of the cluster psd in TBI condition exhibited much higher than in PDB broth, in which eight genes including psdA - F , psdJ , and psdN were selected for investigation (Fig. 4 D). The transcription levels of psdA , psdE and psdF increased remarkably in 3.3, 12.9, and 31.1-fold compared with that of PDB condition, respectively (Fig. 4 D). The transciptional analyses of Psd family genes indicated that TBI broth activated the biosynthesis of PSDs significantly. Investigation on the biosynthesis of metabolite chartarlactam K Phenylspirodrimanes are unique meroterpenoids featuring a phenylpropylfuran skeleton attached to spirocyclic drimane and a benzene ring moiety [ 40 ]. The genetic basis for the biosynthesis of ascochlorin was well determined previously, and the early-stage biosynthesis of PSDs should be highly identical to that of ascochlorin [ 18 ]. In the light of the molecular structures of stachybisbin B and proposed gene functions of the gene cluster psd , a metabolic pathway of chartarlactam K was envisioned [ 9 ] (Scheme 1 ). Domains of the megasynthase PsdA are programmed and generate polyketide chains and an offloading thioesterase domain is used for thioester hydrolyzation and chain release. The following steps involve the prenyltransferase PsdC to accomplish the sesquiterpene chain transferring and an NRPS-like reductase PsdB responsible for aldehyde formation to generate precursor ilicicolin B [ 9 , 18 ]. In ascochlorin pathway, the intermediate ilicicolin B occurs halogenation catalyzed by AscD and AscE-catalyzed epoxidation for the generation of ilicicolin A, followed by terpene cyclase AscF-mediated cyclization and multiple oxidations [ 18 ]. In contrast, the intermediate ilicicolin B might be epoxidized and cyclized to afford the spirocyclic intermediate F1839-I, which was sequentially catalyzed by an uncharacterized flavin monooxygenase and a terpene cyclase in a different manner [ 22 , 33 , 41 ] (Scheme 1 ). Subsequently, an additional oxygenation installs the hydroxyl group in the compound F1839-I. Then, the generated intermediate mer-NF5003E is then subjected to oxidative modification, leading to the formation of the precursor stachybotrydial (bearing an O-phthalaldehyde group) [ 22 ]. The intermediate stachybotrylactam might be transformed from the precursor stachybotrydial by three successive steps including C22-carboxylation, amination, and dehydration [ 41 ]. Subsequently, an additional oxygenation reaction installs the C-2 hydroxyl group in the intermediate F1839-A [ 33 ]. The final reaction might be catalyzed by O-acetyltransferase PsdJ and then furnishes the acetyl group to yield chartarlactam K (Scheme 1 ). In addition, based on the PSDs isolated from the strain CPCC 401591, we believed that the intermediate stachybotrydial containing O-phthalaldehyde moiety exhibits high reactivity and could be condensed with varied amino acids to synthesize structurally diverse N-containing derivatives ( Figure S1 , Supporting Information) [ 41 ]. Considering the diverse class of PSDs, we preferred that multi-step redox cascade occurs during the biosynthetic process [ 33 , 41 ]. This proposal hints to the possibility that redox enzymes might exhibit relatively broad substrate scope during PSDs biosynthesis in vivo . DISCUSSION To the best of our knowledge, this is the first report to characterize the gene cluster psd functioning in biosynthesis of phenylspirodrimanes from Stachybotrys species [2]. This genus has been considered to be a treasure for PSDs discovery, a multitude of bioactive derivatives bearing complexified structural modifications have been isolated and gained increasing interest recently [2, 22, 32]. Stachybotrys is a distinctive fungal genus due to its metabolic versatility and capability to produce diversified secondary metabolites [2, 26, 27]. In this report, genome guided bioinformatic mining of the fungus CPCC 401591 revealed the presence of an uncharacterized gene cluster psd . It carries the conserved locus encoding PsdA, PsdB, and PsdC ensemble, which exhibited higher identities with their counterparts responsible for producing ascochlorin and stachybisbin B, respectively [1, 17]. This finding indicated that PsdA/PsdB/PsdC ensemble is believed to be involved in the conversion of shared intermediate ilicicolin B, which is considered to be an on-pathway precursor in the biosynthesis of many fungal meroterpenoids [1, 12, 17, 20]. A more detailed cluster blast bioinformatic analysis was conducted on genome-sequenced filamentous fungi from NCBI database. Interestingly, all these ilicicolin B-forming genes are associated with the three-gene ensemble. This ensemble consists of genes that encode an orsellinic acid synthase, a UbiA-like prenyltransferase, and an NRPS-like reductase [1, 17]. To further investigate the phylogeny, three conserved proteins were selected for Multi-Locus Sequence Typing (MLST) phylogenetic tree construction. As shown in Figure 5 , markedly, the species Stachybotrys clustered as a well-supported branch, which constitute an important genus for producing PSDs [6, 12, 20-23, 33, 35, 42]. In addition, the biosynthetic pathways with ilicicolin B serving as pathway intermediate are widely distributed in fungal strains, exemplified as stachybisbins from closely related S . bisbyi PYH05-7, ascochlorin and ascofuranone from Acremonium egyptiacum F-1392 [1, 17]. This further implies mining other fungal strains clustered in different branch might supply the opportunities to discover novel ilicicolin B-derived meroterpenoids. Nature’s power as a chemist is well represented by the structures of PSD derivatives, which are featured by a distinct spirocyclic drimane fused to a phenyl substituent, and particularly a characteristic spirofuran ring framework [12, 20, 22, 42]. Nevertheless, no report has described the genetic bases of this class of hybrid metabolites [12]. A more detailed investigation of the gene cluster psd revealed that encoding genes are scattered and distributed separately. Further genome mining of the sequenced S . chartarum IBT 7711 and S . chlorohalonata IBT 40285 also revealed the presence of clusters similar to the cluster psd [17, 43]. Some strains of S . chartarum reportedly produces PSDs, such as chartarutines [44], stachybotrins [45], distachydrimanes [28], bistachybotrysins [7, 23, 45], and therefore, this cluster is also expected to be responsible for the production of phenylspirodrimanes in S . chartarum [12]. Considering the presence of several unrelated genes in the cluster psd and previous OSMAC experimental data, therefore, we tried to distinguish the redundant genes using transcriptomic analyses. Notably, among the co-localized twenty-seven open reading frames in the gene cluster psd , thirteen down-regulated, not expressed, or silent genes were efficiently differentiated. Although the experimental evidence was preliminary, mapping the differential expression profiles could shed light on identification of candidate genes responsible for PSDs biosynthesis. Regarding the metabolic pathway in generating F1839-I from intermediate ilicicolin B, two key reaction steps might be included, e . g ., epoxidation catalyzed by flavin monooxygenase, and cyclization catalyzed by membrane-bound terpene cyclase [1, 12, 17, 28, 40]. Nevertheless, these encoded genes could not be obtained in this cluster [17]. With transcriptome sequencing data, two separate gene clusters are obtained that most likely participated in the catalytic process, and this need to be further characterized through gene deletions. Ultimately, the underlying mechanism of PSDs biosynthesis would be uncovered in the near future. Using a genome mining strategy, we clarified the gene cluster psd functioning in phenylspirodrimanes biosyntheses in Stachybotrys sp. The biosyntheses of PSDs and ascochlorin share the common pathway up to the generation of the pathway intermediate ilicicolin B. Also, a bioinformatic analysis of the BGC psd was performed. Notably, RNA-sequencing based transcriptomic profiling facilitated to decipher intriguing characteristics of PSDs biosyntheses and eliminate the unrelated genes. This study thus contributes to broadening the knowledge on biosynthesizing this group of meroterpenoids. We have established the method for the genetic manipulation of the fungus Stachybotrys , and further manipulation of encoding genes of the psd locus will be conducted in our laboratory. Declarations AUTHOR INFORMATION Corresponding Authors Tao Zhang - Institute of Medicinal Biotechnology , Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China; Email: [email protected] Liyan Yu - Institute of Medicinal Biotechnology , Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China; Email: [email protected] . Author Contributions T.Z. conceived the study, obtained initial funding. T.Z., and L.Y. supervised the work to completion. T.Z., H.L., and B.S. performed experiments and analyzed the data. L.B., W.H., X.F. and L.W. advised on the design and partial interpretation of data. T.Z., and H.L. wrote the draft manuscript. T.Z., and L.Y. edited the draft manuscript. All authors reviewed the final manuscript. Funding This work was financially supported by the CAMS Innovation Fund for Medical Sciences (2021-I2M-1-055), the National Natural Science Foundation of China (No. 31872617), National Microbial Resource Center (No. NMRC-2025-3), and the central level, scientific research institutes for basic R & D fund business (3332018097). Notes The authors declare no competing financial interest. Supporting Information Experiment details and spectroscopic data. This material is available free of charge via the Internet at http://pubs.acs.org. References Bhowmik P, Baezid HM, Arabi, II. 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Wu Z, Li Y, Ma M, Chen J. Salicylic acid derivatives and phenylspirodrimanes from the sponge-associated fungus Hansfordia sinuosae . J Asian Nat Prod Res 2018, 20:985-991. Jagels A, Stephan F, Ernst S, Lindemann V, Cramer B, Hubner F, Humpf HU. Artificial vs natural Stachybotrys infestation-comparison of mycotoxin production on various building materials. Indoor Air 2020, 30:1268-1282. Kawahara T, Itoh M, Izumikawa M, Kagaya N, Sakata N, Tsuchida T, Shin-Ya K. New phenylspirodrimane metabolites MBJ-0030, MBJ-0031, and MBJ-0032 isolated from the soil fungal strain Stachybotrys sp. f23793. Biosci Biotechnol Biochem 2020, 84:1570-1575. Yang B, Long J, Pang X, Lin X, Liao S, Wang J, Zhou X, Li Y, Liu Y. Structurally diverse polyketides and phenylspirodrimanes from the soft coral-associated fungus Stachybotrys chartarum SCSIO41201. J Antibiot (Tokyo) 2021, 74:190-198. Hinkley SF, Mazzola EP, Fettinger JC, Lam YF, Jarvis BB. Atranones A-G, from the toxigenic mold Stachybotrys chartarum . Phytochemistry 2000, 55:663-673. Sasamura S, Kobayashi M, Muramatsu H, Yoshimura S, Kinoshita T, Ohki H, Okada K, Deai Y, Yamagishi Y, Hashimoto M. Bioconversion of FR901459, a novel derivative of cyclosporin A, by Lentzea sp. 7887. J Antibiot (Tokyo) 2015, 68:511-520. Jagels A, Hovelmann Y, Zielinski A, Esselen M, Kohler J, Hubner F, Humpf HU. Stachybotrychromenes A-C: novel cytotoxic meroterpenoids from Stachybotrys sp. Mycotoxin Res 2018, 34:179-185. Fujioka T, Yao K, Hamano K, Hosoya T, Kagasaki T, Furukawa Y, Haruyama H, Sato S, Koga T, Tsujita Y. Epi-cochlioquinone A, a novel acyl-CoA: cholesterol acyltransferase inhibitor produced by Stachybotrys bisbyi . J Antibiot (Tokyo) 1996, 49:409-413. Rong X, He W, Guo Z, Li X, Wang L, Gao K, Yu L, Zhang T. Isolation and antitumor activity of phenylspirodrimane derivatives from the fungus Stachybotrys sp CPCC 401591. Nat Prod Res Dev 2023, 35:1348-1356. Rong X, Guo Z, He W, Cai G, Gong K, Wang L, Yu L, Zhang T, Gao K. Secondary metabolites exhibiting antitumor bioactivities from the fungus Stachybotrys sp CPCC 401591. Mycosystema 2023, 42:1611-1621. Araki Y, Awakawa T, Matsuzaki M, Cho R, Matsuda Y, Hoshino S, Shinohara Y, Yamamoto M, Kido Y, Inaoka DK, et al . Complete biosynthetic pathways of ascofuranone and ascochlorin in Acremonium egyptiacum . Proc Natl Acad Sci U S A 2019, 116:8269-8274. Li C, Matsuda Y, Gao H, Hu D, Yao XS, Abe I. Biosynthesis of LL-Z1272beta: discovery of a new member of NRPS-like enzymes for aryl-aldehyde formation. Chembiochem 2016, 17:904-907. Zhao J, Feng J, Tan Z, Liu J, Zhang M, Chen R, Xie K, Chen D, Li Y, Chen X, Dai J. Bistachybotrysins A-C, three phenylspirodrimane dimers with cytotoxicity from Stachybotrys chartarum . Bioorg Med Chem Lett 2018, 28:355-359. Lin S, Huang J, Zeng H, Tong Q, Zhang X, Yang B, Ye Y, Lin S, Huang J, Zeng H, et al . Distachydrimanes A–F, phenylspirodrimane dimers and hybrids with cytotoxic activity from the coral-derived fungus Stachybotrys Chartarum . Chinese Chemical Letters 2022, 33:4587-4594. Liew MXX, Nakajima Y, Maeda K, Kitamura N, Kimura M. Regulatory mechanism of trichothecene biosynthesis in Fusarium graminearum . Front Microbiol 2023, 14:1148771. Rong X, Zhang L, He W, Guo Z, Lv H, Bai J, Yu L, Zhang T. Exploration of diverse secondary metabolites from Penicillium brasilianum by co-culturing with Armillaria mellea . Appl Microbiol Biotechnol 2024, 108:462. He W, Rong X, Lv H, Zhang L, Bai J, Wang L, Yu L, Zhang T. Genetically-modified activation strategy facilitates the discovery of sesquiterpene-derived metabolites from Penicillium brasilianum . Synth Syst Biotechnol 2025, 10:391-400. Li R, Zhu H, Ruan J, Qian W, Fang X, Shi Z, Li Y, Li S, Shan G, Kristiansen K, et al. De novo assembly of human genomes with massively parallel short read sequencing. Genome Res 2010, 20:265-272. Blin K, Shaw S, Vader L, Szenei J, Reitz ZL, Augustijn HE, Cediel-Becerra JDD, de Crecy-Lagard V, Koetsier RA, Williams SE, et al . antiSMASH 8.0: extended gene cluster detection capabilities and analyses of chemistry, enzymology, and regulation. Nucleic Acids Res 2025, 53:W32-W38. Zhang T, Cai G, Rong X, Wang Y, Gong K, Liu W, Wang L, Pang X, Yu L. A combination of genome mining with an OSMAC approach facilitates the discovery of and contributions to the biosynthesis of melleolides from the Basidiomycete Armillaria tabescens . J Agric Food Chem 2022, 70:12430-12441. Zhang T, Pang X, Zhao J, Guo Z, He W, Cai G, Su J, Cen S, Yu L. Discovery and activation of the cryptic cluster from Aspergillus sp. CPCC 400735 for asperphenalenone biosynthesis. ACS Chem Biol 2022, 17:1524-1533. Zhang T, Wan J, Zhan Z, Bai J, Liu B, Hu Y. Activation of an unconventional meroterpenoid gene cluster in Neosartorya glabra leads to the production of new berkeleyacetals. Acta Pharm Sin B 2018, 8:478-487. Zhang T, Gu G, Liu G, Su J, Zhan Z, Zhao J, Qian J, Cai G, Cen S, Zhang D, Yu L. Late-stage cascade of oxidation reactions during the biosynthesis of oxalicine B in Penicillium oxalicum . Acta Pharm Sin B 2023, 13:256-270. Gao SS, Zhang T, Garcia-Borras M, Hung YS, Billingsley JM, Houk KN, Hu Y, Tang Y. Biosynthesis of heptacyclic duclauxins requires extensive redox modifications of the phenalenone aromatic polyketide. J Am Chem Soc 2018, 140:6991-6997. Zhang T, Cai G, Rong X, Xu J, Jiang B, Wang H, Li X, Wang L, Zhang R, He W, Yu L. Mining and characterization of the PKS-NRPS hybrid for epicoccamide A: a mannosylated tetramate derivative from Epicoccum sp. CPCC 400996. Microb Cell Fact 2022, 21:249. Dayras M, Sfecci E, Bovio E, Rastoin O, Dufies M, Fontaine-Vive F, Taffin-de-Givenchy E, Lacour T, Pages G, Varese GC, Mehiri M. New phenylspirodrimanes from the sponge-associated fungus Stachybotrys chartarum MUT 3308. Mar Drugs 2023, 21. Tribelhorn K, Twaruzek M, Kosicki R, Straubinger RK, Ebel F, Ulrich S. A chemically defined medium that supports mycotoxin production by Stachybotrys chartarum enabled analysis of the impact of nitrogen and carbon sources on the biosynthesis of macrocyclic trichothecenes and stachybotrylactam. Appl Environ Microbiol 2023, 89:e0016323. Zhao J, Feng J, Tan Z, Liu J, Chen R, Xie K, Zhang D, Li Y, Yu L, Chen X, Dai J. Stachybotrysins A-G, phenylspirodrimane derivatives from the fungus Stachybotrys chartarum . J Nat Prod 2017, 80:1819-1826. Semeiks J, Borek D, Otwinowski Z, Grishin NV. Comparative genome sequencing reveals chemotype-specific gene clusters in the toxigenic black mold Stachybotrys . BMC Genomics 2014, 15:590. Li Y, Liu D, Cen S, Proksch P, Lin WH. Isoindolinone-type alkaloids from the sponge derived fungus Stachybotrys chartarum . Tetrahedron 2014, 70:7010-7015. Wang Y, Chen K, Xing Q, Zhang T, Xu Y. Stachybotrins G and H, two new phenylspirodrimane derivatives from the fungus Stachybotrys chartarum . Planta Med 2025. Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Supportinginformation.docx Scheme1.jpg Scheme 1 Proposed scheme of chartarlactam K biosynthesis. toc.jpg Cite Share Download PDF Status: Published Journal Publication published 29 Oct, 2025 Read the published version in Microbial Cell Factories → Version 1 posted Editorial decision: Revision requested 18 Aug, 2025 Reviews received at journal 17 Aug, 2025 Reviews received at journal 17 Aug, 2025 Reviewers agreed at journal 10 Aug, 2025 Reviewers agreed at journal 08 Aug, 2025 Reviewers invited by journal 07 Aug, 2025 Editor assigned by journal 06 Aug, 2025 Submission checks completed at journal 06 Aug, 2025 First submitted to journal 04 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7289101","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":498208919,"identity":"56291c46-9646-4491-8365-d7104b9aaa92","order_by":0,"name":"Hui Lv","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Lv","suffix":""},{"id":498208920,"identity":"8fc0d596-c0e2-4a20-ba72-ce4b0d89b3db","order_by":1,"name":"Bailin Song","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Bailin","middleName":"","lastName":"Song","suffix":""},{"id":498208921,"identity":"c1b3e2db-20f4-428e-ab37-0ddc822e67f4","order_by":2,"name":"Lingjin Bu","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Lingjin","middleName":"","lastName":"Bu","suffix":""},{"id":498208922,"identity":"361aeb25-7756-45bf-8ff4-cbbcb919fe04","order_by":3,"name":"Wenni He","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Wenni","middleName":"","lastName":"He","suffix":""},{"id":498208923,"identity":"fd228ed1-8601-4f91-9db4-a8ef165c6d74","order_by":4,"name":"Lu Wang","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Wang","suffix":""},{"id":498208925,"identity":"bd52acdf-77e0-45e1-a205-24567d434fda","order_by":5,"name":"Liyan Yu","email":"","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":false,"prefix":"","firstName":"Liyan","middleName":"","lastName":"Yu","suffix":""},{"id":498208926,"identity":"2d73e102-7073-4836-92d6-7cc095cf7a59","order_by":6,"name":"Tao Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYPACGx5+9sbGhx9I0JImI9lzuNlYggQth20MbqS3CfAQo5ZfIvnYxy8VzDySMx+2MUgw2MnpNhDQIjkjLXm2zBk2Hn7pxLYHBQzJxmYHCGgxOHPGmFmyjYdHcnZiu4EEw4HEbYS02J85/xmoRYLH4OZBIEmMFgP2HmbGj20GPAY3GInUInG8zZiZ4UwCj2RPIjCQDYjwC38z82PGHxX/7fnZjz98+KHCTo6gFhBgRkSHARHKQYDxB5EKR8EoGAWjYIQCAGUqPpls6a8jAAAAAElFTkSuQmCC","orcid":"","institution":"Chinese Academy of Medical Sciences \u0026 Peking Union Medical College","correspondingAuthor":true,"prefix":"","firstName":"Tao","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-08-04 09:08:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7289101/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7289101/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12934-025-02853-3","type":"published","date":"2025-10-29T15:58:36+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88924590,"identity":"8232252b-c797-4483-9496-9465bd501695","added_by":"auto","created_at":"2025-08-12 18:32:30","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":86353,"visible":true,"origin":"","legend":"\u003cp\u003eChartarlactam K and selected fungal meroterpenoids putatively derived from the ilicicolin B.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7289101/v1/1aec58702bcc9e52481fc5ee.jpg"},{"id":88924399,"identity":"9cd623b1-e55d-421d-a08b-a40996b7a5b4","added_by":"auto","created_at":"2025-08-12 18:24:30","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":119306,"visible":true,"origin":"","legend":"\u003cp\u003eA) Genetic organization of the phenylspirodrimanes (PSDs) biosynthetic locus in the \u003cem\u003eStachybotrys \u003c/em\u003esp. CPCC 401591genome. B) Putative function of ORFs within the BGC\u003cem\u003e ps\u003c/em\u003e\u003cu\u003e\u003cem\u003ed\u003c/em\u003e\u003c/u\u003e involved in PSDs biosynthesis.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7289101/v1/18324af9a84cfa26af6aeb8b.jpg"},{"id":88924592,"identity":"15ede847-e620-4b08-bb25-03d4b0905140","added_by":"auto","created_at":"2025-08-12 18:32:30","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":72922,"visible":true,"origin":"","legend":"\u003cp\u003eGene deletion of \u003cem\u003epsdA\u003c/em\u003e and characterization of \u003cem\u003eΔpsdA \u003c/em\u003emutants. HPLC profiles of organic extracts obtained from the WT and \u003cem\u003eΔpsdA \u003c/em\u003emutants of \u003cem\u003eStachybotrys\u003c/em\u003e sp. The positions of stachybotrylactam and chartarlactam F are indicated in the chromatograms, a peak labeled by \u003cstrong\u003e*\u003c/strong\u003e appears at the same retention time as chartarlactam F in deletion strains. This compound exhibited different UV absorbance and EIC values (Figure S3, Supporting Information). The elution was monitored at 230 nm and shown on the same Y-scale. HPLC-MS analysis of \u003cem\u003eΔpsdA \u003c/em\u003emetabolites in positive ion mode. Stachybotrylactam, EIC at \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e 386.32, [M+H]\u003csup\u003e+\u003c/sup\u003e; \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e 771.28, [2M+H]\u003csup\u003e+\u003c/sup\u003e. Chartarlactam F, EIC at \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e 386.35, [M+H]\u003csup\u003e+\u003c/sup\u003e; \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e 771.26, [2M+H]\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7289101/v1/f30dac46a4ae91d17de74bd5.jpg"},{"id":88924401,"identity":"012ef51f-e790-4ee6-baa5-64f54bdf6ffa","added_by":"auto","created_at":"2025-08-12 18:24:30","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":79894,"visible":true,"origin":"","legend":"\u003cp\u003eMining transcriptome data for phenylspirodrimanes production. A) HPLC profiles of organic extracts obtained from the strain \u003cem\u003eStachybotrys\u003c/em\u003e sp. CPCC 401591 in two fermentation broth. The extracts were analyzed by measuring UV absorbance spectra at 230 nm on an Agilent 1260 Infinity system. B) Analysis of DEGs. FoldChange (FC) shows the ratio of transcriptional levels (TBI/PDB); log\u003csub\u003e2\u003c/sub\u003e(FC) represents the value of differential folds; and -log\u003csub\u003e10\u003c/sub\u003e(P-adjust) shows the levels of significance. C) GO enrichment of up-regulated genes in TBI fermentation broth. D) Differentiated expression levels of the PSDs biosynthetic gene cluster. The error bars are shown as ± standard deviation (n = 3). ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7289101/v1/9bc06bf37e1b1bb79f0ae340.jpg"},{"id":88924407,"identity":"59c030c3-4fa0-4f3e-8533-fdd215d2701c","added_by":"auto","created_at":"2025-08-12 18:24:30","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":142155,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of fungal three-gene ensemble coding proteins retrieved from characterized or uncharacterized ilicicolin B-forming pathways. The ilicicolin B-forming ensemble from \u003cem\u003eStachybotrys\u003c/em\u003e sp. CPCC 401591, \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebisbyi \u003c/em\u003ePYH 05-7, and \u003cem\u003eAcremonium egyptiacum\u003c/em\u003eF-1392 was highlighted in red or blue, respectively. The scale represents changes per site; numbers at branches are bootstrap values. For accession numbers and further details, the reader is referred to the Supporting Information Table S3.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7289101/v1/171dfcf649455222a40bcd02.jpg"},{"id":95041269,"identity":"58e01a7d-7c09-408b-81e1-08be3f8838e1","added_by":"auto","created_at":"2025-11-03 16:11:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1372484,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7289101/v1/72f08e46-2ecf-4047-bb90-566e51e8304b.pdf"},{"id":88924408,"identity":"7744d33b-8473-4ff4-bd99-95d91bdaa2fe","added_by":"auto","created_at":"2025-08-12 18:24:30","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":946395,"visible":true,"origin":"","legend":"","description":"","filename":"Supportinginformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7289101/v1/c0b58f8a0e6e80bb77e4f069.docx"},{"id":88924405,"identity":"918211bf-d381-42ed-bc26-4a8b89942fdc","added_by":"auto","created_at":"2025-08-12 18:24:30","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":127531,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1 \u003c/strong\u003eProposed scheme of chartarlactam K biosynthesis.\u003c/p\u003e","description":"","filename":"Scheme1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7289101/v1/d407d140dfc397849f0c2078.jpg"},{"id":88924403,"identity":"0d2f4f54-cf89-4ddc-8d52-fa4606e8b809","added_by":"auto","created_at":"2025-08-12 18:24:30","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":41906,"visible":true,"origin":"","legend":"","description":"","filename":"toc.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7289101/v1/78e0761e63eac64835b78654.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eCharacterization of the unconventional meroterpenoid gene cluster from \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eStachybotrys \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003esp. CPCC 401591 for phenylspirodrimanes biosynthesis\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003ePhenylspirodrimanes (PSDs), belonging to meroterpenoids, represent a structurally unique family of fungal secondary metabolites [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Naturally occurring PSDs with structural diversity have attracted increasing interest from research communities due to their diverse bioactivities, \u003cem\u003ee\u003c/em\u003e.\u003cem\u003eg\u003c/em\u003e., anti-tumor, antibacterial, anti-diabetic, antiviral, anti-hyperlipidemic, anti-inflammatory, and anti-osteoporosis properties [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Monomeric and dimeric PSDs comprise the largest group with over 120 compounds bearing a phenylpropylfuran skeleton, fused by spirocyclic drimane with a benzene ring \u003cem\u003evia\u003c/em\u003e a spirofuran moiety [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The structural differences among PSD dimers are attributed to various dimerization patterns of two monomers to form different linkage units. The linkage units include alkyl diamine chain [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], nitrogen-containing heterocycle [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], 6/7 oxygen heterocycle [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]-spiroketal aromatic skeleton [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], C\u0026thinsp;\u0026minus;\u0026thinsp;C coupled bridge with a cyclopentanone core [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and C\u0026thinsp;\u0026minus;\u0026thinsp;C coupled connection through two furan rings [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Many PSDs, such as bistachybotrysins M, S, and U, selectively inhibited the proliferation of human tumor cell lines HCT116, BGC 823, Daoy, NCI-H460, and HepG2 with IC\u003csub\u003e50\u003c/sub\u003e values in the range of 1.8\u0026ndash;3.5 \u003cem\u003e\u0026micro;\u003c/em\u003emol/L, making them potential drug leads [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePhenylspirodrimanes were mainly isolated from the fungi of the \u003cem\u003eStachybotrys\u003c/em\u003e genus, and closely related strains exemplified as \u003cem\u003eMemnoniella echinata\u003c/em\u003e and \u003cem\u003eHansfordia sinuosae\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. \u003cem\u003eStachybotrys\u003c/em\u003e is a genus including approximately 60 species and is widely distributed, which has been isolated from plant substrates, soil, rhizomes, sponges, and corals [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Species from this genus are prolific producers of a variety of structurally diversified metabolites, including phenylspirodrimanes, atranones, trichothecenes, dolabellane diterpenes, isoindolinone, cyclosporins, cochlioquinones, xanthones, and alachalasins [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan additionalcitationids=\"CR20 CR21 CR22 CR23\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Recently, postgenomic analyses have accelerated the biosynthetic studies on prenylated aryl-aldehyde of SMs, including stachybisbin B and ascofuranone, and the formation of intermediate ilicicolin B (LL-Z1272β) is associated with a three-gene ensemble that encodes proteins including non-reducing polyketide synthase (NR-PKS), UbiA-family prenyltransferase, and nonribosomal peptide synthetase (NRPS)-like reductase [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Biosynthetically, PSDs are believed to be generated from orsellinic acid and farnesyl diphosphate \u003cem\u003evia\u003c/em\u003e an on-pathway intermediate ilicicolin B (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, no molecular basis for the biosynthesis of phenylspirodrimanes has been characterized.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003ePreviously, we isolated the compound chartarlactam K from the wetland fungus \u003cem\u003eStachybotrys\u003c/em\u003e sp. CPCC 401591, along with seven phenylspirodrimane derivatives (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e, supporting file) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Herein, we investigated an unconventional biosynthetic cluster in the strain CPCC 401591. Intriguingly, using a combination of bioinformatic analyses, gene deletion, and RNA sequencing-based transcriptomic profiling, a plausible metabolic pathway of chartarlactam K was also proposed. Deciphering the biosynthesis and identification of the biocatalysts for the formation of PSDs could broaden our knowledge of fungal meroterpenoid biosynthesis. Our study could facilitate future applications for generation of bioactive molecules through metabolic engineering.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cb\u003eStrains and culture conditions\u003c/b\u003e\u003c/p\u003e\n\u003cp\u003eThe fungus \u003cem\u003eStachybotrys\u003c/em\u003e sp. CPCC 401591 and the deletant used in this study were deposited in the China Pharmaceutical Culture Collection Center (CPCC) [24]. The fungi were routinely cultivated on potato dextrose agar (BD, USA) and preserved in 15% glycerol at − 80°C. For genomic DNA (gDNA) extraction, the fungus was cultured in liquid potato dextrose broth (PDB) for one week. For RNA sequencing, the wild-type fungus was cultivated in PDB broth and TBI broth for seven days, respectively [29]. Small-scale fermentation of the parental strain CPCC 401591 and the deletant \u003cem\u003eΔpsdA\u003c/em\u003e was performed using rice medium (100 g rice/100 mL of deionized water in a 500 mL Erlenmeyer flask) for thirty days [30, 31]. The cultivation temperature of the strains is 30°C. DNA cloning was conducted using \u003cem\u003eEscherichia coli\u003c/em\u003e DH5α as the host. The derived engineered \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e cells were incubated in Luria-Bertani (LB) medium supplemented with kanamycin (50 \u003cem\u003eµ\u003c/em\u003eg/mL) at 37°C.\u003c/p\u003e\n\u003cp\u003e\u003cb\u003eSequencing and bioinformatic tools\u003c/b\u003e\u003c/p\u003e\n\u003cp\u003eThe gDNA from the strain CPCC 401591 was extracted using E.Z.N.A.® Fungal DNA Mini Kit (Omega, USA) as per the manufacturer’s instructions [30]. Genome sequencing was performed at Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China) using the Illumina HiSeq 2500 system. Sequence assembly was performed with SOAPdenovo 1.05 [32]. The systematic bioinformatic analysis of biosynthetic gene clusters (BGCs) was conducted using the antiSMASH platform [33]. Gene annotations of the BGCs coding sequences were performed on FGENESH platform () and then verified manually for BLAST alignments in the NCBI database.\u003c/p\u003e\n\u003cp\u003e\u003cb\u003eRNA-sequencing and Real-time PCR analysis\u003c/b\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA extraction was performed using a PureLink™ RNA Mini Kit (Invitrogen, USA). gDNA was further removed using RNase-free DNase I (Takara, Japan), and the EasyPure® RNA purification kit (TransGen, China) was employed for RNA preparation. RNA concentration was determined using a Nanodrop 2000/2000c spectrometer (Thermo Fisher Scientific, USA). The cDNA synthesis was recovered from RNA (1,250 ng) using HiScript III RT SuperMix (Vazyme, China) as described by the manufacturer. Quantitative PCR was conducted using SYBR qPCR assay (Vazyme, China) according to the standard manual. The primers used in this study are listed in Table S2. The relative quantification of mRNAs was then normalized against the levels of the β-tubulin gene according to the 2\u003csup\u003e−ΔΔCT\u003c/sup\u003e method [31, 34, 35]. Data was calculated with the average value from triplicate experiments. RNA-sequencing was accomplished at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China), and an Illumina NovaSeq 6000 sequencer was employed.\u003c/p\u003e\n\u003cp\u003e\u003cb\u003eGeneral techniques for DNA manipulation\u003c/b\u003e\u003c/p\u003e\n\u003cp\u003eTo construct the disruption cassette of the gene \u003cem\u003epsdA\u003c/em\u003e, the homologous arms of the gene \u003cem\u003epsdA\u003c/em\u003e were amplified from gDNA of the parental strain using primer sets (\u003cem\u003epsdA\u003c/em\u003e-Up-F/R and \u003cem\u003epsdA\u003c/em\u003e-Dn-F/R, respectively) listed in \u003cb\u003eTable S1 (Supporting Information)\u003c/b\u003e. PCR amplifications were performed using Phanta Flash Super-Fidelity DNA Polymerase (Vazyme, China). The plasmid pAg1-ble was digested with the \u003cem\u003eKpn\u003c/em\u003eI/\u003cem\u003ePac\u003c/em\u003eI enzyme sets. Then, the downstream amplicon \u003cem\u003epsd\u003c/em\u003e-Dn was ligated into the linearized pAg1-ble vector, in which the Clonexpress Ultra One Step Cloning Kit was employed (Vazyme, China). In addition, the upstream amplicon \u003cem\u003epsd\u003c/em\u003e-Up was ligated into \u003cem\u003eSbf\u003c/em\u003eI-digested linear fragment pAg1-ble-Dn to generate the deletion plasmid pAg1-ble-\u003cem\u003eΔpsdA\u003c/em\u003e, in which the \u003cem\u003ein vitro\u003c/em\u003e recombination cloning strategy was employed (Vazyme, China).\u003c/p\u003e\n\u003cp\u003e\u003cb\u003eFungal transformation and generation of the deletant\u003c/b\u003e \u003cb\u003eΔpsdA\u003c/b\u003e\u003c/p\u003e\n\u003cp\u003eFresh spores of \u003cem\u003eStachybotrys\u003c/em\u003e sp. were harvested from PDA plates, which were cultured for 7–8 days at 30°C. The deletant of the strain CPCC 401591 was created using the standard protoplast-polyethylene glycol (PEG) strategy previously reported [36–38]. The transformants were inoculated into selective PDA medium (200 \u003cem\u003eµ\u003c/em\u003eg/mL zeocin) with stationary incubation for 7 days. Diagnostic PCRs were further utilized to confirm the genotype of transformants [37, 39]. The specific primers for the construction of deletion cassettes and deletants verification are listed in \u003cb\u003eTable S1\u003c/b\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cb\u003eFermentation, extraction, LC-MS, and GNPS analysis\u003c/b\u003e\u003c/p\u003e\n\u003cp\u003eThe mutant and the wild-type strain CPCC 401591 were initially cultivated on PDA plates for 7 days. Subsequently, 1×10\u003csup\u003e8\u003c/sup\u003e fungal spores were inoculated in 100 mL PDB broth and incubated on a rotary shaker at 200 rpm to afford a seed culture. Then, 10 mL seed culture was inoculated in rice fermentation medium and cultured at 30°C for thirty days, respectively. The fermented material was extracted three times with ethyl acetate, and the organic layer was concentrated to dryness under vacuum. The extract was redissolved in 1 mL acetonitrile, and the sample (5 \u003cem\u003eµ\u003c/em\u003eL) was analyzed by high-performance liquid chromatography analysis (Agilent InfinityII 1260, Agilent Eclipse SB-C18 column, 5 \u003cem\u003eµ\u003c/em\u003em, 4.6 × 250 mm).\u003c/p\u003e\n\u003cp\u003eLC-MS/MS measurements were performed using Thermo Fisher Scientific LTQ Orbitrap\u003csup\u003eXL\u003c/sup\u003e mass spectrometer equipped with a heated electrospray ion source (HESI). Chromatographic separation was acquired using a reversed-phase Agilent Eclipse SB-C18 column (5 \u003cem\u003eµ\u003c/em\u003em particle size, 4.6×250 mm). The elution was conducted with a linear gradient of 10 − 100% acetonitrile-water (v/v) at a flow rate of 1.0 mL/min\u003csup\u003e− 1\u003c/sup\u003e for 25 min, followed by a 5 min wash with 100% acetonitrile for 5 min. MS/MS spectra were achieved using electrospray ionization in positive ion mode. The optimized detection parameters were set as follows: ion spray voltage, 4.5 kV; ion source temperature: 550°C; capillary temperature, 220°C; scan range of \u003cem\u003em\u003c/em\u003e/\u003cem\u003ez\u003c/em\u003e: 150 − 2000 Da, profile mode; a high collision energy of 45 eV was employed for fragmentation. HPLC solvent gradients and MS scan functions were controlled using the Xcalibur data system (Thermo Fisher Scientific).\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cb\u003eMining and bioinformatic analysis of the gene cluster\u003c/b\u003e \u003cb\u003epsd\u003c/b\u003e \u003cb\u003ein\u003c/b\u003e \u003cb\u003eStachybotrys\u003c/b\u003e \u003cb\u003esp.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo characterize the genetic locus responsible for phenylspirodrimanes biosynthesis, we initially performed the whole genome sequencing of the strain \u003cem\u003eStachybotrys\u003c/em\u003e sp. The Illumina HiSeq 2500 sequencing of the fungus generated 276 scaffolds, which concluded 38.44\u0026nbsp;million non-redundant bases. In the course of antiSMASH-based genome mining endeavors in the fungus, one unique meroterpenoid biosynthetic cluster was acquired, which encodes a conserved (\u0026gt;\u0026thinsp;74.69% identity) non-reducing polyketide synthase (NR-PKS, PsdA), adenylate-forming reductase (PsdB), and UbiA-family prenyltransferase (PsdC) ensemble. It has been reported that a 5-methyl orsellinic acid encoding NR-PKS, a prenyltransferase, and an NRPS-like reductase could function collaboratively to produce ilicicolin B, an on-pathway intermediate during the process of ascochlorin and stachybisbin B biosyntheses [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. This cluster is designated as \u003cem\u003epsd\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cb\u003eTable S2\u003c/b\u003e, Supplementary Information).\u003c/p\u003e\u003cp\u003eFurthermore, investigation of the bilateral regions of the skeleton forming genes \u003cem\u003epsdA\u003c/em\u003e, \u003cem\u003epsd\u003c/em\u003eB, and \u003cem\u003epsdC\u003c/em\u003e supported the discovery of other genes encoding post-modification enzymes. The enzymes included one 3-dehydroshikimate dehydratase, one short-chain dehydrogenase/reductase, one oxidoreductase, two methyltransferases, one O-acetyltransferase, one MFS-type transporter, and one pathway-specific transcriptional regulatory factor that correspond to the structural features of phenylspirodrimanes. Nevertheless, the gene cluster \u003cem\u003epsd\u003c/em\u003e was inserted by many genes that encode proteins involved in primary metabolism or obviously unrelated to the biosynthesis of the PSDs, exemplified as gene 08153 (coding glycosyl hydrolase), gene 08166 (coding alpha-L-arabinofuranosidase), gene 08170 (coding neutral protease). Therefore, these redundant genes were easily eliminated. In the light of the PsdA, PsdB, and PsdC encoded by the cluster \u003cem\u003epsd\u003c/em\u003e exhibited highly similar identities with cluster \u003cem\u003estb\u003c/em\u003e and \u003cem\u003easc\u003c/em\u003e, involved in biosynthesizing L-Z1272β and ascochlorin, respectively [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. We hypothesize that the BGC \u003cem\u003epsd\u003c/em\u003e might be responsible for phenylspirodrimanes biosynthesis.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCharacterization of the cluster\u003c/b\u003e \u003cb\u003epsd\u003c/b\u003e \u003cb\u003eresponsible for PSDs biosynthesis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo verify the participation of PsdA in the generation of phenylspirodrimanes, deletion of the NR-PKS coding gene \u003cem\u003epsdA\u003c/em\u003e was performed. We established a protoplast transformation-based gene disruption experiment to support the proposal that \u003cem\u003epsdA\u003c/em\u003e is functioning in PSDs biosynthesis [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The construction of the knockout plasmid was conducted using the scaffold of pAg1-ble by replacing the bilateral fragments of the BleR selective cassette with homologous arms of the target gene \u003cem\u003epsdA\u003c/em\u003e. Next, after protoplast transformation experiment, of thirty-eight putative zeocin\u003csup\u003e+\u003c/sup\u003e \u003cem\u003eStachybotrys\u003c/em\u003e transformants analyzed, the mutant \u003cem\u003eΔpsdA\u003c/em\u003e-16 was verified by diagnostic PCRs characterization with four primer sets. With one Zeo\u003csup\u003eR\u003c/sup\u003e specific primer set, the mutant gave two amplicons of 1,300 bp and 1,392 bp in size, respectively, while the WT had no amplicons (\u003cb\u003eFigure S2\u003c/b\u003e, Supporting Information). Further confirming using one primer set resided in the deletion region of the gene \u003cem\u003epsdA\u003c/em\u003e, two amplicons with 4,553 bp and 4,533 bp in size were recovered from WT, while no amplicons were obtained from the mutant (\u003cb\u003eFigure S2\u003c/b\u003e, Supporting Information).\u003c/p\u003e\u003cp\u003eThe thus-acquired crude extracts from \u003cem\u003eΔpsdA\u003c/em\u003e and the parental strain were analyzed using Liquid Chromatography-Mass Spectrometry (LC-MS) analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Knock-out of the \u003cem\u003epsdA\u003c/em\u003e gene completely abolished the generation of the compounds stachybotrylactam (\u003cem\u003et\u003c/em\u003e\u003csub\u003e\u003cem\u003eR\u003c/em\u003e\u003c/sub\u003e=15.15 min) and chartarlactam F (\u003cem\u003et\u003c/em\u003e\u003csub\u003e\u003cem\u003eR\u003c/em\u003e\u003c/sub\u003e=16.45 min), thereby setting up the linkage between the \u003cem\u003epsd\u003c/em\u003e biosynthetic cluster and the yielding of the PSDs. At the same HPLC conditions, the peaks of chartarlactam F and stachybotrylactam detected in the parental strain (WT) that two target compounds were further verified along with the evidence of \u003cem\u003em/z\u003c/em\u003e\u0026thinsp;=\u0026thinsp;386.3 [M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e and \u003cem\u003em/z\u003c/em\u003e\u0026thinsp;=\u0026thinsp;771.2 [2M\u0026thinsp;+\u0026thinsp;H]\u003csup\u003e+\u003c/sup\u003e using LC-MS/MS analysis. However, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the strain \u003cem\u003eΔpsdA\u003c/em\u003e lacked these characteristic molecular ion peaks, indicating the absence of the target two compounds in metabolic profiling of the mutant (\u003cb\u003eFigure S3\u003c/b\u003e, Supporting Information). Collectively, the experimental evidence further corroborated with our prediction regarding the function of the gene cluster \u003cem\u003epsd\u003c/em\u003e, confirming that the gene \u003cem\u003epsd\u003c/em\u003eA is a key gene for PSDs biosynthesis in \u003cem\u003eStachybotrys\u003c/em\u003e sp.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePreliminary investigation of the genetic basis for PSDs biosynthesis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo reveal the expression level of the gene cluster \u003cem\u003epsd\u003c/em\u003e during PSDs biosynthesis on TBI and PDB broth, transcriptome sequencing was performed to characterize the differentially expressed genes (DEGs) of intergroup comparison (TBI/PDB) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). There were 3,874 DEGs, in which the up-regulated and down-regulated genes were 2,053 and 1,821, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Gene Ontology (GO) enrichment analysis manifested that the up-regulated genes were approximately engaged in biological processes including lipid metabolism, organophosphate catabolism, and secondary metabolites biosynthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The KEGG enrichment data demonstrated that the up-regulated genes were significantly enriched in primary metabolic pathways participating in the processes of fatty acid, amino acid, starch, sucrose, glycolysis and gluconeogenesis (\u003cb\u003eFigure S4\u003c/b\u003e, Supporting Information). These pathways were related to primary metabolism, including carbon metabolic reactions for strain growth and secondary metabolism. In addition, significant enrichment was also observed in the biosynthetic pathways of terpenoids and mycotoxins.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eNext, when we further examined the cluster \u003cem\u003epsd\u003c/em\u003e, one phenomenon observed is that there are many dispensable genes within the cluster. Therefore, transcriptomic analyses of the encoded genes might be helpful to distinguish genes directly involved in PSDs biosynthesis. The expression level of \u003cem\u003epsdA\u003c/em\u003e (gene 08149) and \u003cem\u003epsdN\u003c/em\u003e (gene 08175, coding methyltransferase) in TBI sample were significantly up-regulated than PDB condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cb\u003eFigure S5\u003c/b\u003e, and \u003cb\u003eTable S2\u003c/b\u003e, Supporting Information). Further deciphering the genes 08145\u0026ndash;08148 and 08176\u0026ndash;08180, gene 08148 encoded a hypothetical protein and was significantly down-regulated, while genes 08145\u0026ndash;08147, gene 08176, and gene 08180 exhibited no detectable expression level in both conditions. Moreover, the genes (08177\u0026ndash;08179) were annotated as phosphatase, mannitol dehydrogenase and accessory effector CFEM5, respectively. These three genes are obviously not associated with secondary metabolism and could be excluded from further analysis. Therefore, we speculated that \u003cem\u003epsdA\u003c/em\u003e and \u003cem\u003epsdN\u003c/em\u003e should be located on the left and right boundaries of the cluster \u003cem\u003epsd\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSimilarly, statistical analyses revealed that seven genes (\u003cem\u003epsdA\u003c/em\u003e, \u003cem\u003epsdC\u003c/em\u003e-\u003cem\u003eF\u003c/em\u003e, \u003cem\u003epsdJ\u003c/em\u003e and \u003cem\u003epsdN\u003c/em\u003e) were significantly up-regulated (FC\u0026thinsp;\u0026gt;\u0026thinsp;2, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in TBI condition, demonstrating that corresponding coded proteins should participate in PSDs generation. Also, seven genes (\u003cem\u003epsdB\u003c/em\u003e, and \u003cem\u003epsdG\u003c/em\u003e-\u003cem\u003eI\u003c/em\u003e, and \u003cem\u003epsdK\u003c/em\u003e-\u003cem\u003eM\u003c/em\u003e) from both culture conditions exhibited high expression level in TPM (transcripts per million) values and moderately up-regulated (1\u0026thinsp;\u0026lt;\u0026thinsp;FC\u0026thinsp;\u0026lt;\u0026thinsp;2) in TBI sample, indicating these genes might contribute in PSDs biosynthesis or metabolites transport. Based on the transcriptome data, it was supposed that \u003cem\u003epsdI\u003c/em\u003e might serve as regulatory factor, which could trigger the catalysis of proteins encoded by the cluster \u003cem\u003epsd\u003c/em\u003e. In contrast, eleven genes displayed no detectable expression level in both conditions, which are unlikely involved in biosynthesizing PSDs (\u003cb\u003eFigure S5\u003c/b\u003e, \u003cb\u003eTable S2\u003c/b\u003e, Supporting Information). Real-time reverse-transcription PCR further demonstrated that transcriptional level of the cluster \u003cem\u003epsd\u003c/em\u003e in TBI condition exhibited much higher than in PDB broth, in which eight genes including \u003cem\u003epsdA\u003c/em\u003e-\u003cem\u003eF\u003c/em\u003e, \u003cem\u003epsdJ\u003c/em\u003e, and \u003cem\u003epsdN\u003c/em\u003e were selected for investigation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The transcription levels of \u003cem\u003epsdA\u003c/em\u003e, \u003cem\u003epsdE\u003c/em\u003e and \u003cem\u003epsdF\u003c/em\u003e increased remarkably in 3.3, 12.9, and 31.1-fold compared with that of PDB condition, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The transciptional analyses of Psd family genes indicated that TBI broth activated the biosynthesis of PSDs significantly.\u003c/p\u003e\u003cp\u003e\u003cb\u003eInvestigation on the biosynthesis of metabolite chartarlactam K\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePhenylspirodrimanes are unique meroterpenoids featuring a phenylpropylfuran skeleton attached to spirocyclic drimane and a benzene ring moiety [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The genetic basis for the biosynthesis of ascochlorin was well determined previously, and the early-stage biosynthesis of PSDs should be highly identical to that of ascochlorin [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In the light of the molecular structures of stachybisbin B and proposed gene functions of the gene cluster \u003cem\u003epsd\u003c/em\u003e, a metabolic pathway of chartarlactam K was envisioned [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] (Scheme\u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Domains of the megasynthase PsdA are programmed and generate polyketide chains and an offloading thioesterase domain is used for thioester hydrolyzation and chain release. The following steps involve the prenyltransferase PsdC to accomplish the sesquiterpene chain transferring and an NRPS-like reductase PsdB responsible for aldehyde formation to generate precursor ilicicolin B [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In ascochlorin pathway, the intermediate ilicicolin B occurs halogenation catalyzed by AscD and AscE-catalyzed epoxidation for the generation of ilicicolin A, followed by terpene cyclase AscF-mediated cyclization and multiple oxidations [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In contrast, the intermediate ilicicolin B might be epoxidized and cyclized to afford the spirocyclic intermediate F1839-I, which was sequentially catalyzed by an uncharacterized flavin monooxygenase and a terpene cyclase in a different manner [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSubsequently, an additional oxygenation installs the hydroxyl group in the compound F1839-I. Then, the generated intermediate mer-NF5003E is then subjected to oxidative modification, leading to the formation of the precursor stachybotrydial (bearing an O-phthalaldehyde group) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The intermediate stachybotrylactam might be transformed from the precursor stachybotrydial by three successive steps including C22-carboxylation, amination, and dehydration [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Subsequently, an additional oxygenation reaction installs the C-2 hydroxyl group in the intermediate F1839-A [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The final reaction might be catalyzed by O-acetyltransferase PsdJ and then furnishes the acetyl group to yield chartarlactam K (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In addition, based on the PSDs isolated from the strain CPCC 401591, we believed that the intermediate stachybotrydial containing O-phthalaldehyde moiety exhibits high reactivity and could be condensed with varied amino acids to synthesize structurally diverse N-containing derivatives (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e, Supporting Information) [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Considering the diverse class of PSDs, we preferred that multi-step redox cascade occurs during the biosynthetic process [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. This proposal hints to the possibility that redox enzymes might exhibit relatively broad substrate scope during PSDs biosynthesis \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eTo the best of our knowledge, this is the first report to characterize the gene cluster \u003cem\u003epsd\u0026nbsp;\u003c/em\u003efunctioning in biosynthesis of phenylspirodrimanes from\u0026nbsp;\u003cem\u003eStachybotrys\u0026nbsp;\u003c/em\u003especies [2]. This genus has been considered to be a treasure for PSDs discovery, a multitude of bioactive derivatives bearing complexified structural modifications have been isolated and gained increasing interest recently [2, 22, 32]. \u003cem\u003eStachybotrys\u003c/em\u003e is a distinctive fungal genus due to its metabolic versatility and capability to produce diversified secondary metabolites [2, 26, 27]. In this report, genome guided bioinformatic mining of the fungus\u0026nbsp;CPCC 401591\u0026nbsp;revealed the presence of an uncharacterized gene cluster \u003cem\u003epsd\u003c/em\u003e. It carries the conserved locus encoding PsdA, PsdB, and PsdC ensemble, which exhibited higher identities with their counterparts responsible for producing ascochlorin and stachybisbin B, respectively\u0026nbsp;[1, 17].\u0026nbsp;This finding indicated that PsdA/PsdB/PsdC ensemble is believed to be involved in the conversion of shared intermediate ilicicolin B, which is considered to be an on-pathway precursor in the biosynthesis of many fungal meroterpenoids\u0026nbsp;[1, 12, 17, 20].\u0026nbsp;A more detailed cluster blast bioinformatic analysis was conducted on genome-sequenced filamentous fungi from NCBI database. Interestingly, all these ilicicolin B-forming genes are associated with the three-gene ensemble. This ensemble consists of genes that encode an orsellinic acid synthase, a UbiA-like prenyltransferase, and an NRPS-like reductase\u0026nbsp;[1, 17]. To further investigate the phylogeny, three conserved proteins were selected for Multi-Locus Sequence Typing (MLST) phylogenetic tree construction. As shown in \u003cstrong\u003eFigure 5\u003c/strong\u003e, markedly, the species \u003cem\u003eStachybotrys\u003c/em\u003e clustered as a well-supported branch, which constitute an important genus for producing PSDs\u0026nbsp;[6, 12, 20-23, 33, 35, 42]. In addition, the biosynthetic pathways with ilicicolin B serving as pathway intermediate are widely distributed in fungal strains, exemplified as stachybisbins from closely related \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ebisbyi\u003c/em\u003e PYH05-7, ascochlorin and ascofuranone from\u003cem\u003e\u0026nbsp;Acremonium egyptiacum\u003c/em\u003e F-1392\u0026nbsp;[1, 17]. This further implies mining other fungal strains clustered in different branch might supply the opportunities to discover novel ilicicolin B-derived meroterpenoids.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNature’s power as a chemist is well represented by the structures of PSD derivatives, which are featured by a distinct spirocyclic drimane fused to a phenyl substituent, and particularly a characteristic spirofuran ring framework [12, 20, 22, 42].\u0026nbsp;Nevertheless, no report has described the genetic bases of this class of hybrid metabolites\u0026nbsp;[12]. A more detailed investigation of the gene cluster \u003cem\u003epsd\u0026nbsp;\u003c/em\u003erevealed that encoding genes are scattered and distributed separately. Further genome mining of the sequenced \u003cem\u003eS\u003c/em\u003e.\u003cem\u003e\u0026nbsp;chartarum\u003c/em\u003e IBT 7711 and \u003cem\u003eS\u003c/em\u003e. \u003cem\u003echlorohalonata\u003c/em\u003e IBT 40285 also revealed the presence of clusters similar to the cluster \u003cem\u003epsd\u0026nbsp;\u003c/em\u003e[17, 43]. Some strains of \u003cem\u003eS\u003c/em\u003e.\u003cem\u003e\u0026nbsp;chartarum\u0026nbsp;\u003c/em\u003ereportedly produces PSDs, such as chartarutines\u0026nbsp;[44], stachybotrins\u0026nbsp;[45], distachydrimanes\u0026nbsp;[28], bistachybotrysins\u0026nbsp;[7, 23, 45], and therefore, this cluster is also expected to be responsible for the production of phenylspirodrimanes in \u003cem\u003eS\u003c/em\u003e.\u003cem\u003e\u0026nbsp;chartarum\u003c/em\u003e [12]. Considering the presence of several unrelated genes in the cluster \u003cem\u003epsd\u0026nbsp;\u003c/em\u003eand previous OSMAC experimental data,\u0026nbsp;therefore, we tried to distinguish the redundant genes using transcriptomic analyses. Notably, among the co-localized twenty-seven open reading frames in the gene cluster\u003cem\u003e\u0026nbsp;psd\u003c/em\u003e, thirteen down-regulated, not expressed, or silent genes were efficiently differentiated. Although the experimental evidence was preliminary, mapping the differential expression profiles could shed light on identification of candidate genes responsible for PSDs biosynthesis. Regarding the metabolic pathway in generating F1839-I from intermediate\u0026nbsp;ilicicolin B, two key reaction steps might be included, \u003cem\u003ee\u003c/em\u003e.\u003cem\u003eg\u003c/em\u003e., epoxidation catalyzed by flavin monooxygenase, and cyclization catalyzed by membrane-bound terpene cyclase\u0026nbsp;[1, 12, 17, 28, 40]. Nevertheless, these encoded genes could not be obtained in this cluster\u0026nbsp;[17]. With transcriptome sequencing data, two separate gene clusters are obtained that most likely participated in the catalytic process, and this need to be further characterized through gene deletions. Ultimately, the underlying mechanism of PSDs biosynthesis would be uncovered in the near future.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eUsing a genome mining strategy, we clarified the gene cluster \u003cem\u003epsd\u003c/em\u003e functioning in phenylspirodrimanes biosyntheses in\u0026nbsp;\u003cem\u003eStachybotrys\u003c/em\u003e sp. The biosyntheses of PSDs and ascochlorin share the common pathway up to the generation of the pathway intermediate ilicicolin B. Also, a bioinformatic analysis of the BGC\u003cem\u003e\u0026nbsp;psd\u0026nbsp;\u003c/em\u003ewas performed. Notably, RNA-sequencing based transcriptomic profiling facilitated to decipher intriguing characteristics of PSDs biosyntheses and eliminate the unrelated genes. This study thus contributes to broadening the knowledge on biosynthesizing this group of meroterpenoids. We have established the method for the genetic manipulation of the fungus\u0026nbsp;\u003cem\u003eStachybotrys\u003c/em\u003e, and further manipulation of encoding genes of the \u003cem\u003epsd\u003c/em\u003e locus will be conducted in our laboratory.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAUTHOR INFORMATION\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorresponding Authors\u003c/p\u003e\n\u003cp\u003eTao Zhang -\u0026nbsp;\u003cem\u003eInstitute of Medicinal Biotechnology\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Chinese Academy of Medical Sciences \u0026amp; Peking Union Medical College, Beijing 100050, China;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eEmail:\u0026nbsp;[email protected]\u003c/p\u003e\n\u003cp\u003eLiyan Yu -\u0026nbsp;\u003cem\u003eInstitute of Medicinal Biotechnology\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Chinese Academy of Medical Sciences \u0026amp; Peking Union Medical College, Beijing 100050, China;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eEmail:\u0026nbsp;[email protected].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT.Z. conceived the study, obtained initial funding. T.Z., and L.Y. supervised the work to completion. T.Z., H.L., and B.S. performed experiments and analyzed the data. L.B., W.H., X.F. and L.W. advised on the design and partial interpretation of data. T.Z., and H.L. wrote the draft manuscript. T.Z., and L.Y. edited the draft manuscript. All authors reviewed the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by the CAMS Innovation Fund for Medical Sciences\u0026nbsp;(2021-I2M-1-055),\u0026nbsp;the National Natural Science Foundation of China (No. 31872617), National Microbial Resource Center (No. NMRC-2025-3),\u0026nbsp;and the central level, scientific research institutes for basic R \u0026amp; D fund business (3332018097). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNotes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing\u0026nbsp;financial interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupporting Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExperiment details and spectroscopic data. This material is available free of charge via the Internet at http://pubs.acs.org.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBhowmik P, Baezid HM, Arabi, II. Assessment of antidiabetic activity of three phenylspirodrimanes from fungus \u003cem\u003eStachybotrys chartarum\u003c/em\u003e MUT 3308 by ADME, QSAR, molecular docking and molecular dynamics simulation studies against protein tyrosine phosphatase 1B (PTP1B). \u003cem\u003eJ Biomol Struct Dyn \u003c/em\u003e2024, 42:10210-10224.\u003c/li\u003e\n\u003cli\u003eIbrahim SRM, Choudhry H, Asseri AH, Elfaky MA, Mohamed SGA, Mohamed GA. \u003cem\u003eStachybotrys chartarum\u003c/em\u003e-a hidden treasure: secondary metabolites, bioactivities, and biotechnological relevance. \u003cem\u003eJ Fungi (Basel) \u003c/em\u003e2022, 8.\u003c/li\u003e\n\u003cli\u003eLi Y, Wu C, Liu D, Proksch P, Guo P, Lin W. 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Stachybotrins G and H, two new phenylspirodrimane derivatives from the fungus \u003cem\u003eStachybotrys chartarum\u003c/em\u003e. \u003cem\u003ePlanta Med \u003c/em\u003e2025.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Phenylspirodrimanes, genome mining, gene deletion, transcriptome, biosynthesis, Stachybotrys","lastPublishedDoi":"10.21203/rs.3.rs-7289101/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7289101/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Phenylspirodrimanes (PSDs) are a unique class of meroterpenoids that have been extensively studied due to their diverse biological activities. These metabolites are supposedly biosynthesized through the farnesylation of orsellinate. However, the molecular basis has not yet been elucidated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Herein, using a combination of genome mining, bioinformatic alignment, and gene deletion approach, we characterized the dedicated gene cluster \u003cem\u003epsd\u003c/em\u003e responsible for biosynthesizing PSDs in the fungus \u003cem\u003eStachybotrys\u003c/em\u003e sp. CPCC 401591. RNA sequencing-based transcriptomic analyses broadened our understanding of the genetic basis, regulation, and mechanisms of PSDs biosynthesis. Furthermore, based on the chemical structures of PSD derivatives characterized and deduced gene functions of the cluster \u003cem\u003epsd\u003c/em\u003e, a hypothetical metabolic pathway for biosynthesizing chartarlactam K was proposed. These results revealed the underlying mechanism of phenylspirodrimanes generation, and these findings might facilitate downstream metabolic engineering studies of PSDs or the production of new chemical entities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e Genome discovery and gene deletion experiments unveiled a previously uncharacterized biosynthetic gene cluster \u003cem\u003epsd\u003c/em\u003e involved in the biosynthesizing PSD derivatives in \u003cem\u003eStachybotrys\u003c/em\u003e sp. CPCC 401591. In addition, based on the gene cluster data and transcriptomic analyses, a hypothetical biosynthetic pathway of chartarlactam K was put forward.\u003c/p\u003e","manuscriptTitle":"Characterization of the unconventional meroterpenoid gene cluster from Stachybotrys sp. 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