A hybrid assembly mechanism employs nonreducing polyketide synthase-like logic for partially reduced polyketide formation
Xianyan Zhang, Chuanteng Ma, Ziguang Deng, Xingtao Ren, Kaijin Zhang, Wenxue Wang, Luning Zhou, Yongchun Zhu, Guojian Zhang, Qian Che, Tianjiao Zhu, Hongan Long, Bo Dong, Dehai Li
TL;DR
A new type of fungal enzyme, PbPKS1, was discovered that uses a unique assembly mechanism to produce partially reduced polyketides, expanding our understanding of these enzymes.
Contribution
The discovery of a non-canonical partially reducing polyketide synthase (nPR-PKS) with a novel domain arrangement and assembly mechanism.
Findings
PbPKS1 produces monohydroxybenzoic acids and pyrones through a nonreducing polyketide synthase-like mechanism.
PbPKS1 belongs to a new nPR-PKS family that likely evolved from nonreducing polyketide synthases via gene recombination.
The enzyme features a product template domain for cyclization and a thioesterase domain for product release.
Abstract
Fungal iterative type I polyketide synthases (iPKSs) are commonly classified into nonreducing (NR-), partially reducing (PR-), and highly reducing (HR-) polyketide synthases based on their assembly mechanisms and domain structures. These iPKSs have been considered functionally and evolutionarily distinct, characterized by clear boundaries. However, emerging genomic analyses suggest that the diversity of iPKSs in fungi is far from fully understood. Here, we describe the discovery and characterization of PbPKS1 from a marine-derived fungus Penicillium brocae HDN12-143, which exhibits an atypical domain organization arranged as KR-KS-AT-PT-ACP1-ACP2-CMeT-TE. Heterologous expression of PbPKS1 resulted in the production of two monohydroxybenzoic acids and two pyrones. In vivo and in vitro characterizations demonstrated that PbPKS1 has the capability to synthesize Cα-methylated partially…
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Taxonomy
TopicsMicrobial Natural Products and Biosynthesis · Plant biochemistry and biosynthesis · Fungal and yeast genetics research
Introduction
Type I polyketide synthases (PKSs) are multifunctional mega-enzymes that play a crucial role in the biosynthesis of structurally diverse and bioactive natural products denoted as polyketides (Keatinge-Clay 2012). Possibly derived from common ancestors, type I PKSs share a catalytic logic similar to that of fatty acid synthases (FASs) in primary metabolism, catalyzing the condensation of simple short-chain carboxylic acid precursors to form either linear or cyclic polyketides (Smith and Tsai 2007). Based on the reuse of catalytic modules, type I PKSs are subdivided into iterative PKSs (iPKSs) and assembly line PKSs (also referred to as modular PKSs), wherein iPKSs iteratively catalyze multiple chain elongation cycles employing the same set of enzymatic domains (Grininger 2023; Herbst et al. 2018; Nivina et al. 2019). Notably, assembly line PKSs predominate in bacteria, while iPKSs are primarily found in fungi, although several bacterial iPKSs have also been identified (Ahlert et al. 2002; Chen and Du 2016; Wang et al. 2020). Fungi-derived iPKSs are the basis for the production of enormously diverse and bioactive fungal polyketides, including the pharmaceutical drugs lovastatin and mycophenolic acid (Campbell and Vederas 2010; Regueira et al. 2011). Furthermore, since they bridge the functions of FASs and assembly line PKSs, fungal iPKSs have become central to recent advancements in polyketide research (Grininger 2020; Herbst et al. 2018; Tsai 2018).
In the biosynthetic logic of iPKSs, the minimal enzyme for polyketide chain assembly requires the collaboration of a ketosynthase (KS) domain, a malonyl-CoA:ACP acyltransferase (AT) domain, and an acyl carrier protein (ACP) domain (Hertweck 2009). Moreover, the β-ketoacyl intermediate bound to ACP can be further modified through extra processes involving ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains, to yield an acyl intermediate with different levels of reduction, and an in-line C-methyltransferase (CMeT) domain for the exclusive methylation of C_α_-methylene (Cox 2023; Zhang and Liu 2016). Based on domain architecture and assembly mechanisms, iPKSs can further be categorized into nonreducing (NR-), partially reducing (PR-), and highly reducing (HR-) types (Fig. 1) (Cox 2007; Cox et al. 2018; Nicholson et al. 2001). This classification reflects the evolutionary divergence of iPKSs, resulting in distinct structural and functional characteristics. HR-PKSs, containing fully reducing domains (KR, DH, and ER) in addition to the minimal PKS domains, are responsible for generating diverse linear and cyclic nonaromatic compounds (Cox 2023). Conversely, NR-PKSs lack the β-keto processing domains found in HR-PKSs but possess additional unique domains, a starter unit acyltransferase (SAT) for initial unit loading (Ahuja et al. 2012; Crawford et al. 2006; Crawford et al. 2008a, b) and a product template (PT) domain for cyclization and aromatic ring formation (Crawford et al. 2009; Li et al. 2010; Liu et al. 2015). Usually, NR-PKSs also possess a C-terminal off-loading domain, such as a thioesterase (TE), a Claisen-like cyclase (CLC), or a reductase (R) (Liu et al. 2017; Little and Hertweck 2022). Compared to the diverse NR- and HR-PKSs, the PR-PKSs are only associated with the production of 6-methylsalicyclic acid (6-MSA) in fungi (Fujii et al. 1996), although the PR-PKSs contribute more products in bacteria (Sun et al. 2012). All of the identified PR-PKSs both in bacteria and fungi so far possess a settled domain organization of KS-AT-TH-KR-ACP, without the DH and ER domains and only KR for initial β-keto reduction (Kage et al. 2015; Soehano et al. 2014). As a distinctive characteristic, the PR-PKSs use the thioester hydrolase (TH) domain to catalyze the hydrolysis and release the polyketide chain from the ACP as opposed to the TE domain in NR-PKSs (Moriguchi et al. 2010). It is noteworthy that the PT in NR-PKSs and TH in PR-PKSs share homology with the DH domain, indicating the divergence of DH domain function among the different classes of iPKSs.Fig. 1. Classification, domain architecture, and representative products of fungal iterative polyketide synthases. SAT starter unit acyltransferase, KS ketosynthase, AT malonyl-CoA:ACP acyltransferase, ACP acyl carrier protein, PT product template, TE thioesterase, TH thioester hydrolase, KR ketoreductase, DH dehydratase, ER enoylreductase, CMet C-methyltransferase
Over the past 2 decades, research into the genetics and enzymology of iPKSs has underscored their adaptability and capacity to produce structurally diverse polyketides. Achievements in genome mining have led to the discovery of novel PKSs as well as unexpected products, some with unique release modes or new initiation units (Bunnak et al. 2019; Liu et al. 2023; Winter et al. 2015). Despite these strides, advances in genome sequencing technology revealed that the full potential of iPKSs remains underexplored. In this study, we present the discovery and characterization of a novel iPKS, PbPKS1, from a marine-derived fungus Penicillium brocae HDN12-143. PbPKS1 features a distinct domain organization of KR-KS-AT-PT-ACP_1_-ACP_2_-CMeT-TE. The unconventional domain rearrangement of PbPKS1 offers the prospect of different products and synthesis mechanisms. Notably, it incorporates a PT domain and a C-terminal TE domain, which are typically associated with NR-PKSs. This unique assembly mechanism of PbPKS1 deviates from the canonical PR-PKSs (cPR-PKSs), thus, introducing a new class of iPKSs. Structural domain and evolutionary analyses suggest that this iPKS class, termed non-canonical PR-PKS (nPR-PKS), may have evolved from NR-PKSs through a domain substitution of KR and SAT. This study sheds light on a previously unrecognized class of iPKS, providing new insights into the evolutionary dynamics and functional diversity of fungal iPKSs.
Materials and methods
Strains and culture conditions
The fungus Penicillium brocae HDN12-143 was grown on potato dextrose agar medium (PDA, BD) at 28 °C for 6 days to extract genome DNA (gDNA). The fungus Aspergillus versicolor HDN11-84 was cultured under the same conditions. The heterologous expression host Aspergillus nidulans LO8030 was cultured at 37 ℃ for 3–4 days on solid CD medium (6 g/L NaNO_3_, 1.52 g/L KH_2_PO_4_, 0.52 g/L MgSO_4_·7H_2_O, 0.52 g/L KCl, 10 g/L glucose, 1 mL/L trace elements solution, 20 g/L agar) with supplementation of 5 mmol/L uracil, 10 mmol/L uridine, 0.125 mg/L riboflavin, and 0.5 mg/L pyridoxine for sporulation. Saccharomyces cerevisiae BJ5464-NpgA was used for in vivo yeast homologous recombination. Escherichia coli BL21(DE3) and BAP1 were used for protein expression; Escherichia coli XL-1 and DH5α were used for DNA manipulation.
Bioinformatics analysis
The homologous NR-PKS gene AvOrsA in A. versicolor HDN11-84 was traced by performing LocalBLAST search (Altschul et al. 1997) in its genome database using NR-PKS OrsA (NCBI accession No. Q5AUX1) as a query sequence. The domain organization of PbPKS1 was analyzed by InterPro (InterPro (ebi.ac.uk)). For constructing phylogenetic trees of the KS domain, a total of 718 biosynthetic gene clusters containing PKS were obtained from the MIBiG database (https://mibig.secondarymetabolites.org/, Version 3.1) and the amino acid sequences of KS domain were extracted from the GBK files. For constructing phylogenetic trees of the KR domain, in addition to the KR domains from PKS, we screened microbial FAS sequences from the NCBI database and extracted the amino acid sequence of the KR domain. The sequences of KS (a total of 1302) and KR (a total of 1029) domains were analyzed and obtained by InterPro. Multiple sequence alignment (MSA) was performed by MAFFT v7.515 and trimmed using trimAl v1.4.1 (Capella-Gutierrez et al. 2009) with the parameter “-automated1” to remove poorly aligned regions. Sequence alignments of PbPKS1-PT and PbPKS1-TE domains (Fig. S24–S25) with their homologous to locate active or cofactor-binding sites were conducted by MEGA7.0 and visualized by ESPript 3.0 (ESPript 3 (ibcp.fr)) (Gouet et al. 1999). Maximum-likelihood phylogenetic reconstruction was performed using RAxML-NG v8.2.12 based on the LG + G8 + F model and visualized by ChiPlot 2 (Kozlov et al. 2019; Xie et al. 2023) and Adobe Illustrator.
General DNA manipulation techniques
P. brocae HDN12-143 and A. versicolor HDN11-84 gDNA were extracted using the CTAB method (Tang et al. 2017). To get the intron-free PbPKS1 or AvOrsA, the mycelia of A. nidulans transformant strains harboring target genes were grinded after freezing with liquid nitrogen and solubilized in TRIzol® Reagent (Ambion, USA); the cDNA was obtained after RNA purification and reverse transcription-polymerase chain reactions (RT-PCR) using Direct-zol™ RNA MicroPrep (Zymo Research, USA) and HiScript III 1st Strand cDNA Synthesis Kit (Vazyme Biotech Co. Ltd, China), respectively. All primers, as well as gene fragments difficult to obtain by PCR, were synthesized at Sangon Biotech Co., Ltd. (China). PCR reactions were performed using the Q5® High-Fidelity DNA Polymerases (New England Biolabs, USA) and Hieff Canace® Gold High-Fidelity DNA Polymerase (Yeasen Biotechnology Co. Ltd, China) according to the manufacturer’s instructions. DNA restriction enzymes were used as recommended by the manufacturer (New England Biolabs, USA).
Plasmids construction
The primers used in this study are listed in Table S1. The plasmids are summarized in Table S2. The PCR fragments of PbPKS1 and AvOrsA genes were amplified from P. brocae HDN12-143 and A. versicolor HDN11-84 gDNA, respectively. Then, the PCR products were purified and co-transformed with Not I/Pac I-digested pANU into S. cerevisiae BJ5464-NpgA for in vivo recombination to get pANU-PbPKS1 and pANU-AvOrsA yeast transformants.
To construct the plasmids for PbPKS1-PT and PbPKS1-TE domain mutation, the synthetic fragments containing mutation sites H_1446_A and S_2487_A were co-transformed with other normally amplified PbPKS1 fragments and linearized pANU into S. cerevisiae BJ5464-NpgA, generating pANU-PbPKS1-DHm and pANU-PbPKS1-TEm, respectively. To construct the PbPKS1-DH, PbPKS1-TE domain complementation plasmids, the PbPKS1-DH* and PbPKS1-TE gene fragments with initiation codon and domain linkers were inserted into BamH I/Pac I-digested pANR to generate pANR-PbPKS1-DH* and pANR-PbPKS1-TE.
For deconstruction protein expression plasmid construction, corresponding intron-free gene fragments with homologous recombination sequences (25–40 bp upstream and downstream) were inserted into Nde I/Xba I-digested pColdI or Nde I/Xba I-digested pColdI-MBP vectors using Seamless Assembly Cloning Kit (CloneSmarter Technologies Inc., USA).
Heterologous expression in A. nidulans
To prepare the protoplasts, the spores of A. nidulans LO8030 were transformed into 30 mL of CD liquid medium with supplementation of 5 mmol/L uracil, 10 mmol/L uridine, 0.125 mg/L riboflavin, and 0.5 mg/L pyridoxine at 37 °C, 220 r/min. After incubation, the mycelia were harvested by centrifugation and washed twice with 15 mL of osmotic medium (1.2 mol/L MgSO_4_, 10 mmol/L NaPO_4_, pH 5.8). Then, the mycelia were transferred into 10 mL of osmotic buffer containing 30 mg lysing enzymes from Trichoderma harzianum (Sigma-Aldrich®, Germany) and 20 mg Yatalase (Takara Bio, JAPAN) in a sterile 150 mL Erlenmeyer flask. The flask was kept in a shaker at 28 °C, 80 r/min for 6 h, and the cells were collected and overlaid gently by 10 mL of protoplast trapping buffer (0.6 mol/L sorbitol, 0.1 M Tris–HCl, pH 7.0). The tube was centrifuged at 4,000 r/min for 15 min, and then the protoplasts were collected from the interface of the two buffers and washed using 20 mL of STC buffer (1.2 mol/L sorbitol, 10 mmol/L CaCl_2_, 10 mmol/L Tris–HCl, pH 7.5). After collection by centrifugation, the protoplasts were resuspended in 1 mL STC buffer.
For each transformation, the constructed plasmids used for heterologous expression, domain mutation, or complementation were dissolved in STC buffer and added to 100 μL protoplast resuspension. The mixture was incubated for 60 min on ice, then 600 μL of PEG solution at pH 7.5 (60% PEG4000, 50 mmol/L CaCl_2_ and 50 mmol/L Tris–HCl) was added. The mixture was gently spread on the regeneration dropout solid medium (CD medium with 1.2 mmol/L sorbitol and appropriate supplements) and incubated at 37 °C for about 3 days to obtain transformants. The transformants were cultured on solid CD medium for 2–3 days at 37 °C for sporulation. The A. nidulans strains constructed in this work are listed in Table S3.
Fermentation of A. nidulans transformants
The spores of A. nidulans transformants were inoculated on CD-ST solid medium and cultivated for 4 days at 28 °C. The fermentation medium was extracted with ethyl acetate (EtOAc). After centrifugation, the organic phase was collected and dried by SpeedVac, and then solid residue was dissolved in methanol for analysis or compounds’ isolation.
Expression and purification of PbPKS1 and AvOrsA deconstruction proteins from E. coli
Deconstruction proteins were heterologously expressed from E. coli BL21(DE3) or BAP1; all strains used for protein expression are listed in Table S3. Different E. coli strains were grown overnight in LB medium with 100 µg/mL ampicillin at 37 °C, 220 r/min. 6 mL of the starter culture was inoculated into 600 mL fresh LB medium with 100 µg/mL ampicillin and incubated at 37 °C until the OD_600_ reached 0.4–0.6. After the cultivation system cooled to room temperature, protein expression was induced with 150 µmol/L isopropyl-β-D-thiogalactoside (IPTG) at 16 °C, 220 r/min for 20 h. The cells were harvested by centrifugation at 4000 r/min, 4 °C for 10 min and frozen at -80 °C for further use.
Purification of other proteins follows identical procedures, with the example of PbPKS1-PT detailed here. The E. coli cells harboring pColdI-PbPKS1-PT were resuspended in 20 mL buffer A (50 mmol/L Tris–HCl, 500 mmol/L NaCl, 10% glycerol, pH 7.5) and disrupted by sonication on ice. After removing cellular debris, the soluble protein was further purified using Ni–NTA affinity chromatography (HisTrap™ excel, Cytiva, USA) at 4 °C. The recombinant His_6_-tagged fusion protein was obtained after washing with elution buffer (containing 100 mmol/L imidazole in buffer A). The purified protein PbPKS1-PT was passed through a PD-10 desalting column (Sephadex™ G-25 M, Cytiva, USA) and eluted with buffer C. Concentrated protein was obtained after using a 30-kDa ultrafiltration centrifugal tube. All the proteins were quickly frozen in liquid nitrogen and saved at − 80 °C. The purity of proteins was checked by SDS-PAGE (Figs. S21, S29), and protein concentration was determined by NanoPhotometer® (Implen, Germany).
In vitro reconstitution of PbPKS1 and AvOrsA
The reaction of PbPKS1 reconstitution was performed in a 50 µL mixture with 10 μmol/L PbPKS1-KR-KS-AT, 10 μmol/L PbPKS1-PT, 10 μmol/L PbPKS1-ACP_1_-ACP_2_-MT-TE, 2 mmol/L acyl-CoA, 2 mmol/L n-propionyl CoA, 2 mmol/L malonyl-CoA, 2 mmol/L SAM and 1 mmol/L NADPH in buffer C (pH 7.5) solution and was incubated at 28 °C for 8 h. Meanwhile, the PbPKS1 reconstitution reaction was performed with 10 μmol/L PbPKS1-KR, 10 μmol/L PbPKS1-KS-AT, 10 μmol/L PbPKS1-PT, and 10 μmol/L PbPKS1-ACP1-ACP2-MT-TE deconstruction proteins; the other substrates, cofactors and reaction conditions were the same as above. The control assay was performed without any proteins (Fig. S23).
The reaction of AvOrsA reconstitution was performed in the 50 µL mixture with 10 μmol/L AvOrsA-SAT-KS-AT, 10 μmol/L AvOrsA-PT, 10 μmol/L AvOrsA-ACP_1_-ACP_2_-TE, 2 mmol/L acyl-CoA and 2 mmol/L malonyl-CoA in buffer C (pH 7.5) solution and was incubated at 28 °C for 8 h. The control assay was performed without any proteins.
In vitro AvOrsA-PT domain swapping
The reaction was performed in a 50 µL reaction mixture with 10 μmol/L AvOrsA-SAT-KS-AT, 10 μmol/L PbPKS1-PT, 10 μmol/L AvOrsA-ACP_1_-ACP_2_-TE, 2 mmol/L acyl-CoA and 2 mmol/L malonyl-CoA in buffer C (pH 7.5) solution, and was incubated at 28 °C for 8 h. The control assay was performed without PbPKS1-PT, with other conditions the same. All the reactions described above were quenched by mixing with equal volume of methanol and the products were analyzed by LC–MS.
Analysis of metabolites
LC–MS was performed on Waters ACQUITY H-Class UPLC-MS system equipped with a PDA detector and an SQD2 mass spectrometer (MS) detector using a reversed-phase C18 column (ACQUITY UPLC® BEH, 1.7 µm, 2.1 mm × 50 mm, Waters). The general analysis methods in this work were performed with a linear gradient of 5%–99% MeCN-H_2_O with 0.02% formic acid over 15 min followed by 99% MeCN for 3 min with a flow rate of 0.5 mL/min.
Results
Discovery and functional verification of PbPKS1
Penicillium species possess a capacity for secondary metabolic production and are prominent producers of polyketides. We performed genome mining on our local database and a marine-derived fungus Penicillium brocae HDN12-143 (accession number: OQ874515) which harbors a total of nineteen iPKS genes (PbPKS1-19, Table S1), including seven NR-PKSs and eleven HR-PKSs. Specifically, there is a gene encoding a unique PKS, PbPKS1 (accession No. PP941848), which displays a domain organization distinguished from any other known iPKSs, defined as KR-KS-AT-DH*-ACP_1_-ACP_2_-CMeT-TE (Fig. 2A). Typically, the KR domain resides within the interior of PR- and HR-PKSs, while PbPKS1 processes an unusual N-terminal KR domain. Moreover, a structure with two tandem ACP domains is mostly observed in NR-PKSs, but rarely in PR- and HR-PKS. All these abnormalities predict that PbPKS1 may produce nontrivial products. To identify the corresponding products, we expressed the PbPKS1 gene in a commonly used host, Aspergillus nidulans LO8030 (Chiang et al. 2022). Subsequent LC–MS analysis of the transformant AN-PbPKS1 revealed the production of four metabolites (Fig. 2B, trace ii). These compounds were isolated and determined through nuclear magnetic resonance (NMR) and mass spectrometry (MS) data as 2-hydroxy-3,6-dimethylbenzoic acid (also called JBIR-26, 1a), 4-hydroxy-3,6-dimethyl-2-pyrone (2a), marilactone (2b), (Izumikawa et al. 2008; Almeida et al. 2013; Smetanina et al. 2017), and a novel compound 1b (Fig. 2C).Fig. 2. PbPKS1 is a novel fungal iPKS with an unprecedented domain architecture. A Domain architecture of PbPKS1. B LC–MS analysis of culture extracts from A. nidulans strains expressing PbPKS1 and its mutants. Extracted ion chromatograms (EICs) correspond to m/z 165 [M + H]⁺ (1a), m/z 179 [M + H]⁺ (1b), m/z 139 [M + H]⁺ (2a), and m/z 153 [M + H]⁺ (2b). C Structures of compounds isolated from A. nidulans expressing PbPKS1
Based on their structural features, these compounds can be categorized into two groups: tetraketides 1a/1b, which possess an aromatic ring scaffold, and triketides 2a/2b, which contain an α-pyrone scaffold. The discrepancy between 1a and 1b or 2a and 2b lies in their potential use of different starter units. However, recent research on the NR-PKS UttA from Aspergillus ustus indicated that this discrepancy may stem from the CMeT domain catalyzing methylation at the outset of the starter unit (Zheng et al. 2020). According to the biosynthetic logic of iPKS, the formation of tetraketides 1a/1b involves a β-keto reduction process catalyzed by the KR domain in the second round of extension, analogous to the biosynthesis of 6-MSA. However, it appears that the KR domain is dispensable for the formation of triketides 2a/2b. Consequently, compounds 2a and 2b should be the premature products released early from the assembly line during the formation of 1a and 1b before the KR domain exerts its function.
PbPKS1-DH* domain mediates the cyclization of 1a and 1b
The distinctive domain arrangement of PbPKS1 and its associated compounds indicated an intriguing assembly mechanism of iPKS, differing from the current paradigms. To further elucidate the assembly mechanism of PbPKS1, we investigated the functionality of structural domains beyond the minimal PKS. Given the varying roles of the DH domain in NR-, PR-, and HR-PKSs, we initially focused on the function of the DH* domain in PbPKS1 (PbPKS1-DH*). Phylogenetic analysis of known DH domains revealed that PbPKS1-DH* clustered within PT domains associated with NR-PKS, leading to the hypothesis that PbPKS1-DH* functions as a PT domain (Fig. 3A).Fig. 3. Function verification of PbPKS1-DH* domain. A Phylogenetic analysis of PbPKS1-DH* domain. B Proposed function of PbPKS1-DH* domain. C In vitro function verification of PbPKS1-DH* domain
To evaluate the role of PbPKS1-DH* in the assembly line, we created a DH*-inactive strain AN-PbPKS1(DHm)* through site-specific mutagenesis, substituting His_1446_ with Ala in A. nidulans. LC–MS analysis demonstrated that the H_1446_A mutation completely abolished the production of 1a and 1b, while the yields of compounds 2a and 2b were reduced but still present (Fig. 2B, trace iii). Furthermore, co-expression of the wild-type PbPKS1-DH* with PbPKS1(DHm)* restored the production of 1a and 1b (Fig. 2B, trace iv). These findings suggested that the PbPKS1-DH* is only involved in the biosynthesis of aromatic compounds 1a/1b, rather than pyrones 2a/2b.
Due to the failure to express full-length or truncated PbPKS1 (Fig. S22), it was difficult for us to prove the function of the PbPKS1-DH* domain. Alternatively, we pursued a protein deconstruction and equivalent domain swapping strategy (Crawford et al. 2008a, b). Given the similarity in the cyclization position (C2-C7) between PbPKS1 and OrsA (AN7909) (Feng et al. 2019; Sanchez et al. 2010; Wei et al. 2022), a characterized NR-PKS catalyzing the formation of orsellinic acid (OSA,** 3**), the PbPKS1-DH* domain may be functionally equivalent with OrsA-PT (Fig. 3B) and can be used as an alternative. To test this hypothesis, we cloned a homologous gene of OrsA from Aspergillus versicolor, designated AvOrsA (accession No. PP941849). LC–MS analysis confirmed that expression of AvOrsA resulted in the production of 3 and 4 in A. nidulans as reported previously (Fig. S26) (Feng et al. 2019). We then reconstructed the production of 3 and** 4** in vitro. The deconstructed proteins of AvOrsA-SAT-KS-AT, AvOrsA-PT and AvOrsA-ACP_1_-ACP_2_-TE were successfully expressed in Escherichia coli BAP1 containing a sfp gene from Bacillus subtilis (Pfeifer et al. 2001) and purified by affinity chromatography as C-terminal His_6_-tag fusions. When the complete structural domain construction proteins of AvOrsA were incubated with acetyl-CoA and malonyl-CoA in vitro, the production of 3 and** 4** was detected (Fig. 3C, trace ii). Removal of AvOrsA-PT from the reaction system resulted in the elimination of the production of 3 and 4 (Fig. 3C, trace i); whereas, supplementation with purified PbPKS1-DH* restored the production of both compounds (Fig. 3C, trace iii). These results suggested that the PbPKS1-DH* domain can effectively substitute for the AvOrsA-PT domain and exhibit intramolecular in-line C2–C7 type aldol cyclization activity. Thereafter, the DH* domain of PbPKS1 was redefined as a PT domain.
PbPKS1-TE domain is required for the release of both 1a/1b via hydrolysis and 2a/2b via lactonization
Next, we investigated the function of the C-terminal PbPKS1-TE domain. The TE domain is typically situated at the end of NR-PKS, and is less common in PR- and HR-PKS. After elongation, the TE domain facilitates the liberation of ACP-bound intermediates from PKS enzymes and ensures correct PKS products are formed (Horsman et al. 2016; Xu et al. 2013). The PbPKS1 protein contains a full-length C-terminal TE domain with a conserved catalytic triad (Ser-His-Asp), indicating the enzymatic activity of PbPKS1-TE (Fig. S24) (Caswell et al. 2022; Liu et al. 2017). To confirm the role of PbPKS1-TE in the biosynthesis of compounds 1a, 2a, 1b and 2b, we disrupted the TE domain by mutating the conserved active site Ser_2487_ in the “GXSXG” motif to Ala. The expression of TE-inactive PbPKS1(TEm) in A. nidulans completely abolished the production of all four compounds (Fig. 2B, trace v). Subsequent complementation with the native PbPKS1-TE in A. nidulans harboring PbPKS1(TEm) restored the production of 1a, 1b, 2a and 2b (Fig. 2B, trace vi). These results indicated that the PbPKS1-TE is essential not only for the hydrolysis of 1a/1b but also for the lactonization of 2a/2b, serving as a dual-function enzyme.
Shorter chain length α-pyrone compounds are usually unintended byproducts of iPKSs, spontaneously detaching from the ACP domain without TE domain involvement (Kahlert et al. 2021; Tsai et al. 2001; Vagstad et al. 2012). However, the formation of derailment products 2a/2b appears to be related to the PbPKS1-TE domain. Similar results were also observed in fungal NR-PKS TerA, where mutation of TerA-TE abolished both correct and derailment products (Kahlert et al. 2021). The dual-function TE may act as a corrective mechanism to halt the premature production of default products and ensure the efficient synthesis of the desired products.
Proposed assembly mechanism of PbPKS1
Taken together, we propose the following assembly mechanism for PbPKS1 (Fig. 4). Initially, the PbPKS1-AT domain loads the starter unit onto the KS domain following decarboxylation and optional methylation. Subsequently, an additional molecule of malonyl-CoA is loaded onto the ACP domain as the extension unit. The KS domain facilitates iterative decarboxylative Claisen condensation to form the triketide intermediate 5. This is followed by C_α_-methylation mediated by the CMeT domain and selective reduction of the keto group by the KR domain, producing intermediate 6. Another elongation process is then initiated by the KS domain, generating the tetraketide intermediate 7. The PbPKS1-PT domain catalyzes C2–C7 intramolecular cyclization, while the TE domain facilitates the hydrolysis of the ACP-bound intermediate** 8**, ultimately releasing 1a and 1b. A divergence occurs during the second elongation, where the KR domain is not involved. Instead, the TE domain catalyzes the premature release of the triketide intermediate** 9**, resulting in the formation of derailment products 2a and 2b through lactonization.Fig. 4. Proposed biosynthetic mechanism of PbPKS1
Divergence and evolution of the nPR-PKS
In order to search iPKSs with the same assembly pattern as PbPKS1, we performed a genome mining analysis of available fungal genomes from the National Center for Biotechnology Information (NCBI) database as well as in-house fungal genome sequences. This search reveals several iPKSs with the same structural domain arrangement distributed in several species of filamentous fungi. For better understanding the evolution and differentiation of this kind of iPKSs, a phylogenetic tree of KS domains from PbPKS1 homologs and fungal iPKSs from the NCBI database was constructed (Grininger 2020; Nivina et al. 2019). The analysis showed that KS domains from PbPKS1 and its homologs are more closely related to those from NR-PKS, suggesting that this kind of iPKSs may have diverged and evolved from NR-PKS (Fig. S27). Taken together, we therefore refer to PbPKS1 and its homologs as non-canonical PR-PKSs (nPR-PKSs). Furthermore, consistent with prior reports, it was observed that fungal cPR-PKSs are more closely related to bacteria-derived PR-PKSs than fungal NR- and HR-PKSs according to the genealogy of KS domains, providing support for an origin from actinobacteria via horizontal gene transfer (Kroken et al. 2003).
Considering the distinct features of nPR-PKSs and NR-PKSs, which are characterized by the replacement of the SAT domain with the N-terminal KR domain, we hypothesize that homologous recombination between structural domains has driven the divergence of NR-PKS to nPR-PKS. To elucidate the genealogy of the KR domains in nPR-PKS, we conducted a phylogenetic analysis comparing KR domains from iPKSs, modular PKSs, and FAS. Our results indicate that the KR domains from nPR-PKS show the closest affinity with those from fungal cPR-PKS, suggesting that nPR-PKS may have acquired the KR domain from cPR-PKS (Fig. S28). Taken together, the nPR-PKS likely evolved from the fungi NR-PKs through a fortuitous domain substitution with cPR-PKS.
Discussion
PbPKS1 represents a novel class of fungal iPKSs
Fungal-derived iPKSs exhibit the capacity to produce a variety of bioactive natural products. Compared to type I modular PKSs in bacteria, iPKSs are far more intricate and necessitate the specific coordination of different structural domains throughout the iterative procedure. Nicholson et al. introduced the classification of iPKS as NR-, PR-, and HR-PKS in 2000 (Nicholson et al. 2001), based on the understanding of fungal iPKSs, and this classification has been extensively used ever science. Although structural domain compositions and assembly mechanisms vary significantly among different types of iPKSs, each exhibits a distinct domain organization and assembly model within its group. Despite considerable advances in the study of iPKSs in recent years, no iPKS has been identified that extends beyond current classifications or traditional assembly modes.
In this work, we identified a non-canonical iPKS named PbPKS1, which features a KR-KS-AT-PT-ACP_1_-ACP_2_-CMeT-TE domain organization that deviates from previous classifications in structural domain composition. Heterologous expression of PbPKS1 demonstrated its role in producing partially reducing compounds 1a/1b and derailment pyrones 2a/2b. In vivo and in vitro experiments indicated that PbPKS1 utilizes the PT domain to catalyze the intramolecular C2–C7 aldol cyclization of reduced ACP-bound intermediates and the TE domain for product release.
Given the unique domain arrangement and assembly mechanism, PbPKS1 is distinguished from the well-known canonical PR-PKS (cPR-PKS) (Fig. 1). In cPR-PKS biosynthetic programming for the production of 6-MSA, the inner KR domain reduces the keto group, followed by a subsequent aldol cyclization to form the aromatic ring through dehydration (Moriguchi et al. 2008). Finally, the TH domain catalyzes the release of 6-MSA via hydrolysis (Moriguchi et al. 2010). In contrast, PbPKS1 employs an assembly mechanism akin to NR-PKS, where the PT domain drives aromatic scaffold formation and the TE domain facilitates product release. This unique assembly mode of PbPKS1 is distinct from canonical PR-PKSs but resembles NR-PKSs more closely. Notably, PbPKS1 features an N-terminal KR domain instead of the SAT domain found in NR-PKSs, facilitating keto reduction in the second extender and resulting in partially reducing products. This hybrid architecture establishes nPR-PKS as a fourth iPKS class, diverging from both PR-PKSs and NR-PKSs in domain topology and catalytic logic.
Cis-acting domain integration as a uniquely fungal strategy for polyketide diversification
Previous research on the biosynthesis of actinomycete-derived maduropeptin (10), an enediyne antitumor antibiotic, reveals the formation of partially reduced polyketides with C_α-methylation, which requires a cassette of three enzymes (Fig. 5A) (Ling et al. 2010). Maduropeptin contains a 3,6-dimethylsalicylic acid (1a) moiety, which is derived from 6-MSA catalyzed by the cPR-PKS MdpB. Subsequently, 6-MSA is activated as a CoA thioester by a CoA ligase MdpB2. The stand-alone methyltransferase MdpB1 acts on the CoA-tethered 6-MSA and catalyzes the Cα-methylation to form the 3,6-dimethylsalicylic acid moiety, which is ultimately incorporated into the enediyne core. Unlike cPR-PKSs, which lack intrinsic methyltransferase activity, nPR-PKSs uniquely harbor an embedded methyltransferase (MT) domain. This structural innovation enables autonomous biosynthesis of C_α-methylated partially reduced polyketides within a single enzymatic system.Fig. 5. Biosynthesis of atypical partially reduced polyketides via trans-acting modes in A actinobacteria and B fungi
Furthermore, recent studies have revealed a novel iPKSs architecture in fungi. This architecture comprises a typical NR-PKS (TerA) and a truncated HR-PKS fragment (TerB) containing inactive DH⁰ and CMet⁰ domains alongside a functional KR domain (Fig. 5B) (Kahlert et al. 2021; Ugai et al. 2020). This partnership produces 6-hydroxymellein (11) through a distributed mechanism: TerA synthesizes the polyketide chain, TerB-KR reduces the C9 carbonyl, TerA-PT catalyzes C2-C7 cyclization, and TerA-TE performs lactonization. This represents a fungal strategy borrowing a trans-acting mode, where an NR-PKS cooperates with a trans-acting KR domain to produce partially reduced polyketides.
In contrast to the two aforementioned trans-acting PKS systems, nPR-PKSs integrate a cis-acting KR domain within the NR-PKS scaffold. This integration enables the autonomous production of methylated, atypical partially reduced polyketides by a single enzyme. This consolidated mechanism represents a unique fungal evolutionary innovation for the efficient biosynthesis of structurally diverse, non-canonical polyketides.
The origin and evolution of nPR-PKS
Our findings indicate that nPR-PKSs likely share a closer phylogenetic relationship with NR-PKSs, as evidenced by their analogous assembly mechanisms. This hypothesis was substantiated through evolutionary tree analysis. Phylogenetic evidence indicates KS domains of nPR-PKSs cluster within the NR-PKSs clade, supporting NR-PKS ancestry. However, KR domain genealogy reveals acquisition from fungal cPR-PKSs, implying SAT-to-KR domain substitution via recombination. This modular “domain swap” forged chimeric enzymes that retain NR-PKS cyclization/release machinery while gaining PR-PKS reductive capacity. Strikingly, this endogenous innovation contrasts sharply with the horizontal gene transfer origin proposed for fungal cPR-PKSs, whose KS domains show greater affinity to bacterial homologs. These results validate nPR-PKSs as the authentic, endogenously evolved PR-PKS lineage unique to fungi, distinct from horizontally acquired counterparts. Such recombination events represent fungal-specific adaptations to expand polyketide structural diversity, distinct from strategies reliant on discrete trans-acting enzymes. This paradigm underscores modular domain recombination as a potent mechanism for PKS functional diversification within filamentous fungi.
Conclusions
In conclusion, we have identified and characterized a novel class of iPKSs with a unique structural domain composition that diverges from the well-known NR-, PR-, and HR-PKSs. Our findings reveal that PbPKS1 exhibits the capability to synthesize Cα-methylated partially reducing polyketides and exploits the PT domain to catalyze the formation of the aryl ring and the TE domain for the release of the product. This assembly line pattern of PbPKS1 resembles that of NR-PKSs, representing a new paradigm of fungal PR-PKS. In addition, further evolutionary analysis also indicated that this family of nPR-PKS may have derived from NR-PKSs. Our work not only expanded the diversity of genotypes and assembly patterns of iPKSs, but also offers new insights into the evolution of iPKSs.
Supplementary Information
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