A hybrid assembly mechanism employs nonreducing polyketide synthase-like logic for partially reduced polyketide formation

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 reducing polyketides, yet involved a NR-PKS-like assembly mechanism, featuring a product template (PT) domain for aldol cyclization and a C-terminal thioesterase (TE) domain for product release. Phylogenetic analysis suggests that PbPKS1 belongs to a non-canonical PR-PKS (nPR-PKS) family, which is a minor grouping across the fungal kingdom, and possibly evolved from an NR-PKS through gene recombination. The discovery of nPR-PKS not only expands the diversity of iPKSs but also provides new insights into the evolutionary development of fungal iPKSs.

Keywords

Iterative polyketide synthases (iPKSs) / Product template / Nonreducing PKS (NR-PKS) / Partially reducing PKS (PR-PKS) / Domain recombination

References

[1] Ahlert J, Shepard E, Lomovskaya N, Zazopoulos E, Staffa A, Bachmann BO, Huang K, Fonstein L, Czisny A, Whitwam RE, Farnet CM, Thorson JS. The calicheamicin gene cluster and its iterative type I enediyne PKS. Science, 2002, 297: 1173-1176

[2] Ahuja M, Chiang YM, Chang SL, Praseuth MB, Entwistle R, Sanchez JF, Lo HC, Yeh HH, Oakley BR, Wang CC. Illuminating the diversity of aromatic polyketide synthases in Aspergillus nidulans. J Am Chem Soc, 2012, 134: 8212-8221

[3] Almeida C, Eguereva E, Kehraus S, Konig GM. Unprecedented polyketides from a marine sponge-associated Stachylidium sp. J Nat Prod, 2013, 76: 322-326

[4] Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res, 1997, 25: 3389-3402

[5] Bunnak W, Wonnapinij P, Sriboonlert A, Lazarus CM, Wattana-Amorn P. Heterologous biosynthesis of a fungal macrocyclic polylactone requires only two iterative polyketide synthases. Org Biomol Chem, 2019, 17: 374-379

[6] Campbell CD, Vederas JC. Biosynthesis of lovastatin and related metabolites formed by fungal iterative PKS enzymes. Biopolymers, 2010, 93: 755-763

[7] Capella-Gutierrez S, Silla-Martinez JM, Gabaldon T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics, 2009, 25: 1972-1973

[8] Caswell BT, de Carvalho CC, Nguyen H, Roy M, Nguyen T, Cantu DC. Thioesterase enzyme families: functions, structures, and mechanisms. Protein Sci, 2022, 31: 652-676

[9] Chen H, Du L. Iterative polyketide biosynthesis by modular polyketide synthases in bacteria. Appl Microbiol Biotechnol, 2016, 100: 541-557

[10] Chiang YM, Lin TS, Wang CCC. Total heterologous biosynthesis of fungal natural products in Aspergillus nidulans. J Nat Prod, 2022, 85: 2484-2518

[11] Cox RJ. Polyketides, proteins and genes in fungi: programmed nano-machines begin to reveal their secrets. Org Biomol Chem, 2007, 5: 2010-2026

[12] Cox RJ. Curiouser and curiouser: progress in understanding the programming of iterative highly-reducing polyketide synthases. Nat Prod Rep, 2023, 40: 9-27

[13] Cox RJ, Skellam E, Williams K (2018) Biosynthesis of fungal polyketides. In: Anke, T., Schüffler, A. (eds) Physiology and genetics. The Mycota, vol 15. Springer, Cham, pp 385–412

[14] Crawford JM, Dancy BC, Hill EA, Udwary DW, Townsend CA. Identification of a starter unit acyl-carrier protein transacylase domain in an iterative type I polyketide synthase. Proc Natl Acad Sci, 2006, 103: 16728-16733

[15] Crawford JM, Thomas PM, Scheerer JR, Vagstad AL, Kelleher NL, Townsend CA. Deconstruction of iterative multidomain polyketide synthase function. Science, 2008, 320: 243-246

[16] Crawford JM, Vagstad AL, Ehrlich KC, Townsend CA. Starter unit specificity directs genome mining of polyketide synthase pathways in fungi. Bioorg Chem, 2008, 36: 16-22

[17] Crawford JM, Korman TP, Labonte JW, Vagstad AL, Hill EA, Kamari-Bidkorpeh O, Tsai SC, Townsend CA. Structural basis for biosynthetic programming of fungal aromatic polyketide cyclization. Nature, 2009, 461: 1139-1143

[18] Feng C, Wei Q, Hu C, Zou Y. Biosynthesis of diphenyl ethers in fungi. Org Lett, 2019, 21: 3114-3118

[19] Fujii I, Ono Y, Tada H, Gomi K, Ebizuka Y, Sankawa U. Cloning of the polyketide synthase gene atX from Aspergillus terreus and its identification as the 6-methylsalicylic acid synthase gene by heterologous expression. Mol Gen Genet, 1996, 253: 1-10

[20] Gouet P, Courcelle E, Stuart DI, Metoz F. ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics, 1999, 15: 305-308

[21] Grininger M. The role of the iterative modules in polyketide synthase evolution. Proc Natl Acad Sci, 2020, 117: 8680-8682

[22] Grininger M. Enzymology of assembly line synthesis by modular polyketide synthases. Nat Chem Biol, 2023, 19: 401-415

[23] Herbst DA, Townsend CA, Maier T. The architectures of iterative type I PKS and FAS. Nat Prod Rep, 2018, 35: 1046-1069

[24] Hertweck C. The biosynthetic logic of polyketide diversity. Angew Chem Int Ed Engl, 2009, 48: 4688-4716

[25] Horsman ME, Hari TP, Boddy CN. Polyketide synthase and non-ribosomal peptide synthetase thioesterase selectivity: logic gate or a victim of fate?. Nat Prod Rep, 2016, 33: 183-202

[26] Izumikawa M, Ukai H, Takagi M, Ueda HR, Shin-Ya K. JBIR-26, a novel natural compound from Streptomyces sp. AK-AH76, regulates mammalian circadian clock. J Antibiot Tokyo, 2008, 61: 756-758

[27] Kage H, Riva E, Parascandolo JS, Kreutzer MF, Tosin M, Nett M. Chemical chain termination resolves the timing of ketoreduction in a partially reducing iterative type I polyketide synthase. Org Biomol Chem, 2015, 13: 11414-11417

[28] Kahlert L, Villanueva M, Cox RJ, Skellam EJ. Biosynthesis of 6-hydroxymellein requires a collaborating polyketide synthase-like enzyme. Angew Chem Int Ed Engl, 2021, 60: 11423-11429

[29] Keatinge-Clay AT. The structures of type I polyketide synthases. Nat Prod Rep, 2012, 29: 1050-1073

[30] Kozlov AM, Darriba D, Flouri T, Morel B, Stamatakis A. RAxML-NG: a fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference. Bioinformatics, 2019, 35: 4453-4455

[31] Kroken S, Glass NL, Taylor JW, Yoder OC, Turgeon BG. Phylogenomic analysis of type I polyketide synthase genes in pathogenic and saprobic ascomycetes. Proc Natl Acad Sci, 2003, 100: 15670-15675

[32] Li Y, Xu W, Tang Y. Classification, prediction, and verification of the regioselectivity of fungal polyketide synthase product template domains. J Biol Chem, 2010, 285: 22764-22773

[33] Ling J, Horsman GP, Huang SX, Luo Y, Lin S, Shen B. Enediyne antitumor antibiotic maduropeptin biosynthesis featuring a C-methyltransferase that acts on a CoA-tethered aromatic substrate. J Am Chem Soc, 2010, 132: 12534-12536

[34] Little RF, Hertweck C. Chain release mechanisms in polyketide and non-ribosomal peptide biosynthesis. Nat Prod Rep, 2022, 39: 163-205

[35] Liu L, Zhang Z, Shao CL, Wang JL, Bai H, Wang CY. Bioinformatical analysis of the sequences, structures and functions of fungal polyketide synthase product template domains. Sci Rep, 2015, 5: 10463

[36] Liu L, Zhang Z, Shao CL, Wang CY. Analysis of the sequences, structures, and functions of product-releasing enzyme domains in fungal polyketide synthases. Front Microbiol, 2017, 8: 1685

[37] Liu Q, Zhang D, Gao S, Cai X, Yao M, Xu Y, Gong Y, Zheng K, Mao Y, Yang L, Yang D, Molnar I, Yang X. Didepside formation by the nonreducing polyketide synthase Preu6 of preussia isomera requires interaction of starter acyl transferase and thioesterase domains. Angew Chem Int Ed Engl, 2023, 62 e202214379

[38] Moriguchi T, Ebizuka Y, Fujii I. Domain-domain interactions in the iterative type I polyketide synthase ATX from Aspergillus terreus. ChemBioChem, 2008, 9: 1207-1212

[39] Moriguchi T, Kezuka Y, Nonaka T, Ebizuka Y, Fujii I. Hidden function of catalytic domain in 6-methylsalicylic acid synthase for product release. J Biol Chem, 2010, 285: 15637-15643

[40] Nicholson TP, Rudd BA, Dawson M, Lazarus CM, Simpson TJ, Cox RJ. Design and utility of oligonucleotide gene probes for fungal polyketide synthases. Chem Biol, 2001, 8: 157-178

[41] Nivina A, Yuet KP, Hsu J, Khosla C. Evolution and diversity of assembly-line polyketide synthases. Chem Rev, 2019, 119: 12524-12547

[42] Pfeifer BA, Admiraal SJ, Gramajo H, Cane DE, Khosla C. Biosynthesis of complex polyketides in a metabolically engineered strain of E. coli. Science, 2001, 291: 1790-1792

[43] Regueira TB, Kildegaard KR, Hansen BG, Mortensen UH, Hertweck C, Nielsen J. Molecular basis for mycophenolic acid biosynthesis in Penicillium brevicompactum. Appl Environ Microbiol, 2011, 77: 3035-3043

[44] Sanchez JF, Chiang YM, Szewczyk E, Davidson AD, Ahuja M, Elizabeth Oakley C, Woo Bok J, Keller N, Oakley BR, Wang CC. Molecular genetic analysis of the orsellinic acid/F9775 gene cluster of Aspergillus nidulans. Mol Biosyst, 2010, 6: 587-593

[45] Smetanina OF, Yurchenko AN, Ivanets EV, Gerasimenko AV, Trinh PTH, Ly BM, Nhut ND, Van TTT, Yurchenko EA, Afiyatullov SS. Aromatic metabolites of marine fungus Penicillium sp. KMM 4672 associated with a brown alga Padina sp. Chem Nat Compd, 2017, 53: 600-602

[46] Smith S, Tsai SC. The type I fatty acid and polyketide synthases: a tale of two megasynthases. Nat Prod Rep, 2007, 24: 1041-1072

[47] Soehano I, Yang L, Ding F, Sun H, Low ZJ, Liu X, Liang ZX. Insights into the programmed ketoreduction of partially reducing polyketide synthases: stereo- and substrate-specificity of the ketoreductase domain. Org Biomol Chem, 2014, 12: 8542-8549

[48] Sun H, Ho CL, Ding F, Soehano I, Liu XW, Liang ZX. Synthesis of (R)-mellein by a partially reducing iterative polyketide synthase. J Am Chem Soc, 2012, 134: 11924-11927

[49] Tang MC, Cui X, He X, Ding Z, Zhu T, Tang Y, Li D. Late-stage terpene cyclization by an integral membrane cyclase in the biosynthesis of isoprenoid epoxycyclohexenone natural products. Org Lett, 2017, 19: 5376-5379

[50] Tsai SC. The structural enzymology of iterative aromatic polyketide synthases: a critical comparison with fatty acid synthases. Annu Rev Biochem, 2018, 87: 503-531

[51] Tsai HF, Fujii I, Watanabe A, Wheeler MH, Chang YC, Yasuoka Y, Ebizuka Y, Kwon-Chung KJ. Pentaketide melanin biosynthesis in Aspergillus fumigatus requires chain-length shortening of a heptaketide precursor. J Biol Chem, 2001, 276: 29292-29298

[52] Ugai T, Minami A, Tanaka S, Ozaki T, Liu C, Shigemori H, Hashimoto M, Oikawa H. Biosynthetic machinery of 6-hydroxymellein derivatives leading to cyclohelminthols and palmaenones. ChemBioChem, 2020, 21: 360-367

[53] Vagstad AL, Hill EA, Labonte JW, Townsend CA. Characterization of a fungal thioesterase having Claisen cyclase and deacetylase activities in melanin biosynthesis. Chem Biol, 2012, 19: 1525-1534

[54] Wang B, Guo F, Huang C, Zhao H. Unraveling the iterative type I polyketide synthases hidden in Streptomyces. Proc Natl Acad Sci USA, 2020, 117: 8449-8454

[55] Wei Q, Wang ZP, Zhang X, Zou Y. Diaryl ether formation by a versatile thioesterase domain. J Am Chem Soc, 2022, 144: 9554-9558

[56] Winter JM, Cascio D, Dietrich D, Sato M, Watanabe K, Sawaya MR, Vederas JC, Tang Y. Biochemical and structural basis for controlling chemical modularity in fungal polyketide biosynthesis. J Am Chem Soc, 2015, 137: 9885-9893

[57] Xie J, Chen Y, Cai G, Cai R, Hu Z, Wang H. Tree visualization by one table (tvBOT): a web application for visualizing, modifying and annotating phylogenetic trees. Nucleic Acids Res, 2023, 51: W587-W592

[58] Xu Y, Zhou T, Zhang S, Xuan LJ, Zhan J, Molnar I. Thioesterase domains of fungal nonreducing polyketide synthases act as decision gates during combinatorial biosynthesis. J Am Chem Soc, 2013, 135: 10783-10791

[59] Zhang W, Liu J. Recent advances in understanding and engineering polyketide synthesis. F1000Res, 2016

[60] Zheng L, Yang Y, Wang H, Fan A, Zhang L, Li SM. Ustethylin biosynthesis implies phenethyl derivative formation in Aspergillus ustus. Org Lett, 2020, 22: 7837-7841

RIGHTS & PERMISSIONS

The Author(s)