Article pubs.acs.org/jnp
pks63787, a Polyketide Synthase Gene Responsible for the Biosynthesis of Benzenoids in the Medicinal Mushroom Antrodia cinnamomea Po-Wei Yu,† Ya-Chih Chang,§ Ruey-Fen Liou,† Tzong-Huei Lee,*,‡ and Shean-Shong Tzean*,† †
Department of Plant Pathology and Microbiology and ‡Institute of Fisheries Science, National Taiwan University, Taipei, Taiwan 106 § College of Pharmacy, Taipei Medical University, Taipei, Taiwan 110 S Supporting Information *
ABSTRACT: Antrodia cinnamomea, a unique resupinate basidiomycete endemic to Taiwan, has potent medicinal activities. The reddish basidiocarps and mycelia generally exhibit abundant metabolites and higher biological activity. To investigate the pigments of A. cinnamomea, polyketide synthase (PKS) genes were characterized based on its partially deciphered genome and the construction of a fosmid library. Furthermore, a gene disruption platform was established via protoplast transformation and homologous recombination. Of four putative polyketide synthase genes, pks63787 was selected and disrupted in the monokaryotic wild-type (wt) strain f101. Transformant Δpks63787 was deficient in the synthesis of several aromatic metabolites, including five benzenoids and two benzoquinone derivatives. Based on these results, a biosynthetic pathway for benzenoid derivatives was proposed. The pks63787 deletion mutant not only displayed a reduced red phenotype compared to the wt strain but also displayed less 1,1-biphenyl-2-picrylhydrazyl free radical scavenging activity. This finding suggests that PKS63787 is responsible for the biosynthesis of pigments and metabolites related to the antioxidant activity of A. cinnamomea. The present study focuses on the functional characterization of the PKS gene, the fluctuations of its profile of secondary metabolites, and interpretation of the biosynthesis of benzenoids.
Antrodia cinnamomea, or Niu-Chang-Chih, is an endemic species of Taiwan.1,2 Recently, a new genus, Taiwanof ungus, was identified, with the protologous T. camphoratus replacing the epithet A. camphorata.3,4 Analysis of the constructed phylogenetic tree’s topology revealed a sister group consisting of A. cinnamomea and A. salmonea distinctly separate from the core clade of Antrodia, thus supporting the establishment of Taiwanof ungus (unpublished data). A. cinnamomea is a wood brown-rot fungus that inhabits the decayed inner heartwood wall of Cinnamomum kanehirae Hayata. The resupinate perennial bitter-tasting fruiting body has been used traditionally for decades in Taiwan as a remedy to treat discomforts such as hangover, abdominal pain, diarrhea, and other diseases.5 These medical observations have motivated investigations of the biological activities and bioactive components of A. cinnamomea. Thus, in addition to folk medicinal uses, numerous therapeutic activities have been documented,6,7 including anti-inflammatory,8 cytotoxic,9,10 antioxidant,11 and hepatoprotective activity.12 However, research on the molecular genetics of related metabolites has been limited.13 Filamentous fungi produce a wide variety of bioactive secondary metabolites,14,15 particularly terpenoids and polyketides.16,17 Fungal polyketides with potent biological activity have long been applied in medical and industrial uses,18 such as © 2016 American Chemical Society and American Society of Pharmacognosy
antifungal griseofulvin, immunosuppressant mycophenolate, and cholesterol-lowering lovastatin.19 The biosynthesis of polyketides follows a highly linear approach involving the coupling of coenzyme A (CoA)-derived building blocks by diverse polyketide synthases (PKSs).20 Fungal PKSs are multienzyme complexes classified as iterative type I PKSs, and their functional domains can be reused in a cyclic manner to perform chain extension and reductive processing. Type I PKSs are divided into three classes according to their various processing domains, which include a nonreducing PKS (NRPKS), a partially reducing PKS (PR-PKS), and a highly reducing PKS (HR-PKS).14 In recent years, fungal genome sequencing has revealed that the number of PKS gene sequences is far greater than originally anticipated.21,22 Although numerous hypothetical and predicted polyketide synthases have been characterized from basidiomycetes, few have been confirmed or heterologously expressed.23,24 Understanding the generation of various polyketides is pivotal to identifying new products and developing new bioactive polyketides.18 The average number of PKS genes per genome Received: September 4, 2015 Published: May 26, 2016 1485
DOI: 10.1021/acs.jnatprod.5b00798 J. Nat. Prod. 2016, 79, 1485−1491
Journal of Natural Products
Article
is 4 in basidiomycetes, making them attractive for screening for novel metabolites.25 The genomes of A. cinnamomea isolates were recently deciphered using next-generation sequencing techniques, and four PKS genes, including three reducing PKSs and one NRPKS, were identified. The functions of these PKS genes are not yet known.26 In the present study, to clarify the function of the NR-PKS gene in A. cinnamomea, we deleted pks63787, which led to a deficiency in the biosynthesis of numerous aromatic compounds. In addition, we propose that benzenoids of A. cinnamomea are derived from the polyketide pathway rather than from L-phenylalanine.6 The established transformation system may not only contribute to the investigation of bioactive metabolites from related genes but also be useful for gene functional verification and genetic engineering to increase valuable metabolites.
Table 1. Yield and Regeneration Rate of Protoplasts from Antrodia cinnamomea under Various Conditions factor mycelium age (days)a
germling age (days)b
enzyme concentration (mg/mL)c digestion time (h)d
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RESULTS AND DISCUSSION Gene Characterization of pks63787. The gene pks63787 was first identified based on the presence of a putative betaketoacyl synthase domain in a cDNA sequence from a previously annotated EST database (unpublished data). The full-length cDNA sequence of pks63787 was then obtained by reverse transcription PCR and 5′- and 3′-rapid amplification of cDNA ends (RACE) (Figure S1). Genomic DNA was obtained by screening a fosmid library of A. cinnamomea T1 and primer walking and submitted to the National Center for Biotechnology Information (NCBI) GenBank (accession number KT460194). Comparison of the genomic and cDNA sequences indicated the existence of 9 exons and 8 introns in this gene. The cDNA sequence contains an open reading frame (ORF) of 6342 bp flanked by a 5′-UTR (untranslated region) of 40 bp and a 3′-UTR of 149 bp. The deduced amino acid sequence of pks63787 includes several conserved domains, including the starter unit acyl-carrier protein transacylase (SAT), betaketoacyl synthase (KS), acyl transferase (AT), product template (PT), acyl-carrier protein (ACP), and thioesterase (TE) domains, indicating that pks63787 encodes a putative NRPKS. A blast search of PKS63787 performed in NCBI GenBank identified a gene (BAO20284.1; E value = 0) encoding a PKS (GfPKS) in Grifola f rondosa, a member of Polyporales with potent medicinal uses.27,28 Quantitative reverse transcription PCR (qRT-PCR) analysis indicated that pks63787 was continuously expressed in different growing periods on potato dextrose agar (PDA) (Figure S1). The annotated transcriptome also revealed general expression of pks63787 in monokaryotic A. cinnamomea strains of different mating types (unpublished data), suggesting that PKS63787related metabolites may be present universally in various strains. Protoplasting of Antrodia cinnamomea. To establish a genetic transformation system for A. cinnamomea, a protoplasting method was developed and optimized to maximize the yield of viable protoplasts. The effects of different parameters on the yield of protoplasts and the protoplast regeneration rate were evaluated, including the use of mycelium or germlings from arthrospores, growth age, the concentration of lysing enzymes, the duration of digestion with lysing enzymes, and the concentration of sucrose. Among the four isotonic buffer systems (potassium chloride, mannitol, sucrose, magnesium sulfate) used in a previous study, sucrose generated the highest protoplast yield and was chosen for use in this study.29 As shown in Table 1, approximately 6.0 × 107 protoplasts/mL
sucrose (M)e
range
protoplast yield (107 /mL)
regeneration rate (%)f
4 5 6 7 3 4 5 12 25 50 1 2 3 4 0.6 0.8 1.0 1.2
