Biotechnol Lett (2014) 36:2303–2310 DOI 10.1007/s10529-014-1609-z
ORIGINAL RESEARCH PAPER
Type III polyketide synthase is involved in the biosynthesis of protocatechuic acid in Aspergillus niger Yangyong Lv • Jing Xiao • Li Pan
Received: 8 May 2014 / Accepted: 3 July 2014 / Published online: 22 July 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Genomic studies have shown that not only plants but also filamentous fungi contain type III polyketide synthases. To study the function of type III polyketide synthase (AnPKSIII) in Aspergillus niger, a deletion strain (delAnPKSIII) and an overexpression strain (oeAnPKSIII) were constructed in A. niger MA169.4, a derivative of the wild-type (WT) A. niger ATCC 9029 that produces large quantities of gluconic acid. Alterations in the metabolites were analyzed by HPLC when the extract of the overexpression strain was compared with extracts of the WT and deletion strains. Protocatechuic acid (PCA; 3,4-dihydroxybenzoic acid, 3.2 mg/l) was isolated and identified as the main product of AnPKSIII when inductively expressed in A. niger MA169.4. The molecular weight of PCA was 154.1 (m/z 153.1 [M-H]-), was detected by ESI– MS in the negative ionization mode, and 1H and 13C NMR data confirmed its structure. Keywords AnPKSIII Aspergillus niger 3,4-hydroxybenzoic acid Polyketide synthase (type III) Protocatechuic acid
Electronic supplementary material The online version of this article (doi:10.1007/s10529-014-1609-z) contains supplementary material, which is available to authorized users. Y. Lv J. Xiao L. Pan (&) School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, People’s Republic of China e-mail: [email protected]
Introduction Filamentous fungi are producers of secondary metabolites (Hoffmeister and Keller 2007) that include polyketides (e.g. aflatoxin), non-ribosomal peptides (e.g. ferricrocin), indole terpenes, and terpenes (Sanchez et al. 2012). Aspergillus is a well known producer of medical drugs, toxins, and widely-used industrial enzymes. In particular, Aspergillus niger (GRAS strain) is recognized as an important producer of enzymes and organic acids (Legisˇa and Mattey 2007). The complete genome sequences of A. niger CBS513.88 (Pel et al. 2007) and ATCC1015 (Andersen et al. 2011) have become available, and this has led to the identification of a large number of polyketide (PKS) and non-ribosomal peptide (NRPS) genes. Aspergillus niger thus has the potential to produce a number of secondary metabolites that have yet to be discovered and structurally elucidated (Ferracin et al. 2012). Most polyketides are synthesized by three classes of polyketide synthases (PKSs), types I, II, and III (Shen 2003). The homodimeric type III PKS enzymes can iteratively catalyze substrate priming, decarboxylation, condensation, cyclization, and aromatization reactions to generate a variety of aromatic polyketide products. These products include chalcones, pyrones, and resorcinols (Austin and Noel 2003). Genome sequencing projects have resulted in the identification of type III PKS homologues from bacteria and fungi that were once believed to be exclusively present in plants (Austin and Noel 2003).
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To date, several putative type III PKSs have been discovered and identified in Aspergillus spp. such as csyA, csyB1, cysB2, and cysB3 from A. oryzae (Seshime et al. 2005, 2010a, b; Hashimoto et al. 2013) and AnPKS from A. niger CBS513.88 (Li et al. 2011). In vitro catalytic activity analysis of AnPKS expressed and purified from Escherichia coli (Li et al. 2011) indicates that AnPKS exhibits a broader substrate specificity than csyA from A. oryzae. However, the metabolite regulated in vivo by type III PKSs of A. niger has not been investigated. In the present work, we deleted and overexpressed the type III polyketide synthase gene (AnPKSIII) of A. niger MA169.4. Alterations in the metabolites were detected by HPLC when the extract of the overexpression strain was compared with extracts of the wild type (WT) and deletion strains. ESI–MS, 1H and 13C NMR data analysis confirmed protocatechuic acid (PCA, 3,4-dihydroxybenzoic acid) as the main product of AnPKSIII when inductively expressed in A. niger MA169.4.
Materials and methods Strains and culture conditions Aspergillus niger strains used in this study are listed in Supplementary Table 1. Strains were maintained on potato/dextrose/agar at 30 °C to obtain spores. Fermentation medium for secondary metabolite analysis was GMPY, a modified version of DPY (Jin et al. 2011). GMPY medium contains 1 % (w/v) glucose, 1 % (w/v) maltose, 1 % (w/v) polypeptone, 0.5 % yeast extract, 0.5 % KH2PO4, 0.05 % MgSO47H2O. When required, 10 mM uridine was added to maintain auxotrophy; 1 % (w/v) maltose was used to induce the glucoamylase promoter (PglaA); 100 lg hygromycin B/ml was used to select for transformants. Aspergillus niger strain MA169.4 was obtained from FGSC (Fungal Genetics Stock Center, Kansas City, Missouri, USA). MA169.4delAnPKSIII (delAnPKSIII), a deletion of the AnPKSIII ORF, contained the hygromycin B gene as a selection marker. MA169.4oeAnPKSIII (oeAnPKSIII) is an overexpression strain under glaA promoter (glucoamylase promoter) control with pyrG (Storms et al. 2005) as a selection marker.
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Construction of deletion and overexpression strains of the AnPKSIII gene All primers used in this study are listed in Supplementary Table 2. To obtain an AnPKSIII disruption plasmid, firstly three fragments were amplified by PCR. The first 1.5-kb upstream fragment was obtained by PCR using the primers PKSIIIup F and PKSIIIup R. The second fragment of 1.8 kb contains the hygromycine B marker gene and was obtained by PCR using the primers PKSIIIhygB-F and PKSIIIhygB-R, and plasmid pHGW (Karimi et al. 2005) was used as a template. The third downstream fragment was obtained by PCR using the primers PKSIIIdown F and PKSIIIdown R. The disruption cassette was constructed by fusion PCR by ligating these fragments. The plasmid pMD20TdelAnPKSIII was generated by ligating the fused fragment to T-Vector pMDTM20 vector using the TA cloning kit (Takara, Japan). A PCR fragment was obtained by using the primers PKSIIIup F and PKSIIIdown R and plasmid pMD20T-delAnPKSIII as a template. This PCR fragment was transformed into A. niger MA169.4. To construct the plasmid for AnPKSIII overexpression, the AnPKSIII ORF was amplified by PCR using A. niger MA169.4 cDNA and the primers PKSIIIoverF and PKSIIIoverR, to which NheI restriction sites were added. The PCR product was inserted into the linearized (NheI digestion) plasmid ANIp7 (Storms et al. 2005). The insert direction was confirmed by PCR using primers PKSIIIupoverF and PKSIIIupoverR or PKSIIIdownoverF and PKSIIIdownoverR, respectively.
Generation of AnPKSIII deletion and overexpression strains Strain MA169.4 was transformed with the deletion fragment and the linearized plasmid ANIp7-AnPKSIII (NdeI digestion). Fungal transformation and transformant validation were performed according to established protocols (Campbell et al. 1989; Zhang et al. 2011). Primers containing P1, P2, P3, P4, and P5 (PKSIIIupoverF) and P6 (PKSIIIdownoverR) were used for verification of the transformants. The hygromycin B resistant and uridine prototrophic transformants were selected and confirmed by PCR.
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RNA isolation and qRT-PCR analysis Total RNA was extracted with RNAiso Plus reagent (Takara, Japan) from mycelia harvested from GMPY medium, following the manufacturer’s protocol. Total RNA was treated with DNase I (Takara) to remove chromosomal DNA. Reverse transcription was performed with the PrimeScript RT reagent kit (Takara) using a random primer mix according to the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was performed using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems) for AnPKSIII using the primer pairs listed in Supplementary Table 1 with gpdA (glyceraldehyde-3-phosphate dehydrogenase) as a reference gene. The real-time qRT-PCR was carried out with three replicates per prepared cDNA sample. Metabolite extraction, isolation, ESI–MS and NMR analysis The AnPKSIII deletion and overexpression strains together with the WT strain were grown in 50 ml GMPY medium at 30 °C, 250 rpm for 2 days. After removal of the mycelia by filtration, culture media were extracted twice with ethyl acetate. Ethyl acetate extracts were evaporated and the resulting residues were dissolved in methanol for further analysis. To separate protocatechuic acid (PCA), the extracts were subjected to silica-gel chromatography using an ODS ˚ , YMC, Japan) and eluting with column (50 lm, 120A petroleum/ethyl acetate. A Gilson GX-281 preparative HPLC system with a ZORBAX PrepHT SB-C18 (21.2 9 150 mm, 5 lm) column was used to separate the biosynthesis products. The solvent was acetonitrile/0.5 % acetic acid at 8 ml/min at 35 °C. PCA was separated using the following program: initial hold for 2 min at 5 % acetonitrile; within 30 min, at 30 % acetonitrile; within 35 min 100 % acetonitrile, hold for 5 min; then 5 % acetonitrile within 41 min, hold for 6 min at 5 % acetonitrile. PCA was further purified using Sephadex LH-20 with methanol as solvent. PCA was then characterized using Bruker (500 MHz) NMR and ESI–MS (Amazon SL, Bruker) instruments. Analytical HPLC was carried out using an Agilent 20RBAX SB-C18 (4.6 9 150 mm, 5 lm) column with acetonitrile (solvent A)/0.5 % acetic acid (solvent B) at 1 ml/min at 35 °C. Separation was performed by the following: initial hold for 1 min at
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10 % A; within 17 min at 100 % A; within 19 min 100 % A; then 10 % A within 20 min, hold for 2 min at 10 % A. Elution was monitored at 254 nm. Determination of protocatechuic acid (PCA) yield A standard curve was constructed based on different concentrations of a PCA standard. Protocatechuic acid in the extract of oeAnPKSIII was detected by analytical HPLC and its yield calculated using the standard curve.
Results and discussion Expression and sequence analysis of AnPKSIII The expression of the AnPKSIII gene in the WT strain was observed in GMPY medium cultivated at 30 °Cfor 48 h. Then the AnPKSIII gene was amplified using 48 h cDNA as a template. Sequence analysis of the AnPKSIII gene from A. niger MA169.4 showed that the AnPKSIII gene is 1231 nucleotides in length, consisting of one intron of 52 nucleotides (from 177 to 218 bp), and encodes a polypeptide of 393 amino acid residues with a calculated molecular mass of 43.2 kDa. The amino acid sequence displayed 24.8–84.7 % identity to those of other type III PKSs of plants and fungi (Supplementary Fig. 1): 24.8 % identity with Medicago sativa CHS2 (Ferrer et al. 1999), 31.1 % identity with A. oryzae csyA (Seshime et al. 2005, 2010a), 33.3 % identity with A. oryzae csyB (Seshime et al. 2005, 2010b; Hashimoto et al. 2013), and 84.7 % identity with A. niger CBS513.88 AnPKS (Li et al. 2013). A comparative analysis of A. niger MA169.4 type III PKS with plant and fungi chalcone synthases (CHSs) showed conservation of the catalytic center residues Cys, His, and Asn at positions 164, 303, and 336 respectively (in Supplementary Fig. 1 the amino acid positions are mentioned with respect to the M. sativa CHS2 sequence and are marked by .). As in plant CHS (Fliegmann et al. 1992) and A. oryzae (Seshime et al. 2005), A. niger MA169.4 type III PKSs can also be divided into four conserved domains (indicated by w and underlined). First, the active site has a highly conserved Cys residue and eight other residues. Second, there is a narrow stretch of residues including the Phe active site in addition to Asp and Gly. The third conserved domain contains the His
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Fig. 1 Construction of AnPKSIII-deletion and AnPKSIII-overexpression strains. a Construction of the AnPKSIII deletion strain. The entire ORF of AnPKSIII was deleted and replaced with a hygB marker. b Confirmation of AnPKSIII gene deletion
by PCR analysis. c Plasmid for AnPKSIII overexpression. The full-length AnPKSIII ORF was under glaA promoter control. d Confirmation of the constructed AnPKSIII-overexpression strain by PCR analysis
active residue, Trp residue, and two Gly residues. The presence of the CHS-family characteristic sequence G (F/l) GPG at the C-terminus with the four conserved residues (Phe, Pro, and two Gly residues) indicates that these amino acids, which serve as a substrate specific recognition site, are also conserved in the fungal CHS-like type III PKS sequences. Taken together, it seems that, although type III PKS enzymes share the conserved Cys–His–Asn catalytic triad, they exhibit fewer sequence similarities, even between A. niger and A. oryzae. The sequence variations may result in their variable inter-genus catalytic functions, which are even greater than interspecies variations (Fujii 2009).
gene in A. niger MA169.4. Deletion of the AnPKSIII gene was performed by inserting the hygB marker into the AnPKSIII ORF in A. niger MA169.4 (Fig. 1a). The AnPKSIII-deleted strain was confirmed using primer pair P1 and P4 by PCR to amplify a 5.2kb (lane 2) DNA fragment compared to the 4.7-kb band (lane1) in the WT strain (Fig. 2b). The deletion strain was also confirmed by PCR using primer pairs P1 and P2, P3 and P4 to amplify 2 kb and 2.1 kb DNA fragments (lanes 5 and 6, respectively) compared to no expected band at 2 kb in the WT strain (lanes 3 and 4) (Fig. 2b). Overexpression of the AnPKSIII gene was under control of the strong A. niger glaA promoter (Fig. 2c). Primer pair P5 and P6 was used to confirm the AnPKSIII-overexpression strain (lane 1, WT; lane 2, overexpression strain). A 2.9 kb band (glaA gene) was detected in the WT strain, while an additional 2 kb band (AnPKSIII gene, indicated by the arrow) was observed in the oeAnPKSIII strain (Fig. 2d).
Construction, verification and qRT-PCR analysis of AnPKSIII mutants A deletion strain and an overexpression strain were constructed to determine the function of the AnPKSIII
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Fig. 2 qRT-PCR analysis of AnPKSIII. The expression of AnPKSIII gene were normalized to the expression level of the endogenous control gene gpdA. a Amplification plot of qRTPCR. The abscissa shows the number of PCR cycles and the
ordinate shows the normalized fluorescence intensity. The cycle threshold (Ct) is the cycle number at which the fluorescence intensity crosses the threshold value (0.2). b RQ (relative quantitation) value of qPCR
The expression level of AnPKSIII under glaA promoter control was induced in maltose induction medium and compared to that of the WT strain. However, in the deletion strain no transcription was observed (Fig. 2a, b). These results show that the expression level of AnPKSIII in WT was low when grown in GMPY medium (Fig. 2a). Genes associated with secondary metabolites are most likely not expressed under the usual laboratory growth conditions, which differ significantly from the conditions that these organisms encounter in their natural environment (Gacek and Strauss 2012). Fungal secondary metabolism genes are controlled by an intricate regulatory network including multiple proteins and complexes that respond to various environmental stimuli involving carbon and nitrogen sources, amino acids in the environment, temperature, light, pH, iron availability, and also stimuli from other organisms (Brakhage 2013). A prime example is the regulation of toxin production in Aspergillus species by the nitrogen source: A. parasiticus and A. nidulans produce the highest levels of aflatoxin and sterigmatocystin (a precursor of aflatoxin) on ammonium and nitrate as the sole nitrogen source, respectively (Feng and Leonard 1998).
deletion strain by HPLC. Four distinct peaks, designated as products A, B, C, and D, were detected upon overexpression of the AnPKSIII gene, of which product A was the major peak whereas no clear differences were observed when the WT strain was compared with the deletion strain (Fig. 3a). In order to further characterize the major product A, we isolated it from the 40 l induction culture of ANIp7-AnPKSIII transformant. ESI–MS indicated that the molecular weight of product A was 154.1 based on the observed ion peaks (m/z 153.1 [M-H]-, 307.1 [2 M-H]-) in the negative ionization mode (Fig. 3b). 1H NMR data (Table 1; Supplementary Fig. 2b) indicated that the H signals were from a benzene ring (6.87 ppm, 7.40–7.45 ppm). 13C NMR data (Table 1; Supplementary Fig. 2a) indicated the presence of -HC=CH- moieties (115.7, 117.3, 122.8, 123.7, 143.7, and 149.3 ppm) in the major product A. These data showed that the structure of product A is protocatechuic acid (PCA, 3,4-dihydroxybenzoic acid) (Fig. 3b). 13C-NMR data for the product produced by overexpression of type III polyketide synthase csyA in A. oryzae indicated the presence of para-disubstitution of the benzene ring [108.8 ppm (C-2,6), 159.3 ppm (C-3,5)]. The results show that protocatechuic acid is an isomer of 3,5-dihydroxybenzoic acid produced by overexpression of type III polyketide synthase csyA in A. oryzae (Seshime et al. 2010a). The yield of protocatechuic acid was 3.2 mg/l in the extract of oeAnPKSIII, calculated using the standard curve (Supplementary Fig. 3).
Analysis of metabolites of the AnPKSIII overexpression strain Next we analyzed the metabolites produced by the AnPKSIII overexpression strain, WT strain, and the
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a
b
Fig. 3 HPLC, ESI–MS, and structural formula analysis of product A. a HPLC analysis of WT, delAnPKSIII, and oeAnPKSIII strains and PCA standard. b Molecular weight and structural formula of product A. ESI–MS m/z 153.1 [M-H]-, 307.1 [2 M-H]-
Table 1 1H (500 MHz) and 13C NMR (125 MHz) spectral data for product A in deuterium oxide Position
dC
1
122.8
2
115.7
3
143.7
4 5
149.3 117.3
6
123.7
7
171.2
dH
Ref (Wang et al. 2009) dC
7.45–7.43 (1H, m)
115.8
Ref (Wang et al. 2009) dH
123.2 7.43 (1H, br s)
146.1 6.87 (1H, t, J = 8.0 Hz) 7.42–7.40 (1H, m)
151.5 117.7 123.9
6.78 (1H, d, J = 7.9 Hz) 7.42 (1H, d, J = 7.9 Hz)
170.2
Protocatechuic acid can also be synthesized via the shikimic acid pathway in E. coli and B. subtilis (Kra¨mer et al. 2003) in which phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) condense to form 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP). Dehydroquinate synthase converts DAHP into 3-dehydroquinate (DHQ). DHQ dehydratase catalyzes the elimination of H2O to obtain 3-dehydroshikimate (DHS). DHS dehydratase converts DHS into protocatechuic acid. Thus, DHS dehydratase is
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vital for the formation of protocatechuic acid as found with E. coli and Bacillus sp. (Draths and Frost 1994; Williams et al. 2012). It is encoded by asbF/aroZ genes. However, a homologous gene of aroZ/asbF was not found in A. niger in the data-base of Aspergillus (www.aspgd.org) and KEGG (www. genome.jp/kegg/). In addition, the catalytic characteristic of type III polyketide synthase enzymes is that they iteratively condense a starter CoA ester substrate with units derived from malonyl-CoA and cyclize the linear polyketide intermediates to produce compounds with ring structures. Since protocatechuic acid is an isomer of 3,5-dihydroxybenzoic acid, produced by overexpression of type III polyketide synthase csyA in A. oryzae, we inferred that the pathway of protocatechuic acid synthesis is similar to that proposed in A. oryzae. Product A observed in A. niger MA169.4 culture was different from the compounds catalyzed by AnPKS in vitro (Li et al. 2011). Another example is the compounds catalyzed by type III polyketide synthase csyA from A. oryzae. Overexpression of csyA under the control of the a-amylase promoter in A. oryzae leads to the identification of 3,5-dihydroxybenzoic acid (Seshime et al. 2010a) as the major product, which is not found among the compounds catalyzed by csyA from A. oryzae in vitro (Yu et al.
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2010). The explanation for this contradictory result between in vivo and in vitro data from both csyA and AnPKSIII is that a proper expression system is required for the functional analysis of PKSs, even in the relatively small type III PKSs (Seshime et al. 2010a). The AnPKSIII gene variations between A. niger MA169.4 and CBS513.88 might also contribute to the different molecular structures of the product compounds.
Conclusion To date, type III polyketide synthases of A. niger have only been studied in vitro (Li et al. 2011). In this study we have created a deletion and an overexpression strain of the A. niger MA169.4 type III polyketide synthase AnPKSIII gene. Upon overexpression of the AnPKSIII gene, protocatechuic acid was isolated and identified as the main product in A. niger MA169.4. With further comprehensive annotation of the secondary metabolite biosynthesis genes (Inglis et al. 2013) and an efficient expression system (Chiang et al. 2013), the food-safety-grade A. niger strains could be prospective candidates as secondary metabolite producers in the future. Acknowledgments We wish to acknowledge the financial support by the 863 (Hi-tech Research and Development Program of China) program under contract No. 2012AA022108, the Guangdong Natural Science Foundation (S2012030006235), and the Guangdong Provincial Department of Science and Technology Research Project (No. 2012B010900028).
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ORIGINAL RESEARCH PAPER
Type III polyketide synthase is involved in the biosynthesis of protocatechuic acid in Aspergillus niger Yangyong Lv • Jing Xiao • Li Pan
Received: 8 May 2014 / Accepted: 3 July 2014 / Published online: 22 July 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Genomic studies have shown that not only plants but also filamentous fungi contain type III polyketide synthases. To study the function of type III polyketide synthase (AnPKSIII) in Aspergillus niger, a deletion strain (delAnPKSIII) and an overexpression strain (oeAnPKSIII) were constructed in A. niger MA169.4, a derivative of the wild-type (WT) A. niger ATCC 9029 that produces large quantities of gluconic acid. Alterations in the metabolites were analyzed by HPLC when the extract of the overexpression strain was compared with extracts of the WT and deletion strains. Protocatechuic acid (PCA; 3,4-dihydroxybenzoic acid, 3.2 mg/l) was isolated and identified as the main product of AnPKSIII when inductively expressed in A. niger MA169.4. The molecular weight of PCA was 154.1 (m/z 153.1 [M-H]-), was detected by ESI– MS in the negative ionization mode, and 1H and 13C NMR data confirmed its structure. Keywords AnPKSIII Aspergillus niger 3,4-hydroxybenzoic acid Polyketide synthase (type III) Protocatechuic acid
Electronic supplementary material The online version of this article (doi:10.1007/s10529-014-1609-z) contains supplementary material, which is available to authorized users. Y. Lv J. Xiao L. Pan (&) School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, People’s Republic of China e-mail: [email protected]
Introduction Filamentous fungi are producers of secondary metabolites (Hoffmeister and Keller 2007) that include polyketides (e.g. aflatoxin), non-ribosomal peptides (e.g. ferricrocin), indole terpenes, and terpenes (Sanchez et al. 2012). Aspergillus is a well known producer of medical drugs, toxins, and widely-used industrial enzymes. In particular, Aspergillus niger (GRAS strain) is recognized as an important producer of enzymes and organic acids (Legisˇa and Mattey 2007). The complete genome sequences of A. niger CBS513.88 (Pel et al. 2007) and ATCC1015 (Andersen et al. 2011) have become available, and this has led to the identification of a large number of polyketide (PKS) and non-ribosomal peptide (NRPS) genes. Aspergillus niger thus has the potential to produce a number of secondary metabolites that have yet to be discovered and structurally elucidated (Ferracin et al. 2012). Most polyketides are synthesized by three classes of polyketide synthases (PKSs), types I, II, and III (Shen 2003). The homodimeric type III PKS enzymes can iteratively catalyze substrate priming, decarboxylation, condensation, cyclization, and aromatization reactions to generate a variety of aromatic polyketide products. These products include chalcones, pyrones, and resorcinols (Austin and Noel 2003). Genome sequencing projects have resulted in the identification of type III PKS homologues from bacteria and fungi that were once believed to be exclusively present in plants (Austin and Noel 2003).
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To date, several putative type III PKSs have been discovered and identified in Aspergillus spp. such as csyA, csyB1, cysB2, and cysB3 from A. oryzae (Seshime et al. 2005, 2010a, b; Hashimoto et al. 2013) and AnPKS from A. niger CBS513.88 (Li et al. 2011). In vitro catalytic activity analysis of AnPKS expressed and purified from Escherichia coli (Li et al. 2011) indicates that AnPKS exhibits a broader substrate specificity than csyA from A. oryzae. However, the metabolite regulated in vivo by type III PKSs of A. niger has not been investigated. In the present work, we deleted and overexpressed the type III polyketide synthase gene (AnPKSIII) of A. niger MA169.4. Alterations in the metabolites were detected by HPLC when the extract of the overexpression strain was compared with extracts of the wild type (WT) and deletion strains. ESI–MS, 1H and 13C NMR data analysis confirmed protocatechuic acid (PCA, 3,4-dihydroxybenzoic acid) as the main product of AnPKSIII when inductively expressed in A. niger MA169.4.
Materials and methods Strains and culture conditions Aspergillus niger strains used in this study are listed in Supplementary Table 1. Strains were maintained on potato/dextrose/agar at 30 °C to obtain spores. Fermentation medium for secondary metabolite analysis was GMPY, a modified version of DPY (Jin et al. 2011). GMPY medium contains 1 % (w/v) glucose, 1 % (w/v) maltose, 1 % (w/v) polypeptone, 0.5 % yeast extract, 0.5 % KH2PO4, 0.05 % MgSO47H2O. When required, 10 mM uridine was added to maintain auxotrophy; 1 % (w/v) maltose was used to induce the glucoamylase promoter (PglaA); 100 lg hygromycin B/ml was used to select for transformants. Aspergillus niger strain MA169.4 was obtained from FGSC (Fungal Genetics Stock Center, Kansas City, Missouri, USA). MA169.4delAnPKSIII (delAnPKSIII), a deletion of the AnPKSIII ORF, contained the hygromycin B gene as a selection marker. MA169.4oeAnPKSIII (oeAnPKSIII) is an overexpression strain under glaA promoter (glucoamylase promoter) control with pyrG (Storms et al. 2005) as a selection marker.
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Construction of deletion and overexpression strains of the AnPKSIII gene All primers used in this study are listed in Supplementary Table 2. To obtain an AnPKSIII disruption plasmid, firstly three fragments were amplified by PCR. The first 1.5-kb upstream fragment was obtained by PCR using the primers PKSIIIup F and PKSIIIup R. The second fragment of 1.8 kb contains the hygromycine B marker gene and was obtained by PCR using the primers PKSIIIhygB-F and PKSIIIhygB-R, and plasmid pHGW (Karimi et al. 2005) was used as a template. The third downstream fragment was obtained by PCR using the primers PKSIIIdown F and PKSIIIdown R. The disruption cassette was constructed by fusion PCR by ligating these fragments. The plasmid pMD20TdelAnPKSIII was generated by ligating the fused fragment to T-Vector pMDTM20 vector using the TA cloning kit (Takara, Japan). A PCR fragment was obtained by using the primers PKSIIIup F and PKSIIIdown R and plasmid pMD20T-delAnPKSIII as a template. This PCR fragment was transformed into A. niger MA169.4. To construct the plasmid for AnPKSIII overexpression, the AnPKSIII ORF was amplified by PCR using A. niger MA169.4 cDNA and the primers PKSIIIoverF and PKSIIIoverR, to which NheI restriction sites were added. The PCR product was inserted into the linearized (NheI digestion) plasmid ANIp7 (Storms et al. 2005). The insert direction was confirmed by PCR using primers PKSIIIupoverF and PKSIIIupoverR or PKSIIIdownoverF and PKSIIIdownoverR, respectively.
Generation of AnPKSIII deletion and overexpression strains Strain MA169.4 was transformed with the deletion fragment and the linearized plasmid ANIp7-AnPKSIII (NdeI digestion). Fungal transformation and transformant validation were performed according to established protocols (Campbell et al. 1989; Zhang et al. 2011). Primers containing P1, P2, P3, P4, and P5 (PKSIIIupoverF) and P6 (PKSIIIdownoverR) were used for verification of the transformants. The hygromycin B resistant and uridine prototrophic transformants were selected and confirmed by PCR.
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RNA isolation and qRT-PCR analysis Total RNA was extracted with RNAiso Plus reagent (Takara, Japan) from mycelia harvested from GMPY medium, following the manufacturer’s protocol. Total RNA was treated with DNase I (Takara) to remove chromosomal DNA. Reverse transcription was performed with the PrimeScript RT reagent kit (Takara) using a random primer mix according to the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) was performed using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems) for AnPKSIII using the primer pairs listed in Supplementary Table 1 with gpdA (glyceraldehyde-3-phosphate dehydrogenase) as a reference gene. The real-time qRT-PCR was carried out with three replicates per prepared cDNA sample. Metabolite extraction, isolation, ESI–MS and NMR analysis The AnPKSIII deletion and overexpression strains together with the WT strain were grown in 50 ml GMPY medium at 30 °C, 250 rpm for 2 days. After removal of the mycelia by filtration, culture media were extracted twice with ethyl acetate. Ethyl acetate extracts were evaporated and the resulting residues were dissolved in methanol for further analysis. To separate protocatechuic acid (PCA), the extracts were subjected to silica-gel chromatography using an ODS ˚ , YMC, Japan) and eluting with column (50 lm, 120A petroleum/ethyl acetate. A Gilson GX-281 preparative HPLC system with a ZORBAX PrepHT SB-C18 (21.2 9 150 mm, 5 lm) column was used to separate the biosynthesis products. The solvent was acetonitrile/0.5 % acetic acid at 8 ml/min at 35 °C. PCA was separated using the following program: initial hold for 2 min at 5 % acetonitrile; within 30 min, at 30 % acetonitrile; within 35 min 100 % acetonitrile, hold for 5 min; then 5 % acetonitrile within 41 min, hold for 6 min at 5 % acetonitrile. PCA was further purified using Sephadex LH-20 with methanol as solvent. PCA was then characterized using Bruker (500 MHz) NMR and ESI–MS (Amazon SL, Bruker) instruments. Analytical HPLC was carried out using an Agilent 20RBAX SB-C18 (4.6 9 150 mm, 5 lm) column with acetonitrile (solvent A)/0.5 % acetic acid (solvent B) at 1 ml/min at 35 °C. Separation was performed by the following: initial hold for 1 min at
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10 % A; within 17 min at 100 % A; within 19 min 100 % A; then 10 % A within 20 min, hold for 2 min at 10 % A. Elution was monitored at 254 nm. Determination of protocatechuic acid (PCA) yield A standard curve was constructed based on different concentrations of a PCA standard. Protocatechuic acid in the extract of oeAnPKSIII was detected by analytical HPLC and its yield calculated using the standard curve.
Results and discussion Expression and sequence analysis of AnPKSIII The expression of the AnPKSIII gene in the WT strain was observed in GMPY medium cultivated at 30 °Cfor 48 h. Then the AnPKSIII gene was amplified using 48 h cDNA as a template. Sequence analysis of the AnPKSIII gene from A. niger MA169.4 showed that the AnPKSIII gene is 1231 nucleotides in length, consisting of one intron of 52 nucleotides (from 177 to 218 bp), and encodes a polypeptide of 393 amino acid residues with a calculated molecular mass of 43.2 kDa. The amino acid sequence displayed 24.8–84.7 % identity to those of other type III PKSs of plants and fungi (Supplementary Fig. 1): 24.8 % identity with Medicago sativa CHS2 (Ferrer et al. 1999), 31.1 % identity with A. oryzae csyA (Seshime et al. 2005, 2010a), 33.3 % identity with A. oryzae csyB (Seshime et al. 2005, 2010b; Hashimoto et al. 2013), and 84.7 % identity with A. niger CBS513.88 AnPKS (Li et al. 2013). A comparative analysis of A. niger MA169.4 type III PKS with plant and fungi chalcone synthases (CHSs) showed conservation of the catalytic center residues Cys, His, and Asn at positions 164, 303, and 336 respectively (in Supplementary Fig. 1 the amino acid positions are mentioned with respect to the M. sativa CHS2 sequence and are marked by .). As in plant CHS (Fliegmann et al. 1992) and A. oryzae (Seshime et al. 2005), A. niger MA169.4 type III PKSs can also be divided into four conserved domains (indicated by w and underlined). First, the active site has a highly conserved Cys residue and eight other residues. Second, there is a narrow stretch of residues including the Phe active site in addition to Asp and Gly. The third conserved domain contains the His
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Fig. 1 Construction of AnPKSIII-deletion and AnPKSIII-overexpression strains. a Construction of the AnPKSIII deletion strain. The entire ORF of AnPKSIII was deleted and replaced with a hygB marker. b Confirmation of AnPKSIII gene deletion
by PCR analysis. c Plasmid for AnPKSIII overexpression. The full-length AnPKSIII ORF was under glaA promoter control. d Confirmation of the constructed AnPKSIII-overexpression strain by PCR analysis
active residue, Trp residue, and two Gly residues. The presence of the CHS-family characteristic sequence G (F/l) GPG at the C-terminus with the four conserved residues (Phe, Pro, and two Gly residues) indicates that these amino acids, which serve as a substrate specific recognition site, are also conserved in the fungal CHS-like type III PKS sequences. Taken together, it seems that, although type III PKS enzymes share the conserved Cys–His–Asn catalytic triad, they exhibit fewer sequence similarities, even between A. niger and A. oryzae. The sequence variations may result in their variable inter-genus catalytic functions, which are even greater than interspecies variations (Fujii 2009).
gene in A. niger MA169.4. Deletion of the AnPKSIII gene was performed by inserting the hygB marker into the AnPKSIII ORF in A. niger MA169.4 (Fig. 1a). The AnPKSIII-deleted strain was confirmed using primer pair P1 and P4 by PCR to amplify a 5.2kb (lane 2) DNA fragment compared to the 4.7-kb band (lane1) in the WT strain (Fig. 2b). The deletion strain was also confirmed by PCR using primer pairs P1 and P2, P3 and P4 to amplify 2 kb and 2.1 kb DNA fragments (lanes 5 and 6, respectively) compared to no expected band at 2 kb in the WT strain (lanes 3 and 4) (Fig. 2b). Overexpression of the AnPKSIII gene was under control of the strong A. niger glaA promoter (Fig. 2c). Primer pair P5 and P6 was used to confirm the AnPKSIII-overexpression strain (lane 1, WT; lane 2, overexpression strain). A 2.9 kb band (glaA gene) was detected in the WT strain, while an additional 2 kb band (AnPKSIII gene, indicated by the arrow) was observed in the oeAnPKSIII strain (Fig. 2d).
Construction, verification and qRT-PCR analysis of AnPKSIII mutants A deletion strain and an overexpression strain were constructed to determine the function of the AnPKSIII
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Fig. 2 qRT-PCR analysis of AnPKSIII. The expression of AnPKSIII gene were normalized to the expression level of the endogenous control gene gpdA. a Amplification plot of qRTPCR. The abscissa shows the number of PCR cycles and the
ordinate shows the normalized fluorescence intensity. The cycle threshold (Ct) is the cycle number at which the fluorescence intensity crosses the threshold value (0.2). b RQ (relative quantitation) value of qPCR
The expression level of AnPKSIII under glaA promoter control was induced in maltose induction medium and compared to that of the WT strain. However, in the deletion strain no transcription was observed (Fig. 2a, b). These results show that the expression level of AnPKSIII in WT was low when grown in GMPY medium (Fig. 2a). Genes associated with secondary metabolites are most likely not expressed under the usual laboratory growth conditions, which differ significantly from the conditions that these organisms encounter in their natural environment (Gacek and Strauss 2012). Fungal secondary metabolism genes are controlled by an intricate regulatory network including multiple proteins and complexes that respond to various environmental stimuli involving carbon and nitrogen sources, amino acids in the environment, temperature, light, pH, iron availability, and also stimuli from other organisms (Brakhage 2013). A prime example is the regulation of toxin production in Aspergillus species by the nitrogen source: A. parasiticus and A. nidulans produce the highest levels of aflatoxin and sterigmatocystin (a precursor of aflatoxin) on ammonium and nitrate as the sole nitrogen source, respectively (Feng and Leonard 1998).
deletion strain by HPLC. Four distinct peaks, designated as products A, B, C, and D, were detected upon overexpression of the AnPKSIII gene, of which product A was the major peak whereas no clear differences were observed when the WT strain was compared with the deletion strain (Fig. 3a). In order to further characterize the major product A, we isolated it from the 40 l induction culture of ANIp7-AnPKSIII transformant. ESI–MS indicated that the molecular weight of product A was 154.1 based on the observed ion peaks (m/z 153.1 [M-H]-, 307.1 [2 M-H]-) in the negative ionization mode (Fig. 3b). 1H NMR data (Table 1; Supplementary Fig. 2b) indicated that the H signals were from a benzene ring (6.87 ppm, 7.40–7.45 ppm). 13C NMR data (Table 1; Supplementary Fig. 2a) indicated the presence of -HC=CH- moieties (115.7, 117.3, 122.8, 123.7, 143.7, and 149.3 ppm) in the major product A. These data showed that the structure of product A is protocatechuic acid (PCA, 3,4-dihydroxybenzoic acid) (Fig. 3b). 13C-NMR data for the product produced by overexpression of type III polyketide synthase csyA in A. oryzae indicated the presence of para-disubstitution of the benzene ring [108.8 ppm (C-2,6), 159.3 ppm (C-3,5)]. The results show that protocatechuic acid is an isomer of 3,5-dihydroxybenzoic acid produced by overexpression of type III polyketide synthase csyA in A. oryzae (Seshime et al. 2010a). The yield of protocatechuic acid was 3.2 mg/l in the extract of oeAnPKSIII, calculated using the standard curve (Supplementary Fig. 3).
Analysis of metabolites of the AnPKSIII overexpression strain Next we analyzed the metabolites produced by the AnPKSIII overexpression strain, WT strain, and the
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a
b
Fig. 3 HPLC, ESI–MS, and structural formula analysis of product A. a HPLC analysis of WT, delAnPKSIII, and oeAnPKSIII strains and PCA standard. b Molecular weight and structural formula of product A. ESI–MS m/z 153.1 [M-H]-, 307.1 [2 M-H]-
Table 1 1H (500 MHz) and 13C NMR (125 MHz) spectral data for product A in deuterium oxide Position
dC
1
122.8
2
115.7
3
143.7
4 5
149.3 117.3
6
123.7
7
171.2
dH
Ref (Wang et al. 2009) dC
7.45–7.43 (1H, m)
115.8
Ref (Wang et al. 2009) dH
123.2 7.43 (1H, br s)
146.1 6.87 (1H, t, J = 8.0 Hz) 7.42–7.40 (1H, m)
151.5 117.7 123.9
6.78 (1H, d, J = 7.9 Hz) 7.42 (1H, d, J = 7.9 Hz)
170.2
Protocatechuic acid can also be synthesized via the shikimic acid pathway in E. coli and B. subtilis (Kra¨mer et al. 2003) in which phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) condense to form 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP). Dehydroquinate synthase converts DAHP into 3-dehydroquinate (DHQ). DHQ dehydratase catalyzes the elimination of H2O to obtain 3-dehydroshikimate (DHS). DHS dehydratase converts DHS into protocatechuic acid. Thus, DHS dehydratase is
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vital for the formation of protocatechuic acid as found with E. coli and Bacillus sp. (Draths and Frost 1994; Williams et al. 2012). It is encoded by asbF/aroZ genes. However, a homologous gene of aroZ/asbF was not found in A. niger in the data-base of Aspergillus (www.aspgd.org) and KEGG (www. genome.jp/kegg/). In addition, the catalytic characteristic of type III polyketide synthase enzymes is that they iteratively condense a starter CoA ester substrate with units derived from malonyl-CoA and cyclize the linear polyketide intermediates to produce compounds with ring structures. Since protocatechuic acid is an isomer of 3,5-dihydroxybenzoic acid, produced by overexpression of type III polyketide synthase csyA in A. oryzae, we inferred that the pathway of protocatechuic acid synthesis is similar to that proposed in A. oryzae. Product A observed in A. niger MA169.4 culture was different from the compounds catalyzed by AnPKS in vitro (Li et al. 2011). Another example is the compounds catalyzed by type III polyketide synthase csyA from A. oryzae. Overexpression of csyA under the control of the a-amylase promoter in A. oryzae leads to the identification of 3,5-dihydroxybenzoic acid (Seshime et al. 2010a) as the major product, which is not found among the compounds catalyzed by csyA from A. oryzae in vitro (Yu et al.
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2010). The explanation for this contradictory result between in vivo and in vitro data from both csyA and AnPKSIII is that a proper expression system is required for the functional analysis of PKSs, even in the relatively small type III PKSs (Seshime et al. 2010a). The AnPKSIII gene variations between A. niger MA169.4 and CBS513.88 might also contribute to the different molecular structures of the product compounds.
Conclusion To date, type III polyketide synthases of A. niger have only been studied in vitro (Li et al. 2011). In this study we have created a deletion and an overexpression strain of the A. niger MA169.4 type III polyketide synthase AnPKSIII gene. Upon overexpression of the AnPKSIII gene, protocatechuic acid was isolated and identified as the main product in A. niger MA169.4. With further comprehensive annotation of the secondary metabolite biosynthesis genes (Inglis et al. 2013) and an efficient expression system (Chiang et al. 2013), the food-safety-grade A. niger strains could be prospective candidates as secondary metabolite producers in the future. Acknowledgments We wish to acknowledge the financial support by the 863 (Hi-tech Research and Development Program of China) program under contract No. 2012AA022108, the Guangdong Natural Science Foundation (S2012030006235), and the Guangdong Provincial Department of Science and Technology Research Project (No. 2012B010900028).
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