Identification of the gene cluster for bistropolone-humulene meroterpenoid biosynthesis in Phoma sp.

Fungal Genetics and Biology 129 (2019) 7–15

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Identification of the gene cluster for bistropolone-humulene meroterpenoid biosynthesis in Phoma sp.

T

Yanan Zhaia,b, Yumei Lic,d, Jinyu Zhangd, Yang Zhangd, Fengxia Rend, Xiaoling Zhangb, ⁎ ⁎ Gang Liub, Xingzhong Liua,b, , Yongsheng Ched,e, a

School of Life Sciences, University of Science and Technology of China, Hefei 230026, China State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China c College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China d State Key Laboratory of Toxicology & Medical Countermeasures, Beijing Institute of Pharmacology & Toxicology, Beijing 100850, China e Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Tropolonic sesquiterpene Phoma sp. Eupenifeldin Humulenol Biosynthetic gene cluster

Eupenifeldin, a bistropolone meroterpenoid, was first discovered as an antitumor agent from the fungus Eupenicillium brefeldianum. We also isolated this compound and a new congener from a strain of Phoma sp. (CGMCC 10481), and evaluated their antitumor effects. Eupenifeldin showed potent in vitro anti-glioma activity. This tropolone-humulene-tropolone meroterpenoid could be originated from two units of tropolone orthoquinone methides and a 10-hydroxyhumulene moiety via hetero-Diels-Alder reactions. To explore the biosynthesis of this class of tropolonic sesquiterpenes, the genome of a eupenifeldin-producing Phoma sp. was sequenced and analyzed. The biosynthetic gene cluster of eupenifeldin (eup) was identified and partially validated by genomic analysis, gene disruption, and product analysis. A nonreducing polyketide synthase EupA, a FAD-dependent monooxygenase EupB, and a non-heme Fe (II)-dependent dioxygenase EupC, were identified as the enzymes responsible for tropolone formation. While the terpene cyclase EupE of an unknown family was characterized to catalyze humulene formation, and a cytochrome P450 enzyme EupD was responsible for hydroxylation of humulene. This study sheds light on the biosynthesis of eupenifeldin, and paves the way to further decipher its biosynthetic pathway.

1. Introduction Meroterpenoids are natural products of mixed-biogenesis, and typically originated from terpenoid and polyketide/shikimate/indole pathways (Geris and Simpson, 2009; Matsuda and Abe, 2016). Tropolonic sesquiterpenes are derived from a sesquiterpene and one or two tropolones, and show profound biological effects (Ainsworth et al., 1995; Ayers et al., 2008; Cai et al., 1998; El-Elimat et al., 2019; Mayerl et al., 1993; Pornpakakul et al., 2007), thereby attracting much attention from synthetic chemists (Adlington et al., 2002; Baldwin et al., 1999). Eupenifeldin is a bistropolone-humulene meroterpenoid first discovered as an antitumor agent from the fungus Eupenicillium brefeldianum (Mayerl et al., 1993), and also showed anthelmintic, antimalarial, and antifungal effects (Ayers et al., 2008; Bunyapaiboonsri et al., 2008). In our chemical investigation of Phoma sp. (CGMCC 10481), we also isolated eupenifeldin (1; Fig. 1A) and a rearranged

congener phomanolide A (2; Fig. 1A) with a unique pentacyclic skeleton (Zhang et al., 2015). Eupenifeldin showed potent antiproliferative effects against three human glioma cell lines, with IC50 values ranging from 0.08 to 0.13 µM, suggesting that it could be used as a lead compound for further modification and evaluation. In addition, putative biosynthetic pathways leading to the formation of 1 and 2 were proposed based on the structures of isolated products and possible intermediate/precursor (Zhang et al., 2015). Specifically, the tropolonehumulene-tropolone meroterpene eupenifeldin could be the heteroDiels-Alder adduct derived from two units of tropolone orthoquinone methides (b′; Fig. 1B) and a humulenol moiety (4′; Fig. 1A) via a key intermediate 3 (El-Elimat et al., 2019), while the rearranged monotropolone sesquiterpene 2 could also be generated from 3 and a derivative of 3-methylorcinaldehyde (a; Fig. 1B) via hetero-Diels-Alder reaction followed by rearrangements (Zhang et al., 2015). Despite of intriguing structures and profound activities, the

⁎ Corresponding authors at: School of Life Sciences, University of Science and Technology of China, Hefei 230026, China (X. Liu). State Key Laboratory of Toxicology & Medical Countermeasures, Beijing Institute of Pharmacology & Toxicology, Beijing 100850, China (Y. Che). E-mail addresses: [email protected] (X. Liu), [email protected] (Y. Che).

https://doi.org/10.1016/j.fgb.2019.04.004 Received 6 February 2019; Received in revised form 15 March 2019; Accepted 4 April 2019 Available online 10 April 2019 1087-1845/ © 2019 Elsevier Inc. All rights reserved.

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Fig. 1. Structures of eupenifeldin, phomanolide A, and their putative precursors and/or intermediates. A. Structures of the compounds isolated from Phoma sp. B. Structures of putative precursors and/or intermediates of eupenifeldin and phomanolide A (Zhang et al., 2015), which were not isolated from Phoma sp.

biosynthesis of bistropolone-humulene meroterpenes remained underexplored (Guo et al., 2018). In biosynthetic study of the fungal tropolone stipitatic acid (Davison et al., 2012), three enzymes, a nonreducing polyketide synthase (NR-PKS) TropA, a FAD-dependent monooxygenase TropB, and a non-heme Fe (II)-dependent dioxygenase TropC, were identified to catalyze formation of troplone backbone (Davison et al., 2012). The enzymes TropB and TropC are required to form the key intermediate tropolone stipitaldehyde (b; Fig. 1B) from a. In another study of fungal meroterpenoid xenovulene A (Raggatt et al., 1997; Schor et al., 2018), a new terpene cyclase AsR6 in Acremonium strictum was characterized to catalyze the conversion from farnesylpyrophosphate (FPP) to humulene (Schor et al., 2018; 4; Fig. 1B), which represents a highly unusual example of terpene cyclases. Considering the unique structural features of eupenifeldin and its potent anti-glioma activity, we first attempted to identify its biosynthetic gene cluster (BGC). The genome of eupenifeldin-producing Phoma sp. was sequenced and analyzed, and results from in silico assays revealed that the fungus was potential producers of diverse secondary metabolites (SMs). The eupenifeldin BGC eup was identified and partially validated by genomic analysis, gene disruption, and product analysis. Three enzymes, EupA–C, highly homologous to TropA–C, were required to form tropolone backbone in Phoma sp., and a terpene cyclase EupE, highly homologous to AsR6, was responsible for humulene formation. In addition, a cytochrome P450 enzyme EupD was identified to catalyze the hydroxylation of humulene. This study provides the first insight into the biosynthesis of bistropolone-humulene meroterpenoid eupenifeldin, and paves the way to further decipher its biosynthetic pathway.

on a rotary shaker at 220 rpm for 7 days to prepare the seed culture. Fermentation was carried out in 20 Fernbach flasks (500 mL), each containing 80 g of rice, and the contents inoculated with the spore inoculum were incubated at 25 °C for 30 d. Rice medium (16% rice powder and 1.5% agar) was used for fungal growth and extraction of total RNA, and TSA medium (1.7% tryptone, 0.3% tryptone soya broth, 0.25% glucose, 0.5% NaCl, 0.25% K2HPO4, 0.24% KH2PO4 and 1.5% agar; pH 7.0) was used for fungal growth, extraction of genomic DNA, and selection of Phoma sp. transformants. Escherichia coli DH5α and E. coli Rosetta (CWBIO, China) used for plasmid propagation and in vitro protein expression, respectively, were grown in Luria–Bertani (LB) medium (1% NaCl, 1% tryptone, and 0.5% yeast extract; pH 7.0) at 37 °C. Agrobacterium tumefaciens AGL-1 (ZOMANBIO, China) was used for A. tumefaciens-mediated transformation (ATMT). Medium used for ATMT of Phoma sp. was described previously (Mullins et al., 2001).

2. Materials and methods

2.3. Genome assembly, annotation, and analysis

2.1. Strains, plasmids, media, and growth conditions

Genome datasets were assembled using softwares Celera Assembler V8.3 and Falcon v0.3.0 (Chin et al., 2016; Myers et al., 2000), and the best assembly was selected. SSPACE_Basic_v2.0 was used to link contigs to scaffolds with long inset size pair-end reads, and the gap was filled using pbjelly2 V15.8.24 (English et al., 2012; Kim et al., 2014). The complete genome was annotated using Augustus V3.2.1 (Stanke et al., 2008), SNAP v2010-07-28 (Johnson et al., 2008), GeneMark-ES V4.21 (Ter-Hovhannisyan et al., 2008), and Genewise V2.20 (Birney et al., 2004). Repeat prediction was identified using RepeatMasker V4-0-6 based on the RepeatMasker library (Bao et al., 2015). Program tRNAscan-SE V1.3.1 was used to identify transfer RNAs (tRNAs) in the genome (Lowe

2.2. Genomic DNA extraction and sequencing Genomic DNA of Phoma sp. was extracted by sodium laurate (Cai et al., 2014), and dissolved in distilled H2O. The quality of obtained sample was assessed by gel-electrophoresis, and the purity was analyzed on a Thermo Scientific Nanodrop 2000 instrument. For genome sequencing, a paired-end DNA library with an average fragment size of 200 bp was constructed, and sequenced on Illumina HiSeq2500 sequencer, while a mate-pair DNA library with an average fragment size of 20 kb was constructed, and sequenced on Pacific Biosciences RS II sequencer (BGI, China).

Strains and plasmids used in this study were listed in Table S1. Phoma sp. (CGMCC 10481), was first isolated from a soil sample collected from the Qinghai-Tibetan plateau, Tibet, P. R. China, identified based on morphology and sequence (Genbank Accession No. KP896486) analysis of the internal transcribed spacer (ITS) region of the rDNA, and used as the wild-type strain (WT). The WT was cultured on potato dextrose agar (PDA) at 25 °C for 10 d to produce spores, which were inoculated in three Fernbach flasks (500 mL), each containing 200 mL media (0.4% yeast extract, 1% malt extract, and 0.4% glucose; pH 6.5). After sterilization, the flasks were incubated at 25 °C 8

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and Eddy, 1997). Ribosomal RNAs (rRNAs) were identified using RNAmmer V1.2 (Lagesen et al., 2007). The total putative proteins were annotated using databases KEGG (Kanehisa et al., 2015), GO (Ashburner et al., 2000), and eggNOG (Huerta-Cepas et al., 2015). The ITS sequences of Phoma sp. and 14 reference fungal species (Table S2) were aligned using Clustal W, based on which a phylogenetic tree was created using MEGA V5.1 (Hall, 2013). Potential BGCs of SMs in the genome of Phoma sp. were predicted by anti-SMASH v3.0.3 (Medema et al., 2011).

n-pentanes, and analyzed on a Shimadzu GC/MS-QP2010 Ultra instrument equipped with a DB-5MS column (30 m × 0.25 mm; 0.25 µm). The oven temperature was set at 75 °C for 1 min, increased by 10 °C/ min to 300 °C, and held at 300 °C for 4 min. The injector temperature was 270 °C. The GC was operated in the splitless mode using helium as the carrier gas (2 mL/min).

2.4. RNA isolation, RT-PCR, and quantitative real-time RT-PCR

Fermented cultures of WT and transformants were extracted with 200 mL ethyl acetate (EtOAc), and the resulting extracts were analyzed on an Agilent Accurate-Mass-Q-TOF LC/MS 6550 instrument equipped with an electrospray ionization (ESI) source (Agilent Eclipse Plus C-18 RRHD column; 1.8 μm; 2.1 × 50 mm). HPLC separation was performed using 0.1% formic acid in H2O (A) and CH3CN (B) as the eluents (20% B for 0.5 min, 20–40% B for 9.5 min, 40–100% B for 20 min, and 100% B for 3 min; 0.3 mL/min). For MS analysis, the fragmentor and capillary voltages were kept at 175 and 3,500 V, respectively. The temperature and flow rate of drying gas were set at 200 °C and 14 L/min, respectively. The pressure of the nebulizer was 35 psi. The temperature and flow rate of sheath gas were set at 350 °C and 11 L/min, respectively. Full-scan spectra were acquired over a scan range of 40–1,700 at 1 spectra/s. All MS experiments were performed in positive ion mode.

2.7. Chemical analysis

The mycelia of Phoma sp. cultured on rice for 3 and 6 days were separately collected, and the total RNAs were isolated using RNeasy Plant Mini Kit (QIAGEN, Germany). The PrimeScript™ RT reagent Kit (TaKaRa, Japan) was used for reverse transcription PCR (RT-PCR), which was performed using 2 × Taq Master Mix (Vazyme, China) with 10 ng cDNA as the template. Quantitative real-time RT-PCR analysis was carried out with the primers eupDRTF/eupDRTR and actinF/actinR as described previously (Li et al., 2013). 2.5. Construction of plasmids and strains All primers used in this study were listed in Table S3. PCR amplification of DNA fragments were performed using Phanta Max Superfidelity DNA polymerase (Vazyme, China), and the amplified fragments corresponding to the upstream and downstream regions of target genes were recombined into plasmid pAg1H3 using ClonExpress MultiS One Step Cloning Kit (Vazyme, China). As an exception, the upstream and downstream regions of gme5038, gme12629, gme12632, gme12633, and gme12636, were digested with restriction enzymes (Thermo Fisher Scientific, USA) and joined into corresponding sites of plasmid pAg1H3 using T4 DNA ligase (Thermo Fisher Scientific, USA). The recombined plasmids were used to construct gene disruption mutants by ATMT (Khang et al., 2006; Long et al., 2012). Hygromycin B-resistant colonies were selected after cultured in TSA medium at 28 °C for 4 days, and the disruption mutants were verified by PCR with inside (IF/IR) and outside (OF/OR) primers of target genes (Table S3). To construct complemented strains, DNA fragments containing target gene sequence and its promoter and terminator regions were amplified and inserted into corresponding sites of plasmid pAg1H3-neo, and the recombined plasmids were introduced into individual disruption mutants by ATMT (Khang et al., 2006; Long et al., 2012). The G418-resistant colonies were selected after cultured in TSA medium at 28 °C for 4 days, and verified by PCR using the neoF/neoR and relevant IF/IR primers (Table S3).

3. Results 3.1. Genome sequence analysis of Phoma sp. The genome of Phoma sp. was assembled, resulting in 33 contigs with 442,708 reads (Table 1). The estimated genome size was 40.1 Mb, in which 13,812 putative proteins were encoded. The sequence has been deposited (GenBank Accession No. RBKR00000000), and general characteristics of the genome were listed in Table 1. A total of 4,477 predicted proteins were assigned into 311 pathways by KEGG database and summarized in 6 parts and 44 subparts (Fig. S1). Since the ITS secondary structure is highly conserved in eukaryotes, its sequence can serve as a valuable barcode marker for fungi (Schoch et al., 2012). Phylogenetic analysis based on the ITS sequences of Phoma sp. (Zhang et al., 2015) and other 14 strains (Table S2) revealed that Phoma sp. was genetically close to the plant pathogenic fungi Ascochyta rabiei and Epicoccum nigrum (Fig. S2). A total of 31 putative BGCs for SMs were identified in Phoma sp., and classified according to putative key enzymes (Fig. S3). To estimate the biosynthetic capabilities of Phoma sp. cultured on rice, transcription of all clusters was analyzed by RT-PCR using the primers listed in Table S3, and those clusters without detectable transcription of key enzymes were considered as silent ones. Results from RT-PCR showed that seven of the predicted clusters remained silent when cultured on rice (Table 2).

2.6. Gene expression in E. coli and enzymatic assay Plasmids pET28a-eupE, pET28a-eupD, pET28a-eupH, and pET28aeupF were constructed using their optimized sequences and introduced into E. coli Rosetta, whereas plasmid pET28a-eupB was constructed using its cDNA sequence. Gene expression was induced by addition of 0.1 mM IPTG when the OD600 values of the cultures reached 0.6. Cells were incubated at 15 °C for 20 h, harvested by centrifugation (10,000 rpm, 5 min), resuspended in binding buffer (50 mM Tris-HCl, 150 mM NaCl, and 20 mM imidazole; pH 8.0), and sonicated on ice for 40 min. Proteins were purified by Ni-NTA agarose chromatography (Thermo Fisher Scientific, USA), and assessed by SDS–PAGE with 12% polyacrylamide gel. Purified proteins were concentrated using Millipore Amicon Ultra Centrifugal Filter (Billerica, MA, USA), and stored in 5% glycerol at −20 °C. Enzymatic assay of EupE was performed by incubating FPP (150 µM) and purified EupE (320 µg) in 300 µL buffer (10 mM HEPES, 5 mM MgCl2, and 5 mM dithiothreitol; pH 8.0) at 30 °C for 30 min (Schor et al., 2018). The reaction products were extracted with 300 µL

Table 1 Genome characteristics of the Phoma sp. Features

Value

Assembly size (Mb) Scaffold N50 (Mb) Scaffold N90 (Mb) GC content (%) Repeat rate (%) Protein-coding genes Exons Introns tRNA genes rRNA genes

40.1 1.5 1.0 50.38 11.06 13,812 35,295 21,483 187 71

Mb, mega base pairs; tRNA, transfer RNA; rRNA, ribosomal RNA. 9

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gme12628 located in the predicted clusters 22 and 7, respectively (Figs. S4 and 2A). Since the first step in tropolone biosynthesis is catalyzed by a NR-PKS TropA, its amino acid sequence was also used as the BLAST query, resulting in identification of a NR-PKS GME12632 encoded by the gene gme12632 located in cluster 7. Since both gme12628 and gme12632 were identified in cluster 7, this cluster is most likely the BGC responsible for eupenifeldin biosynthesis. To verify above speculation, gme12632 was deleted by homologous recombination (HR; Fig. S5), and the disruption mutant Δ12632 was verified by PCR analysis (Fig. S5E). The fragments of 9,843 and 4,036 bp were amplified from WT and Δ12632, respectively, using the primers outside gme12632. While a 500 bp fragment was amplified from WT, but not from Δ12632 when the primers inside gme12632 were used (Fig. S5E). The disruption mutant of gme5038 (Δ5038) was constructed using the same strategy as the control (Fig. S5L). To determine whether Δ5038 and Δ12632 still produce eupenifeldin, the extracts from two disruption mutants and WT were analyzed by HPLC/MS using eupenifeldin as the standard (Fig. 2B), and the results showed that eupenifeldin production was abolished in Δ12632, whereas Δ5038 still produced eupenifeldin (Fig. 2C). The complemented strain 12632CM was constructed by introducing the entire gme12632 gene with its promoter and terminator regions into Δ12632 (Fig. S6E), and HPLC/MS analysis showed that eupenifeldin production was partially restored in 12632CM (Fig. 2C). These results indicated that gme12632 is essential

Table 2 Putative secondary metabolism gene clusters in Phoma sp. Type

Quantity

Transcribed

PKS NRPS TS Hybrid (PKS-NRPS) Hybrid (TS-NRPS) Other Total

16 2 4 2 1 6 31

10 2 4 2 0 6 24

PKS, clusters with polyketide synthase encoding genes; NRPS, clusters with non-ribosomal peptide synthase encoding genes; TS, clusters with terpenoid synthase encoding genes; Hybrid, clusters with two types of synthases encoding genes; Other, clusters with unusual core genes; Transcribed, the number of transcribed clusters when cultured on rice.

3.2. Identification of eupenifeldin biosynthetic gene cluster In biosynthetic study of stipitatic acid, a non-heme Fe (II)-dependent dioxygenase TropC was identified to catalyze formation of the seven-membered ring via oxidative ring expansion (Davison et al., 2012). Therefore, the amino acid sequence of TropC was used as the BLAST query to scan the predicted proteins in Phoma sp. Two homologous proteins were identified with their encoding genes gme5042 and

Fig. 2. Identification of the eupenifeldin biosynthetic gene cluster. (A) Amino acid sequence alignment of TropC (GenBank Accession No. DAA64706.1), GME5042, and GME12628 with homologous sequences indicated with black background. The position of conserved domain is indicated by the line above sequence. (B) Mass spectrum of eupenifeldin. (C) HPLC traces of standard eupenifeldin, and the extracts from WT, the disruption mutants Δ12632 and Δ5038, and the complemented strain 12632CM (UV detection at 254 nm). Peak 1 represents eupenifeldin, which was not detected in the extract of Δ12632. 10

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Fig. 3. Identification of the boundary genes of cluster 7. (A) HPLC traces of standard eupenifeldin, and the extracts from WT, disruption mutant of gme12628 (Δ12628), and the complemented strain 12628CM (UV detection at 254 nm). (B) HPLC traces of standard eupenifeldin, and the extracts from WT, disruption mutant of gme12638 (Δ12638), and the complemented strain 12638CM (UV detection at 254 nm). Peak 1 represents eupenifeldin, which was not detected in the extracts of Δ12628 and Δ12638.

Fig. 4. The biosynthetic gene cluster (BGC) and proposed biosynthetic pathway of eupenifeldin in Phoma sp. (A) Schematic organization of the eupenifeldin BGC. The arrows represent the genes and their direction of transcription. The genes were labeled in different colors based on their putative functions. (B) Proposed biosynthetic pathway for eupenifeldin. Proteins that catalyzed the proposed reactions are indicated. Double arrows indicate that two or more steps are required. Table 3 Putative function analysis of the genes within the eup cluster. Gene

Size (bp/aa)

Putative Function

Gene

Size (bp/aa)

Putative Function

eupA eupB eupC eupD eupE

8315/2682 1704/446 1119/263 970/285 1140/379

NR-PKS FAD-dependent monooxygenase non-heme Fe (II)-dependent dioxygenase cytochrome P450 monooxygenase putative humulene synthase

eupF eupG eupH eupR eupM eupT

1117/352 951/278 1293/430 1238/350 2035/301 4783/1430

putative DAase short-chain dehydrogenase FAD-dependent monooxygenase transcription factor MFS transporter ABC transporter

responsible for humulene biosynthesis in Phoma sp. Since intermediate 3 (Fig. 1A) proposed in the biosynthesis of eupenifeldin and phomanolide A in Phoma sp. is structurally analogous to xenovulene A, the enzymes responsible for its biosynthesis should be similar to those encoded by the genes in BGC of xenovulene A (Schor et al., 2018). Comparison of amino acid sequence alignment between the two clusters revealed that the proteins encoded by gme12628, gme12631, and gme12632 were respectively homologous to those encoded by asL3, asL1, and aspks1 (Fig. S8), which are essential for tropolone formation (Schor et al., 2018). In addition, an unknown protein

for eupenifeldin biosynthesis in Phoma sp. To explore how humulene was generated in Phoma sp., the amino acid sequence of the reported humulene synthase AsR6 (Schor et al., 2018) was used as the BLAST query to search homologous proteins in Phoma sp., and an unknown protein GME12634 showing high homology to AsR6 was identified in cluster 7 (Fig. S7A). The disruption mutant Δ12634 and the complemented strain 12634CM were constructed (Figs. S5G and S6G), and HPLC/MS analysis of their extracts showed that humulene production was abolished in Δ12634, and was restored in 12634CM (Fig. S7C), indicating that gme12634 is 11

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Fig. 5. Identification of the gene responsible for humulene hydroxylation. (A) Transcriptional analysis of gene eupD. The relative abundance of mRNAs was standardized against the level of actin gene. The transcriptional level of eupD in each strain was indicated by the column. Error bars represent the standard deviations from three independent experiments. (B) Mass spectrum of humulenol (4′; Std). (C) Extracted ion chromatogram (EIC) of standard 4′ and the extracts prepared from WT, disruption mutant of eupD (ΔeupD), the complemented strain eupDCM, and the eupD-overexpressed strain eupDOE (m/z 221 [M+H]+]). Peak 4′ represents humulenol, which was not detected in the extract of ΔeupD. (D) HPLC traces of standard humulene (4; Std) and the extracts prepared from WT, disruption mutant ΔeupD, the complemented strain eupDCM, and the eupD-overexpressed strain eupDOE (UV detection at 200 nm). Peak 4 represents humulene.

eupenifeldin biosynthesis in Phoma sp. Collectively, cluster 7 was identified as the BGC for eupenifeldin, and named the eup cluster (GenBank Accession No. MK400120; Fig. 4A).

GME12633 showed high sequence similarity to the putative Diels-Alderase (DAase) AsR5 (Schor et al., 2018), which presumably catalyze formation of the tropolone-humulene skeleton. Considering above information, gme12628 and gme12631–12634 in cluster 7 could be involved in the biosynthesis of intermediate 3 in Phoma sp. To verify the predicted boundary from gme12628 to gme12638 for cluster 7, the nearby genes gme12627 and gme12639 were subjected to BLAST analysis. The results showed that gme12627 encoded a putative transposase protein with a conserved domain typically found in the DDE superfamily endonuclease, and gme12639 encoded a protein homologous to an RNA-directed DNA polymerase from transposon X-element (Table S4). Attempts to knockout these two genes were unsuccessful, implying that they could be vital for the survival of Phoma sp., and are thereby excluded from the cluster. The genes gme12628 and gme12638 were disrupted by HR (Fig. S5A and K), and their complemented strains 12628CM and 12638CM were also constructed (Fig. S6A and K). HPLC/ MS analyses of their extracts showed that eupenifeldin production was abolished in Δ12628 and Δ12638, and restored in 12628CM and 12638CM (Fig. 3A and B), indicating that these two genes are responsible for eupenifeldin biosynthesis in Phoma sp., and are the boundary of cluster 7. Other genes in this cluster were also disrupted by HR, and the resulting disruption mutants were verified by PCR (Fig. S5B–D and S5F–J). The extracts of WT, the disruption mutants Δ12629–Δ12631 and Δ12633–Δ12637, and the complemented strains 12629CM–12631CM and 12633CM–12637CM were analyzed by HPLC/ MS, and the results showed that eupenifeldin production was abolished in all disruption mutants except for Δ12630 (Fig. S9). These results verified that at least 10 genes in the cluster were involved in

3.3. Functional analysis of genes in the eup cluster To explore the biosynthetic pathway of eupenifeldin, the functions of all genes in the eup cluster were analyzed by BLASTp (Table S4), gene disruption, and product analysis (Fig. S9; Table 3). Generation of the putative precursor a could be catalyzed by a NR-PKS encoded by eupA, and dearomatization of a by hydroxylation was likely catalyzed by a FAD-dependent monooxygenase encoded by eupB. In turn, formation of tropolone stipitaldehyde b could be catalyzed by a non-heme Fe (II)dependent dioxygenase encoded by eupC. While generation of sesquiterpene humulene was catalyzed by a terpene cyclase encoded by eupE (Fig. 4B). In addition, a predicted cytochrome P450 monooxygenase encoded by eupD was also essential for eupenifeldin biosynthesis in Phoma sp. (Fig. S9H). In biosynthetic study of the plant sesquiterpene zerumbone, a cytochrome P450 enzyme was demonstrated to catalyze the hydroxylation of humulene (Yu et al., 2011), suggesting that EupD was likely the enzyme to convert humulene to humulenol. A functionally unknown protein encoded by eupF was also essential for eupenifeldin production, which is a putative DAase showing high homology to the proposed AsR5 (Fig. S10; Schor et al., 2018). Disruption of the oxidoreductase encoding genes eupG and eupH also abolished eupenifeldin production, indicating that EupG and EupH were likely the additional decorating enzymes or accessory proteins for the biosynthesis of eupenifeldin or congeners. The deduced product of eupR, which 12

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Fig. 6. GC/MS analysis of in vitro enzymatic reaction products. (A) GC chromatogram of the humulene standard (Sigma, USA) with its mass spectrum inserted. (B) GC chromatogram of the organic phase (n-pentanes) of the reaction mixture with mass spectrum inserted. The peak represents the newly observed product, showing the same molecular weight and fragments as humulene. (C) GC chromatogram of the organic phase (n-pentanes) of the reaction mixture using denaturated EupE. Additional products were not detected in the reaction mixture.

hydroxylation. The recombined plasmid pAg1H3-neo-P12637T (Fig. S6J) was introduced into WT by ATMT to construct the eupD-overexpressed strain eupDOE, which was verified by quantitative real-time RT-PCR (Fig. 5A). The results showed that the transcription of eupD was increased in eupDOE compared to that in WT. The extracts of WT, the disruption mutant ΔeupD, the complemented strain eupDCM, and eupDOE were analyzed by HPLC/MS, and the results showed that humulenol production was abolished in ΔeupD, but restored in eupDCM (Fig. 5C), whereas humulene was detected in the extracts of WT, ΔeupD, and eupDCM (Fig. 5D). In addition, overexpression of eupD increased humulenol production, but decreased the production of humulene (Fig. 5C and D). These results indicated that EupD catalyzes the conversion from humulene to humulenol.

has a specific transcription factor domain found in fungi, could be a regulator for eupenifeldin biosynthesis. While the two predicted transporters, the major facilitator superfamily (MFS) transporter EupM and the ATP-binding cassette (ABC) transporter EupT, are encoded by eupM and eupT, respectively. Combining identified eup cluster with gene function analysis and the isolated putative intermediates (ElElimat et al., 2019; Zhang et al., 2015), a putative biosynthetic pathway for eupenifeldin was proposed (Fig. 4B). 3.4. EupD is required to convert humulene to humulenol Humulenol is rarely encountered as fungal SMs. Based on functional analysis of the genes in eup cluster, a putative cytochrome P450 monooxygenase encoded by eupD was likely responsible for humulene 13

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of EupD. However, incubation of EupD with either EupB or EupH failed to produce the desired products, suggesting the possible existence of other unknown redox partners for EupD, or the necessity for optimizing the conditions of enzymatic reactions. In conclusion, we have identified the BGC of eupenifeldin (eup) in Phoma sp., and predicted the functions for most of the genes in eup cluster. Besides enzymes EupA–C, which are required to form the tropolone backbone in Phoma sp., a terpene cyclase EupE, highly homologous to AsR6, was also identified to catalyze humulene formation. While a cytochrome P450 enzyme EupD was identified for the hydroxylation of humulene. This study provides the first insight into the biosynthesis of tropolone-humulene-tropolone meroterpene eupenifeldin, and paves the way to further decipher its biosynthetic pathway and to generate new congeners for further evaluations.

3.5. In vitro enzymatic activity To clarify the biosynthetic pathway of humulenol, eupE was expressed in E. coli Rosetta (Fig. S11), and the purified EupE was used for the in vitro enzymatic reaction. Based on the results from GC/MS analysis, incubation of EupE with FPP led to the production of humulene (Fig. 6B), indicating that EupE indeed catalyze the conversion from FPP to humulene in Phoma sp. (Fig. 4B). Since EupD belongs to the Class II cytochrome P450 systems, which requires redox partners to obtain electrons from NADPH via a FAD-containing reductase (Hannemann et al., 2007), the FAD-dependent monooxygenases EupB and EupH (Table 3) could be the candidates for the redox partners of EupD. Therefore, eupD, eupB, and eupH were expressed in E. coli Rosetta, and the purified proteins were used for in vitro enzymatic reactions. Unfortunately, incubation of EupD with EupB or EupH failed to produce the desired products when humulene was used as the substrate.

Acknowledgements

4. Discussion

We are grateful to Prof. Seogchan Kang (Penn State University) for providing plasmid pAg1H3. Financial support from the CAMS Innovation Fund for Medical Sciences (2018-I2M-3-005) is gratefully acknowledged.

Meroterpenoids, a unique class of natural products with intriguing structure features and important biological activities, are derived from terpenoid-polyketide or terpenoid-nonpolyketide pathways (Geris and Simpson, 2009; Matsuda and Abe, 2016). Tropolonic sesquiterpene are those generated from a sesquiterpene and one or two tropolone units, and show profound biological effects (Ainsworth et al., 1995; Ayers et al., 2008; Cai et al., 1998; El-Elimat et al., 2019; Mayerl et al., 1993; Pornpakakul et al., 2007). As a typical tropolonic sesquiterpene, eupenifeldin is a tropolone-humulene-tropolone configured antitumor meroterpenoid discovered from different fungal strains (El-Elimat et al., 2019; Mayerl et al., 1993; Zhang et al., 2015). We recently found that eupenifeldin showed potent anti-glioma activity and could serve as a lead for further optimization (Zhang et al., 2015). Therefore, it is urgently needed to elucidate the biosynthetic pathways of eupenifeldin and congeners to address the problem of sample limitations. In this study, the eupenifeldin BGC (eup) was identified and partially validated. Although functions for most of the genes in eup cluster have been predicted, elucidation of the biosynthetic mechanism for eupenifeldin remained a major challenge, especially formation of the tropolone-humulenol-tropolone core via hetero-Diels-Alder reactions. Despite extensive efforts, only a limited number of examples of DAases have been reported in recent years (Lichman et al., 2019; Walsh and Tang, 2018). An unknown enzyme encoded by eupF in eup cluster showed high homology to AsR5 based on sequence comparison and structure alignment (Schor et al., 2018), indicating that it may be a DAase with highly conserved domain, and catalyze similar hetero-DielsAlder reactions. Its encoding gene eupF was expressed in E. coli Rosetta, and the expected protein was obtained in soluble form. Due to unavailability of tropolone orthoquinone methide (b′), in vitro enzymatic reaction was performed using commercially available dehydrolencodin as the diene substrate instead of b′. However, the desired hetero-DielsAlder adducts were not detected. Study towards addressing this issue is currently ongoing. In previous chemical study of Phoma sp., all isolated meroterpenoids were derived from tropolone orthoquinone methide and humulenol (Zhang et al., 2015). To our knowledge, this is the first demonstration of humulenol involved in generating fungal SMs. The biosynthetic pathway from FPP to humulenol was proposed, in which two enzymes, EupE and EupD, were identified to catalyze its formation. EupE is a humulene synthase belonging to an unusual terpene cyclase family with AsR6 as the only known example. Hydroxylation of humulene was documented only in a biosynthetic study of plant sesquiterpene zerumbone (Yu et al., 2011; Zhang et al., 2018). A cytochrome P450 enzyme encoded by eupD in eup cluster was proposed to catalyze the hydroxylation of humulene, which was verified by gene disruption analysis. In addition, two FAD-dependent monooxygenases individually encoded by eupB and eupH, were identified as potential redox partners

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