Biosynthetic Investigation of Phomopsins Reveals a Widespread Pathway for Ribosomal Natural Products in Ascomycetes
Total Page：16
File Type：pdf, Size：1020Kb
Biosynthetic investigation of phomopsins reveals a widespread pathway for ribosomal natural products in Ascomycetes Wei Dinga, Wan-Qiu Liua, Youli Jiaa, Yongzhen Lia, Wilfred A. van der Donkb,c,1, and Qi Zhanga,b,1 aDepartment of Chemistry, Fudan University, Shanghai 200433, China; bDepartment of Chemistry, University of Illinois at Urbana–Champaign, Urbana, IL 61801; and cHoward Hughes Medical Institute, University of Illinois at Urbana–Champaign, Urbana, IL 61801 Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved February 23, 2016 (received for review November 24, 2015) Production of ribosomally synthesized and posttranslationally mod- dehydroaspartic acid, 3,4-dehydroproline, and 3,4-dehydrovaline ified peptides (RiPPs) has rarely been reported in fungi, even (Fig. 1A). though organisms of this kingdom have a long history as a prolific Early isotopic labeling studies showed that Ile, Pro, and Phe were source of natural products. Here we report an investigation of the all incorporated into the phomopsin scaffold (24). Because a Phe phomopsins, antimitotic mycotoxins. We show that phomopsin is a hydroxylase that can convert Phe to Tyr has not been found in fungal RiPP and demonstrate the widespread presence of a path- Ascomycetes, the labeling study seems incongruent with a ribosomal way for the biosynthesis of a family of fungal cyclic RiPPs, which we route to phomopsins, in which Tyr, not Phe, would need to be in- term dikaritins. We characterize PhomM as an S-adenosylmethionine– corporated into the phomopsin structure via a tRNA-dependent dependent α-N-methyltransferase that converts phomopsin A to pathway. Here we report our investigation of the biosynthetic path- an N,N-dimethylated congener (phomopsin E), and show that the way of phomopsins, which explains the paradoxical observations in methyltransferases involved in dikaritin biosynthesis have evolved the early labeling studies and unequivocally demonstrates that pho- differently and likely have broad substrate specificities. Genome mopsins belong to the family of RiPP natural products. Moreover, mining studies identified eight previously unknown dikaritins in our study shows that RiPPs are widespread in fungi, paving the way different strains, highlighting the untapped capacity of RiPP biosyn- for a study of their biological activities and biosynthesis. thesis in fungi and setting the stage for investigating the biolog- ical activities and unknown biosynthetic transformations of this Results and Discussion family of fungal natural products. Characterization of the Phomopsin Biosynthetic Gene Cluster. To study phomopsin biosynthesis, we sequenced the genome of RiPP | mycotoxin | posttranslational modification | methyltransferase | P. leptostromiformis ATCC 26115, a potential phomopsin-producer biosynthesis strain. The sequenced genome was found to encode several puta- tive nonribosomal synthases, but none of these appeared to be a scomycetes constitute the largest phylum of the fungal kingdom suitable candidate for phomopsin biosynthesis. Instead, a protein- Aand are a prolific source of natural products that are of great to-nucleotide Blast (tBlastn) search against the sequenced genome significance to humankind (1–4). Beneficial ascomycetous natural using the YVIPID sequence (the putative precursor peptide se- products include many clinically important drugs and various food quence of phomopsin) as the query resulted in identification of a and industrial chemicals. Many other compounds produced by as- precursor peptide gene, phomA, whose product contains five re- comycetes are toxic and are known as mycotoxins, which pose sub- peats of short peptides with the sequence YVIPID (Fig. 2A). Ex- stantial threats to human food supplies and health (5). Major classes amination of the DNA sequence adjacent to phomA led to of natural products from Ascomycetes include alkaloids (6, 7), ter- penoids (8), polyketides (PKs) (9–12), nonribosomal peptides (NRPs) BIOCHEMISTRY Significance (12–14), and PK-NRP hybrids (15, 16). However, ribosomally syn- thesized and posttranslationally modified peptides (RiPPs), a rapidly growing class of natural products (17), have rarely been found in Ascomycetes are a prolific source of natural products that are of Ascomycetes. It has been known since 2007 that amatoxins and great significance for human health, yet production of riboso- phallotoxins are RiPPs produced by strains of another fungal phylum, mally synthesized and posttranslationally modified peptides Basidiomycetes (which, together with Ascomycetes, compose the (RiPPs), a ubiquitous class of natural products, have rarely been subkingdom Dikarya) (17–19). It also was recently reported that the reported in this fungal phylum. Here we show that phomopsins, ustiloxins produced by Aspergillus flavus and Ustilaginoidea virens be- a family of antimitotic mycotoxins, have a ribosomal origin and long to the RiPP class (20, 21). demonstrate the widespread presence of a fungal RiPP pathway Phomopsins are a group of hexapeptide mycotoxins produced for cyclic peptides that we term dikaritins. The framework de- by the pathogenic Ascomycetes Phomopsis leptostromiformis (class scribed herein provides a foundation for mining for additional Sordariomycetes, order Diaporthales) that infects lupins (Lupinus). dikaritin members and investigating the biological activities and Lupin-based animal food contaminated with phomopsins causes biosynthetic chemistry of this family of fungal natural products. lupinosis, a liver disease of sheep and cattle that is particularly Author contributions: W.A.v.d.D. and Q.Z. designed research; W.D., W.-Q.L., Y.J., Y.L., and problematic in Australia and South Africa but affects other coun- Q.Z. performed research; W.D., W.-Q.L., Y.J., Y.L., W.A.v.d.D., and Q.Z. analyzed data; and tries as well (22). By targeting the vinca domain of tubulin (23), W.A.v.d.D. and Q.Z. wrote the paper. phomopsins exhibit potent antimitotic activity and thus also rep- The authors declare no conflict of interest. resent a potential lead for antitumor drug development. Phomop- This article is a PNAS Direct Submission. sins have a 13-member macrocyclic ring formed by an ether bridge Data deposition: The sequences reported in this paper have been deposited in the Gen- linking an Ile residue to the phenyl ring of a Tyr (Fig. 1A). A similar Bank database (accession nos. KU645826–KU645844). Tyr-containing macrocycle is also present in ustiloxins (Fig. 1B). 1To whom correspondence may be addressed. Email: [email protected] or qizhang@ Unlike ustiloxins, however, phomopsins contain no proteinogenic sioc.ac.cn. amino acids and feature a set of dehydroamino acids found only This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. in this group of compounds, including 2,3-dehydroisoleucine, 2,3- 1073/pnas.1522907113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1522907113 PNAS | March 29, 2016 | vol. 113 | no. 13 | 3521–3526 Downloaded by guest on September 24, 2021 tested the methyltransferase activity of PhomM by incubating the enzyme with SAM, phomopsin A, and S-adenosylhomocysteine (SAH) hydrolase; the latter was included to minimize potential product inhibition (30). HPLC analysis of the reaction mixture clearly showed production of a different compound with a re- tention time very close to that of phomopsin A (Fig. 2D, trace v), and this compound was absent in the control reactions in which either SAM was omitted or the supernatant of the boiled enzyme was used (Fig. 2D, traces iii and iv). HR-LC-MS analysis showed that this compound has a protonated molecular ion at m/z = 803.3010, which is consistent with the expected formula of the + dimethylated phomopsin (phomopsin E) ([M + H] calc. m/z 803.3019; 1.1 ppm error) (Fig. 3A). HR-MS/MS analysis and a comparison of its mass spectrum (Fig. 3A) with that of phomopsin A(SI Appendix, Fig. S2) clearly demonstrated that the newly produced compound is a phomopsin A analog featuring a dime- Fig. 1. Chemical structures of phomopsins (A) and ustiloxins (B). The com- thylated amino group at the N terminus. This result shows that pounds labeled in red are previously unknown N,N-dimethylated dikaritins α identified in this study. PhomM is an -N-methyltransferase involved in phomopsin bio- synthesis. We note that the LC-MS analysis showed that pho- mopsin E was also produced in the fermentation culture of identification of a gene cluster (here designated the phom gene P. leptostromiformis; however, the yield was significantly (∼50-fold) cluster; GenBank accession nos. KU645826–KU645844), whose lower than that of phomopsin A and phomopsin E could not be predicted protein products include a copper-containing tyrosinase detected by absorption analysis (Fig. 2D, trace ii). (PhomQ), an S41 family peptidase (PhomP1), an S-adenosylme- thionine (SAM)-dependent methyltransferase (PhomM), and sev- Unusual Phe-Containing Phomopsins. The product of phomA con- eral functionally unknown proteins (Fig. 2B and SI Appendix,Table tains five repeats of short peptides with the sequence YVIPID (Fig. S1). (The nomenclature used for the phomopsin biosynthetic en- 2A) and thus is reminiscent of the precursor peptides of cyano- zymes in this study is aimed at maintaining consistency with that bactins (31) and several plant-derived RiPPs (32, 33), which also used for ustiloxin biosynthesis.) Based on the sequence of the contain multiple putative core peptides and a leader peptide before putative PhomA precursor peptide and the