Biosynthesis of the mycotoxin tenuazonic acid by a fungal NRPS–PKS hybrid enzyme

Tenuazonic acid (TeA) is a well-known mycotoxin produced by various plant pathogenic fungi. However, its biosynthetic gene has been unknown to date. Here we identify the TeA biosynthetic gene from Magnaporthe oryzae by finding two TeA-inducing conditions of a low-producing strain. We demonstrate that TeA is synthesized from isoleucine and acetoacetyl-coenzyme A by TeA synthetase 1 (TAS1). TAS1 is a unique non-ribosomal peptide synthetase and polyketide synthase (NRPS–PKS) hybrid enzyme that begins with an NRPS module. In contrast to other NRPS/PKS hybrid enzymes, the PKS portion of TAS1 has only a ketosynthase (KS) domain and this domain is indispensable for TAS1 activity. Phylogenetic analysis classifies this KS domain as an independent clade close to type I PKS KS domain. We demonstrate that the TAS1 KS domain conducts the final cyclization step for TeA release. These results indicate that TAS1 is a unique type of NRPS–PKS hybrid enzyme.


Type III
Type I

Supplementary Figure 13. Sequence alignment of the KS domains in types I and III
polyketide synthases (PKSs) and TAS1. KS domain sequences were aligned with MUSCLE, and gaps in the type III PKSs were adjusted to align the catalytic residues. Only the regions including catalytic residues are shown. Catalytic triad residues (orange) and surrounding regions (yellow) are highlighted. TAS1 sequences in the highlighted regions are underlined. Supplementary

OSM1 gene disruption
The PCR primers used are listed in Supplementary Table 4. OSM1 disruptants were constructed as follows. The upstream sequence of OSM1 was amplified from the genomic DNA of M.
oryzae by PCR using ApaI-5′OSM1-F and MluI-5′OSM1-R primers. The 1.0 kb fragment was gel purified, TOPO-cloned, digested with ApaI and MluI, and gel purified (fragment 1). The hygromycin B-resistance gene expression unit was amplified from pCSN45 by PCR with MluI-HYG-F and XbaI-HYG-R primers. The 1.6 kb fragment was gel purified, TOPO-cloned, digested with MluI and XbaI, and gel purified (fragment 2). The downstream sequence of OSM1 was amplified from the genomic DNA of M. oryzae by PCR with XbaI-3′OSM1-F and BsiWI-3′OSM1-R primers. The 5.0 kb fragment was gel purified, TOPO-cloned, digested with XbaI and BsiWI, and gel purified (fragment 3). The vector sequence of pBI121 between the right border and left border was amplified from pBI121 by PCR with ApaI-RB and BsiWI-LB primers. The 8.7 kb fragment was gel purified, TOPO-cloned, digested with ApaI and BsiWI, and gel purified (fragment 4). The 4 fragments were ligated to yield a OSM1 disruption vector, pBI-OSM1::HPH. The A. tumefaciens strain transformed with this plasmid was used for ATMT. OSM1 disruptants were selected by PCR with OSM1-check-F and OSM1-check-R primers, which hybridize upstream and downstream of the deleted OSM1 ORF, respectively. This primer set can amplify 1.7 kb OSM1 ORF from wild-type strains, the 1.6-kb hygromycin B-resistance gene expression unit from OSM1 disruptants, and both fragments from ectopic transformants. The PCR products were digested with EcoRV, yielding 0.7 kb and 1.0 kb fragments from the 1.7 kb OSM1 ORF and an intact 1.6 kb fragment from the hygromycin B-resistance gene expression unit. The ∆osm1-2 and ∆osm1-5 strains were selected as OSM1 gene-disrupted strains ( Supplementary Fig. 2).

Rapid amplification of cDNA ends (RACE) PCR
To determine the ORF of TAS1, we conducted 5′ and 3′ RACE PCR with a SMARTer RACE cDNA Amplification kit (Clontech). Total RNA (1 µg) from M. oryzae was used for cDNA synthesis, and then 5′ and 3′ RACE PCR was conducted with universal and gene-specific primers (for 5′, RACE_GSP-5; for 3′, RACE_GSP-3) according to the manufacturer's instructions. The RACE PCR product was cloned into pCRII-Blunt TOPO using a TOPO PCR cloning kit (Invitrogen) and then transformed into E. coli DH5α. Transformants were selected on an LB plate supplemented with Km (50 µg/ml). The obtained cDNA-inserted plasmid was sequenced.

Artificial intermediate synthesis
To a stirred solution of N-(tert-butoxycarbonyl)-L-isoleucine (92.4 mg, 0.4 mmol) in tetrahydrofuran (10 ml) was added a mixture of