assiana culture, the BbGT1/MT1 but not the BbGT2/MT2 pair was extremely upregulated in cocultures (Fig. S4D). In contrast, the orthologous gene pair MrGT1/MT1 was substantially downregulated, whereas the MrGT2/MT2 pair was upregulated in cocultures compared with these from the pure M. robertsii sample (Fig. S4E).November/December 2021 Volume 12 Issue six e03279-21 mbio.asm.orgChen et al.FIG 3 Intermediate PKD1 site production and cross-modification of 15-HT by the nonclustered methylglucosylation genes. (A) HPLC analysis displaying the production or nonproduction of distinct intermediate compounds soon after gene deletions inside the OE::tenR mutant of B. bassiana (Bb). (B) Verification with the methylglucosylation genes contributing towards the production of compound 1 in B. bassiana. (C) Verification on the cross-modification of 15-HT by M. robertsii (Mr). (D) Substrate feeding assay confirming the conversion of compound 3 to compound 1 by MrGT1. Feeding with compound 4 couldn’t be converted by M. robertsii. (E) Compound conversions by transgenic yeast cells. Compound two or 3 was added towards the media at a final concentration of ten m g/ml for two days.To examine the potential contribution of these two gene pairs for the production in the glycoside PMGP, we deleted these two gene pairs within the OE::tenR strain. HPLC analysis revealed that the BbGT1/MT1 but not the BbGT2/MT2 pair is accountable for PMGP production (Fig. 3B). Not surprisingly, the deletion of BbGT1 also disabled the production of PMGP by the fungus. We also cocultured M. robertsii with all the OE::tenR DBbGT1/ MT1 strain and located proof of your cross-modification of 15-HT, i.e., the catalysis ofNovember/December 2021 Volume 12 Challenge six e03279-21 mbio.asm.orgChemical Biology of Fungal 2-PyridonesFIG 4 Schematic in the biosynthesis with the tenellin-related compounds. The scheme shown in square brackets for tenellin biosynthesis was recommended previously (20). The query marks indicate that the involved enzymes or pathways remain unclear. The compounds labeled in blue are recognized items reported previously, though those labeled in red are novel chemical substances identified within this study. Pretenellins A and B were not detected in this study. SAM, RGS16 list S-adenosylmethionine; CoA, coenzyme A.15-HT to PMGP by M. robertsii (Fig. 3C). Consistently, PMGP was yielded by direct feeding with the WT strain but not the DMrGT1 strain of M. robertsii with 15-HT. Even so, coculturing on the OE::tenR DBbGT1/MT1 and DMrGT1 strains failed to generate detectable PMGP. Moreover, it was confirmed that feeding in the DMrGT1 strain with 15-HT or M. robertsii with compound four (i.e., 1-O-methyl-15-HT) didn’t cause the occurrence of any conversion (Fig. 3D). The feeding of transgenic yeast cells additional confirmed that 15-HT could possibly be converted to PMGP by either BbGT1/MT1 or MrGT1/MT1. Also, a novel peak, 19, appeared within the yeast cultures soon after feeding with 15-HT (Fig. 3E). This compound was purified and structurally identified as a novel compound, pyridovericin-N-O-( b –D-glucopyranoside), i.e., the 4-O-position-unmethylated PMGP (Fig. S1 and Information Sets S1 and S2). The feeding of BbGT1/MT1 transgenic yeast cells with pyridovericin didn’t show any further peak (Fig. 3E). Taken together, the results indicated that BbGT1 and MrGT1 target only the N-OH hydroxyl residue of 15-HT. Intriguingly, on the other hand, BbGT1/ MT1 transgenic yeasts failed to catalyze the methylglucosylation of farinosone B (Fig. S3E). Proposal on the 2-pyridone biosynthetic pathway. Hav
