Reconstruction of the epoxyjanthitrem pathway from Epichloë sp. LpTG-3 strain AR37 in Penicillium paxilli

Using CRISPR-Cas9, our lab overcame the genetic intractability of Epichloë sp. LpTG-3 strain AR37 and defined its epoxyjanthitrem biosynthetic pathway. We categorically demonstrated the genes responsible for the formation of the unique left-hand A and B rings (idtD and idtO) of the epoxyjanthitrem compounds, as well as the acetyl derivatization of the right-hand C10 hydroxyl group of epoxyjanthitrem I and IV (idtA). However, the identity of the precursor to these modifications is disputed, with various research studies implicating either terpendole I or paxilline as the potential precursor. To clarify this key step in the pathway, the Epichloë epoxyjanthitrem pathway was reconstructed using two strategies. Firstly, the two key genes for epoxyjanthitrem production (idtD and idtO) were inserted into an Epichloë strain that contains the genes required to produce terpendole I. Secondly, to confirm no additional genes were required, the pathway was reconstructed in the model organism Penicillium paxilli by substituting P. paxilli paxP and paxQ genes with Epichloë idtP and idtQ, and further including stepwise combinations of the Epichloë idtD and idtO genes. The gene insertions were achieved using a Golden Gate strategy enabling sequential random integration of a minimum subset of genes required to achieve epoxyjanthitrem biosynthesis. This approach showed that the idtP + idtQ insertions produced some paxilline, but the dominant products were 13-desoxypaxilline and terpendole I. Epoxyjanthitriol, the initial epoxyjanthitrem in the biosynthetic pathway, was produced by introduction of just idtD and idtO. Thus, our results strongly suggest that terpendole I is the precursor of epoxyjanthitriol.