Expanded biosynthetic pathway of ergot alkaloids in Claviceps gigantea

The results of our genetic, biochemical, and chemical analyses demonstrate that some isolates of C. gigantea, the ergot pathogen of maize, have more extensive ergot alkaloid biosynthetic capacity than was previously determined.

For the majority of Claviceps species, ergot alkaloids are synthesized via a pathway that begins with tryptophan and passes through lysergic acid, which can then be isolated and modified at this point to create a range of pharmaceutically and industrially useful compounds.

Running parallel to this is another pathway that describes the biosynthesis of alkaloids unique to species C. africana and C. gigantea. These are dihydroergot (hydroxylated) alkaloids, also used in pharmaceuticals like hydergine and with markedly lower toxicity. They also have important implications for agriculture due to the species’ parasitism of maize crops.

These two pathways split into the precursors agroclavin and festuclavin via species-dependent activity of the same enzyme, EasA. Intermediates are thereafter oxidised along parallel pathways, all catalysed by the same enzyme, CloA.

Up until 2017, the ergot species Claviceps gigantea had been thought to end its biosynthesis pathway at dihydrolysergol (DHL), a precursor of the fully oxidized product dihydrolysergic acid (DHLA). However, the enzyme responsible for catalysing this hydroxylation, CloA, is the same for its relative species, C. africana, which is known to produce DHLA as well as products after further hydroxylation. Furthermore other species, namely Aspergillus fumigata (N. fumigata), have successfully been engineered with CloA to produce lysergic acid from agroclavin, in the other pathway.

Researchers at West Virginia University hypothesized this may be due to substrate specificity, and tested this only to find that C. gigantea can indeed oxidise DHL to DHLA. They not only found that CloA had a greater oxidative potential than expected, but detected DHLA as the primary end-product for C. gigantea.

In order to do this, a strain of N. fumigata was engineered (by knocking out the easM gene) to be able to accumulate the substrate and precursor to DHLA, festuclavin. The CloA enzyme needed to catalyse festuclavin to DHL and then to DHLA was taken from C. gigantea and transformed into N. fumigata.

The now CloA-expressing N. fumigata was externally fed the festuclavin substrate, and the resulting produced compounds were identified using HPLC and LC-MS.

In the HPLC analysis, far less DHL was detected in comparison to DHLA and to the standards, indicating that festuclavine is rapidly converted into DHLA with DHL as an intermediate rather than the end-product. In the LC-MS analysis, profiles of DHL and DHLA corresponded to their standards, confirming their identity.

The fact that CloA in C. gigantea is capable of oxidizing festuclavine rapidly to DHLA provides us with a much clearer context to the biosynthesis pathway of dihydroergot alkaloids as well as the potential of CloA itself. This has important implications for its use in synthetic biology, especially as it is the only known fungus to end its pathway at DHLA. Interestingly, C. gigantea still accumulated trace amounts of the DHL intermediate, something that would require more research into which enzymatic processes, if not the CloA reaction, catalyse its production.

Reference

1. Bragg, P.E., Maust, M.D., & Panaccione, D.G. (2017). Ergot Alkaloid Biosynthesis in the Maize (Zea mays) Ergot Fungus Claviceps gigantea. Journal Of Agricultural And Food Chemistry, 65. https://doi.org/10.1021/acs.jafc.7b04272