Requirement of two acyltransferases for 4-O-acylation during biosynthesis of Harzianum A, an antifungal trichothecene produced by Trichoderma arundinaceum

Authors: LINDO, LAURA - University Of Leon; McCormick, Susan; CARDOZA, ROSA - University Of Leon; Busman, Mark; ALEXANDER, NANCY - Retired ARS Employee; Proctor, Robert; GUTIERREZ, SANTIAGO - University Of Leon

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/17/2018
Publication Date: 12/18/2018
Citation: Lindo, L., McCormick, S.P., Cardoza, R.E., Busman, M., Alexander, N.J., Proctor, R.H., Gutierrez, S. 2018. Requirement of two acyltransferases for 4-O-acylation during biosynthesis of Harzianum A, an antifungal trichothecene produced by Trichoderma arundinaceum. Journal of Agricultural and Food Chemistry. 67(2):723-734. http://dx.doi.org/10.1021/acs.jafc.8b05564.

Interpretive Summary: Trichothecenes are fungal toxins that pose a health risk to humans and domestic animals because of their toxicity and because they can contaminate crop plants used for food and feed. The genetic, enzymatic and biochemical processes that lead to the formation of trichothecenes (i.e., trichothecene biosynthesis) have been studied extensively, because understanding these processes should lead to strategies that reduce trichothecene contamination in crops. Studies of multiple fungi have identified nineteen genes that are or are likely to be involved in trichothecene biosynthesis. Here, we determined the function of one of these genes, tri18. Our results indicate that tri18 encodes (i.e., serves a blueprint for) a protein that belongs to a class of enzymes known as acyltransferases. The results also indicate the tri18¬-encoded acyltransferase and another acyltransferase are both required for the same trichothecene biosynthetic reaction. These findings provide the first example of two acyltransferase enzymes being required for the same biochemical reaction in fungi. The findings also provide insight into how enzymes involve in trichothecene biosynthesis vary in function among different fungi. Such information has potential to contribute to the design of novel enzymes that detoxify trichothecenes. The results of this research have potential to contribute to strategies that reduce trichothecene contamination in crops, and thereby the potential to improve the safety of human food and animal feed.

Technical Abstract: Trichothecenes are sesquiterpenoid toxins produced by multiple fungi, including plant pathogens, entomopathogens, and saprotrophs. Most of these fungi have the acyltransferase-encoding gene tri18. Even though its function has not been determined, tri18 is predicted to be involved in trichothecene biosynthesis because of its pattern of expression and its location near other trichothecene biosynthetic genes. Here, molecular genetic, precursor feeding, and analytical chemistry experiments indicate that in the saprotroph Trichoderma arundinaceum the tri18-encoded acyltransferase (TRI18) and a previously characterized acyltransferase (TRI3) are required for conversion of the trichothecene biosynthetic intermediate trichodermol to harzianum A, an antifungal trichothecene analog with an octa-2,4,6-trienedioyl acyl group. On the basis of the results, we propose that TRI3 catalyzes trichothecene 4-O-acetylation, and subsequently, TRI18 catalyzes replacement of the resulting acetyl group with octa-2,4,6-trienedioyl to form harzianum A. Thus, the findings provide evidence for a previously unrecognized two-step acylation process during trichothecene biosynthesis in T. arundinaceum and possibly other fungi.