Flavin Dependent Halogenases and SAM-Dependent Methyltransferases in Sporidesmin Biocatalysis

ABSTRACT

Pseudopithomyces chartarum (also referred to as Pithomyces chartarum) is a fungus commonly found in rye grasses after rain spells in the southern hemisphere, known for producing Sporidesmin, a mycotoxin responsible for acute liver toxicity in livestock. Sporidesmin belongs to the epipolythiodioxopiperazines (ETP) class of natural products and is notable for both its toxic effects and the presence of a chlorine atom in its structure. Its biosynthesis is governed by a cluster of 21 genes, referred to as spd. This research project focuses on two key enzymes within the Sporidesmin biosynthetic pathway: Spd1, a homolog of an S-adenosyl-L-methionine (SAM)-dependent methyltransferase, and Spd4, predicted to be a flavin-dependent halogenase. The Reddick Research Group has an interest in the spd gene cluster for its potential for biocatalytic activity and for the basic understanding of the biosynthetic pathway of Sporidesmin. Spd1 is of interest for its potential as a methylation biocatalyst, while Spd4 may be responsible for the installation of the chlorine atom in Sporidesmin and holds promise as a general halogenation biocatalyst. The purpose of this research was to better understand the biosynthetic pathway and to potentially use the two enzymes as biocatalysts for other reactions. We are doing this through testing the activity of these enzymes on simple structural analogs of Sporidesmin. These reactions could be applied to other various types of chemistry for the uses in green chemistry and other industrial purposes. To investigate these enzymes, Spd4-MBP, Spd1, and Spd1-MBP were cloned and overexpressed in Escherichia coli (E. coli) using Gibson assembly and polymerase chain reaction (PCR). The maltose-binding protein, MBP, was fused to two of the proteins to aid in their folding and solubility during purification due to their fungal origins. The resulting proteins were purified, using immobilized metal affinity chromatography, IMAC, resulting in several milliliters, usually around 6 mg of protein per liter of culture grown, that was then able to be used in subsequent reactions. High-Performance Liquid Chromatography (HPLC) and several enzymatic reactions were used to test Spd4-MBP for the chlorination of a simple indole. Similarly, several enzymatic reactions along with HPLC analysis were conducted on Spd1-MBP for potential methylation products from simple indoles and simple diketopiperazines. These studies have yet to yield any promising results. However, research is ongoing, and modifications are still being made to the investigative process to potentially result in the production of chlorinated or methylated products with good yields and stability.