Trichosporon mycotoxinivorans transformation

The mycotoxin zearalenone (ZON) is produced by various species of plant pathogenic fungi of the genus Fusarium. Toxicologically relevant levels of ZON-contamination can be found as a consequence of Fusarium infection of crop plants (most importantly maize). To protect consumers, the European Commission has enacted harsh maximum tolerated levels of ZON in different food commodities. Yet, in order to still allow utilization of infected material the recommended guideline levels for animal feed are comparably high. Another possibility to utilize contaminated corn would be the use in bioethanol production. Yet, ZON is enriched about 3-fold in the protein-rich byproduct (distillers dried grains with solubles) which is used as feed. Possible solutions of the ZON problem would be the development of transgenic plants, which are capable of detoxifying ZON already in the field, or of engineered microbes that could be used as feed additives or during bioethanol fermentation, or by development of enzyme preparations which can inactivate ZON. The yeast Trichosporon mycotoxinivorans is able to convert ZON efficiently into a non- estrogenic metabolite (ZOM-1). The main goal of this project is to develop a transformation system for this basidiomycete yeast which should allow cloning of the ZON detoxification genes. In previous work an auxotrophic mutant (Arg - ) was identified, which lost the ability to degrade ZON. In this project a transformation system based either on dominant drug resistance markers or based on complementation of the defect in the arginine biosynthesis should be developed. Transformation with clones from a genomic library should allow identification of plasmids which can complement the defect in ZON degradation. The postulated degradation pathway in T. mycotoxinivorans proceeds in two steps. The hypothetical intermediate is currently not available. Thus, a further goal of the project is to generate the intermediate by de novo synthesis to allow testing of its toxicological properties, in particular the estrogenic activity. The availability of the intermediate should also allow do develop strategies for cloning of the gene responsible for the second step. Cloned genes from Trichosporon should provide a basis for transfer of the detoxification ability to other organisms and further applications and product development.

The basidiomycete yeast Trichosporon mycotoxinivorans is capable to detoxify the strongly estrogenic Fusarium mycotoxin zearalenone. In a previous publication we proposed a two-step degradation pathway from zearalenone to the inactive metabolite ZOM-1, but at this time the intermediate could not be detected. Presumably the first reaction, the introduction of an oxygen atom into the macrocycle next to a keto group in a Baeyer-Villiger type reaction is slow, while the hydrolysis of the formed lactone is very rapid, so that the intermediate does not accumulate. The goal of the project was the cloning of the relevant detoxification genes. Since a zearalenone degradationdeficient mutant was available at project start, the intended strategy was to develop a transformation method and to identify the gene by complementation of the mutant. A retrofitting system was developed, which allowed Cre-lox mediated addition of dominant drug resistance markers to excised phage lambda clones of a genomic library. Yet, since the efficiency of the retrofitting process and in particular the transformation frequency of Trichosporon was low, a new strategy was chosen. The whole genome sequences of wild-type Trichosporon mycotoxinivorans and the mutant were determined. The mutant, which was generated by irradiation with neutrons and gamma-rays, contained a large deletion in which a candidate gene with sequence similarity to Baeyer-Villiger monooxygenases was identified. Heterologous expression of this gene in bakers yeast produced transformants with the capability to metabolize zearalenone to a new product. The substance was purified and used for structure elucidation. It was confirmed that it is identical to the postulated intermediate. The purified compound was used to test the estrogenicity. The results showed that already the first step leads to a more than 100-fold reduction in the estrogenic activity.Several possibilities exist for utilization of the identified zearalenone detoxification gene. It could be introduced into host plants of Fusarium, so that e.g. transgenic maize would gain the ability to detoxify zearalenone already at the site of infection. Highly contaminated cereals and maize are frequently diverted to bioethanol production, but this leads to increased concentrations of the stable toxin. Modified yeasts that already detoxify zearalenone during the fermentation could reduce the mycotoxin content of the valuable protein-rich by-product dried distillers grain with solubles which is used as animal feed. Potentially the purified gene product could be used as detoxification enzyme, e.g. as a feed additive.The process for intellectual property rights protection for this invention was initiated.