Jiranek, VladimirFord, ChristopherWilkinson, KerryLi, Jin-Chen2021-11-152021-11-152021https://hdl.handle.net/2440/1331303-Isobutyl-2-methoxypyrazine (IBMP) is a naturally occurring volatile compound with a potent aroma reminiscent of green capsicum. It occurs in a diverse range of organisms, including wine grapes. It is present at trace levels (ng/L) and contributes varietal characters and aroma complexity to grape cultivars such as Cabernet Sauvignon and Sauvignon Blanc, but can mask wine aroma bouquet and even reduce wine quality at elevated concentrations. IBMP concentrations evolve during grape development such that less IBMP is found in fully ripe grapes, whereas early harvested grapes (e.g. picked to produce lower alcohol wines) or grapes from cool climate regions may contain higher levels of IBMP, which may indicate poor quality and are undesirable. IBMP managements in grapes and wines are therefore crucial for quality improvement. Vineyard solutions have been investigated extensively and include canopy management and irrigation control. However, options for postharvest remediation are so far limited in their effectiveness and/or specificity. A solution is needed. The aim of this project is to discover/develop biological methods for remediation of undesirably high levels of IBMP in grapes or wines. Specifically, this project focused on microbiological and enzymatic approaches. Based on the fact that little is known about the biological degradation of IBMP by any organisms or enzymes, this project took an exploratory approach to seeking potential IBMP remediation solutions. From a microbiological perspective, 11 wine-related non- Saccharomyces yeast strains were trialled in sequential inoculation with a commercial Saccharomyces cerevisiae strain (EC1118, Lallemand) in Sauvignon Blanc fermentation spiked with IBMP. According to the instrumental and sensorial analysis of the final wine, IBMP levels did not significantly alter, neither among the various yeast treatments nor during alcoholic fermentation for any single treatment. Nevertheless, sensory studies suggested otherwise, with wines fermented with several strains being evaluated as more pronounced in fruity rather than green characters. This implies a masking of IBMP characters and may prove to be a feasible approach in Sauvignon Blanc winemaking to produce wines with organoleptically less greenness. It should be acknowledged that this result is variety-dependent, and yeasts perform differently in different grape juices (likely with different aroma precursors). Therefore, more trials are required to map the complete profile of yeast and variety interactions for masking green character. A large-scale microbial screen was performed with IBMP provided as a putative sole or key nutrient with the aim of identifying IBMP-degrading microbes. Alternative substrates with similar structures with IBMP were also used for screening due to the fact that rapid and inexpensive determination of IBMP consumption was not available to this project. Results indicated that, unfortunately, no microbes appeared able to significantly degrade IBMP under the screening conditions. Nevertheless, two fungal strains, Rhodotorula mucilaginosa and Rhodotorula glutinis, were shown to degrade one of the alternative substrates, methyl violet. Further investigation of these two strains are needed to study their metabolic abilities against other wine-related compounds. Enzyme engineering was also explored as a potential method for enzymatic remediation. VvOMT3, the O-methyltransferase catalysing the synthesis of IBMP in grapes was selected as the target protein for enzyme engineering due to its presumed structural specificity for substrate binding and catalytic activity. Random mutation was applied on this enzyme with error-prone PCR in the hope that the reaction may be reversed. Heterologous expression of mutant genes in E. coli BL21 was achieved and crude protein extracts were used for enzymatic assay. Unfortunately, no positive mutants were obtained from random mutation process. Nevertheless, some experimental findings and computational analysis, lead to predictions of key structural features relating to enzymatic functions. This data may support vine modification or selective breeding programs to reduce synthesis of IBMP. As part of a commencement of a new line of investigation, a review of recent literature pertaining to demethylases was completed. New strategies and candidate enzymes and organism are being considered for work that could follow on from this PhD. While this project did not yield a definitive IBMP modulation strategy, the findings provided important fundamental knowledge, as well as guidance for future work. Opportunities to utilise novel yeasts to, at least, mask the sensory impacts of IBMP may prove to be particularly fruitful.en3-isobutyl-2-methoxypyrazinewine aromawine microbiologybiological remediationThesis