Bioprospecting the Regional Diversity of Australian Wine Microbiota

Abstract

Like all plants, grapevines are host to a plethora of microorganisms. During the winemaking process, the microflora of the grape surface can be transferred into the fermentation environment. While the yeast Saccharomyces cerevisiae is the main driver of alcoholic fermentation during wine production, it has become apparent that other grape and cellarderived microbes can have a significant impact on fermentation and the resulting wine. This impact is manifested in the sensorial differences between wines resulting from inoculated fermentations and spontaneous ‘wild’ fermentations and is attributed to the various secondary metabolites and enzymes these microorganisms produce. Carbohydrate active enzymes can affect the composition of grape juice by altering the extractability and organoleptic properties of many wine compounds. Of these enzymes, glycoside hydrolases mediate hydrolysis of glycosides and are found ubiquitously through nature. Micro-organisms, especially those associated with plants, use glycoside hydrolases to afford access to plant resources by degrading the plant cell wall and similarly soil microbes utilise these enzymes during degradation and composting of plant material. During winemaking, glycoside hydrolases have the potential to affect the breakdown of complex sugars in plant cell walls, which can aid in juice extraction and clarification. Glycoside hydrolases can also mediate the release of volatile flavour and aroma compounds from glycosyl-linked, non-volatile precursors. These enzymes therefore represent an attractive target for biotechnological applications. A combination of metagenomics and synthetic biology were used to explore the enzymatic potential of two grapevine derived environments, an Chardonnay grape must (CGM) and a mixed varietal grape marc (MVGM), with the aim of identifying novel enzymes with potential applications as winemaking adjuncts. The CGM was specifically selected as a source of fungal microbiota native to the grapevine, whilst the grape marc was chosen as a source of plant decomposing microorganisms from a glucose-limited environment. Whole genome sequencing and bioinformatic techniques were used to probe the genomic landscape of these two environments, identify enzymes of interest, assess the types of microbiota present in the microbial communities and where possible assemble microbial genomes. The two environments contained very distinct microbial communities. As expected, the Chardonnay must community comprised a high portion of fungal genomes whereas the MVGM was dominated almost exclusively by bacteria. Five candidate polygalacturonases, with potential applications in wine clarification, and seven β-glucosidases, with potential applications in wine aroma and flavour, were chosen for heterologous expression in Pichia pastoris and Saccharomyces cerevisiae. Two of the selected polygalacturonases exhibited significant levels of activity at wine pH and were tested for in-wine activity, using recombinant S. cerevisiae strains in fermentation experiments with synthetic grape must. With future testing in real grape juice, these enzymes and S. cerevisiae strains could be leveraged to improve juice extraction and clarification during winemaking.