The microbiology of the winemaking process, which includes inoculated strains of the yeast Saccharomyces cerevisiae and the lactic acid bacterium, Oenococcus oeni, is critical to process efficiency and wine quality. In each case these organisms are required to complete a core conversion (sugar to ethanol or lactate to malate, respectively) as well as make desirable sensory contributions. These activities typically occur under extreme conditions which may include high sugar (osmolarity) and ethanol content and low pH, temperature and nutrient availability. We have used mutant screening strategies and functional genomic approaches to identify the basis of superior yeast performance in the face of these challenges. In addition we have use adaptive evolution to yield yeast with enhance fermentation reliability based on increase nitrogen efficiency, fructophilicity or general robustness. In parallel work, we have isolated and heterologously expressed genes from O. oeni which encode esterases or glucosidases. Characterisation of these gene products has provided insights into their roles within the cell as well as potential contribution to wine.

1. Wine Microbiology for Fermentation Success and Wine Quality Assoc Prof Vladimir Jiranek School of Agriculture, Food and Wine The University of Adelaide, Waite Campus.

3. Concentration  Time  sugars ethanol yeast Key events in winemaking LAB pH malic Fermentation Alcoholic Malolactic lactic

4. + = This transformation involves (requires) two broad activities: Efficient conversions - of sugar to ethanol & CO 2 by yeast - of malic acid to lactic acid by LAB Sensory contributions - by both yeast and bacteria Our focus is on a better understanding of the critical role of microbes in winemaking, improving these organisms to ensure process completion or tailoring their contribution to wine. Sold

5. Fermentation is a harsh environment for yeast Grape Juice -> Wine Low pH (3 - 4) Temperature extremes (<15 - >30 °C ) Competing yeasts and bacteria Toxins 200+ g/L sugar (high osmolarity) leads to ethanol accumulation to 15+% (v/v) Little oxygen (or ‘substitutes’) Modest supply of nutrients (esp N) which often becomes depleted Etc. Ensuring yeast complete fermentation is a challenge, especially as there are multiple possible causes of problem fermentations. We have used multiple approaches to 1) generate yeast which are reliable fermenters and 2) we are seeking a deeper understanding of the yeast response to fermentation conditions.

6. Implications of a depletion of YAN YAN is an essential nutrient (quantitatively 2 nd only to carbon) Largely present as amino acids and ammonium, some peptides Amounts in juice are highly variable (can be supplemented with NH 4 + salts) Deficiency and hence depletion common Results are diverse: ● poor biomass yield ● reduced fermentative activity ● other problems e.g. H 2 S Biomass formation Fermentation rate Agenbach (1987)

7. Implications of a depletion of YAN YAN is an essential nutrient (quantitatively 2 nd only to carbon) Largely present as amino acids and ammonium, some peptides Amounts in juice are highly variable (can be supplemented with NH 4 + salts) Deficiency and hence depletion common Results are diverse: ● poor biomass yield ● reduced fermentative activity ● other problems e.g. H 2 S Biomass formation Fermentation rate Agenbach (1987) Lagunas et al., (1982)

8. Implications of a depletion of YAN YAN is an essential nutrient (quantitatively 2 nd only to carbon) Largely present as amino acids and ammonium, some peptides Amounts in juice are highly variable (can be supplemented with NH 4 + salts) Deficiency and hence depletion common Results are diverse: ● poor biomass yield ● reduced fermentative activity ● other problems e.g. H 2 S Biomass formation Fermentation rate Agenbach (1987) Lagunas et al., (1982) Jiranek et al, (1995) NH 4 + H 2 S

10. Several genes are inversely involved in nitrogen efficiency Disruption leads to superior performance on limited N (ammonium) Wouldn’t expect same results if using complex N source (confirmed) Gardner et al (2005) Gardner et al (2005) wildtype ngr1 gid7 ngr1, gid7 Defined medium, 75 mg FAN/L (NH 4 + ) Residual sugar (complex N) Residual sugar (ammonium)

11. Several genes are inversely involved in nitrogen efficiency Disruption leads to superior performance on limited N (ammonium) Wouldn’t expect same results if using complex N source (confirmed) Alternate screen used (i.e. complex N) Further genes found whose disruption leads to superior NE in limited complex N media Gardner et al (2005) Gardner et al (2005) wildtype ngr1 gid7 ngr1, gid7 Defined medium, 75 mg FAN/L (NH 4 + ) Residual sugar (complex N) Residual sugar (ammonium)

13. Exploiting the lab yeast deletion library Use homozygous diploid deletion library (BY4743) Several auxotrophic markers related to N metabolism (leu, his, ura) Thus the need to supplement is not conducive to studies with limited N Supplementation does produce a similar growth response to the prototrophic Thus possible to use deletion library to identify “fermentation essential” genes Similar screens of libraries have been performed but they have limitations in terms of relevance to wine e.g.: 300 g/L sucrose – growth after 20 h, 30 º C. 300 g/L glucose minimal agar and liquid – OD and colony size. 10% (v/v) ethanol in YPD agar – growth of dilution series Our system uses liquid media, 230 g/L glucose + fructose, pH 3.5, polyphenols (anthocyanins), and requires all sugar to be fermented within a preset time of 7 days Prototrophic ( ■ ) Auxotrophic + supps. (●)

14. Genome-wide screen for genes required for successful fermentation completion Duplicate static ferments CDGJM-PP, 30 °C, 7 days Yeast deletion library ‘ wine’ residual sugar Genes required for fermentation only * OD 600 * 2 nd screen of deletion subset – measured OD 600 Identified 416 genes that were essential for on-time completion of fermentation Of these 101 showed reduced growth (i.e. 34 had ~70% and 67 had ~30% the OD of the parent) and weren’t considered further The remaining 315 were designated essential for fermentation and examined further. While these could be subdivided according to the extent of fermentation, anything more that 2.5 g/L residual sugar is not useful

17. Apart from gene searches, also seek to produce superior strains Useful for industry and also as tool to understand the interaction of yeast with the wine environment. Adaptive evolution approach has been used Have generated strains that are: More ‘robust’ e.g. FM16 vs L2056 More nitrogen efficient B11 vs PDM FM16 L2056 CDGJM (100 mL, 200 g sugar/L, 580 mg N/L, 30  C) 77 h 138 h Juice (various) FM16 same or better CDGJM (100 mL, 250 g sugar/L, 125 mg N/L, 23  C) 240 h 528 h (stuck) Riesling (80 L, 220 g sugar/L, 152 mg N/L, ~18  C) 790 h 1000 h (stuck)

19. AE for more fructophilic strains Evaluated hundreds of candidate evolved strains collected across 50- generation intervals. Triplicate trials via robotic screen (>20K assays). Several very promising strains found. Selected (promising) mutants related to parent (not contaminants) Mutant phenotype stable during passaging Further characterisation underway P 1 2 Mutant 43 Mixed population Parent

20. AE for more fructophilic strains Evaluated hundreds of candidate evolved strains collected across 50- generation intervals. Triplicate trials via robotic screen (>20K assays). Several very promising strains found. Selected (promising) mutants related to parent (not contaminants) Mutant phenotype stable during passaging Further characterisation underway P 1 2 Mutant 43 Mixed population Parent

21. AE for more fructophilic strains Evaluated hundreds of candidate evolved strains collected across 50- generation intervals. Triplicate trials via robotic screen (>20K assays). Several very promising strains found. Selected (promising) mutants related to parent (not contaminants) Mutant phenotype stable during passaging Further characterisation underway P 1 2 Mutant 43 Mixed population Parent

22. Enzymatic activities of lactic acid bacteria Whole LAB cells found to possess a large number of enzymatic activities O. oeni can influence the anthocyanin content of a medium Suggested  -glucosidase activity Search for such activities demonstrated these to be present May be of importance in aroma liberation as well as colour i.e. terpenoids, norisoprenoids, etc. Significant activities of whole cells against number of artificial substrates Suggests presence of multiple activities Used cloning and heterologous expression to try to characterise these further Develop primers to PSU-1 genome seq. Several proteins expressed in E. coli Purified by affinity chromotography Characterisation underway Grimaldi et al (2005)

23. Enzymatic activities of lactic acid bacteria Genes appear to be arranged in an operon: phosphoenolpyruvate dependent phosphotransferase system (PEP-PTS) Involved in uptake, phosphorylation and translocation of β-glucosides across the cytoplasmic membrane. Thus suggests a phosphorylation step is involved. Has 480 residues, a molecular weight of 55.5 kDa Protein has been purified Does in fact appear to be a be phospho-glucosidase Has pH optimum of 5.5 and good activity at 4°C - 40°C Relevance to wine? Capaldo et al (2010)

24. Enzymatic activities of lactic acid bacteria Capaldo et al (2010)

25. Enzymatic activities of lactic acid bacteria Fact that there is a difference compared to other glucosidases is of interest Note that amino acid sequence differences are minimal Does Ser – Ala = substrate difference as in Pectobacterium carovatorum? Using a site-directed mutagenesis to determine basis for preference for phosphorylated glucosides Capaldo et al (2010)

26. Esterases of lactic acid bacteria Again LAB generally poorly characterised in terms of flavour impacts beyond diacetyl or deacidification Changes in ester content post MLF are widely reported ► Esters responsible for the fruity aromas of wines Likely to be of importance in shorter term (lost through hydrolysis) Examination of whole cells reveals activities against multiple ester substrates (pNP linked) Strain dependent Also some evidence of synthesis Again use clone and heter- ologous expression approach. Matthews et al (2007)

27. Sumby et al (2010)

28. Esterases of lactic acid bacteria In work analogous to the glucosidase study, use sequence homologue to amplify putative esterases ex Oenococcus oeni. Number of genes cloned and expressed in E. coli. One of these EstB28 is first esterase to be so characterized ex O. oeni . 912-bp open reading frame encoding a 34.5 kDa protein. Member of family IV of lipolytic enzymes and contains the GDSAG motif common to other lactic acid. Optimum temperature of 40°C, pH of 5.0 and ethanol concentration of 28% (v/v). Specificity for difference substrates varies. Sumby et al (2009)

29. Esterases of lactic acid bacteria Enzyme show good tolerance of wine-like conditions Sumby et al (2009)

31. Acknowledgements Maria Astorga Alana Capaldo Paul Easton Jennie Gardner Antonio Grimaldi Vicky Harris Nick Kalatzis Tommaso Liccioli Angela Matthews Colin McBryde Frank Schmid Krista Sumby Michelle Walker Tristan Warren Paul Grbin Eveline Bartowsky Paul Chamber Miguel de Barros Lopes