1. Evolutionary engineering and genome-wide screening to improve wine microorganisms Professor Vladimir Jiranek Wine Microbiology & Microbial Biotechnology Laboratory

2. Improving yeast and their management Overall goal is to provide superior yeast strains - Tolerant of various and multiple stresses (eg, high sugar, SO 2, EtOH, [CC]) - Less dependent on nutrients (eg limited assimilable nitrogen, vitamins) - Able to operate at temperature extremes (cold and/or heat) - More effective in utilisation of fructose Via evolutionary engineering (Adaptive/Directed Evolution) have generated - Strains of greater ‘robustness’ - Strains with greater affinity for fructose Extensive evaluation of the winemaking properties and the basis for changes Further targets sought via mutant screens - Libraries of laboratory yeast deletion mutants - Assessment of deletants shows critical cell processes and improvement opportunities - The data defines the ‘fermentome’ – ie the set of genes essential for fermentation Am J Enol Vitic (2006) 57:423-430

3. Directed (Adaptive) Evolution applied in number of ways 1.Repeated batch fermentations of a defined (wine-like) medium 200 g/L of sugar, pH 3.5, low inoculum, short aerobic propagation, ~350 generations Ensures isolates can perform at all phases of fermentation Sample and evaluate each 50 generations – identify “FM” strains FM completes fermentation in ~57% of time of parent strain Extensive evaluation and characterisation 2.Continuous fermentation in media containing fructose as only sugar ~ 5 g/L of fructose, pH 3.5, ~150 generations Sample and evaluate at 50 generations – identify strain “T9” Superior utilisation of fructose 1 mL 100 mL 10 L 20 kg 150 L

4. More robust evolved yeast strains FM series strains prove more robust About ~40% quicker in defined media Similar abilities in juices (vs good strains) ▼ Potential use as rescue strains (ferment well & produce less unpleasant aromas) ► Comparison with parent Genome = 200 SNPs in 176 gene ORFs 35 discriminatory metabolites - some in TCA cycle and amino acid metabolism

5. Evolved yeast with improved fructose use Via extended continuous culture with fructose as sole carbon source High-throughput (robotic) screens of 100s of isolates finds interesting strains These offer promise for standard fermentations (late stages) as well as ‘rescue’ yeast for addition to stuck (i.e. fructose-rich) ferments Strain characterisation gives clues to basis for novel phenotype 14 C-fructose reveals a 50% rise in fructose uptake (nears known* ‘fructophilic’ strains) * see Guillaume et al AEM (2007)

6. How to better inform strain improvement? WT Auxotroph + LEU, URA, MET, HIS Auxotroph (leu, ura, met, his) Take advantage of deletion mutant library that spans the yeast genome All non-essential genes represented as individual deletants (~4,800) Has been studied extensively, typically subjected to a single stressor Our interest is in many stresses simultaneously (pH, °C, O 2, YAN, EtOH, etc) Used the homozygous diploid lab strain BY4743 with 3 auxotrophic markers Limitations: Not a wine yeast background Sceening for attributes linked to N nutrition is complicated: But fermentation not affected if add key nutrients to medium ► Conduct as micro-fermentations Key aim: Find genes linked to ferment progress – ie Fermentation Essential Genes

7. Fermentation Essential Genes – the ‘fermentome’ mutants 1 st screen 4 th screen 4835 336 101 72 93 21 Most not essential for fermentation genes with related function ~80

8. Fermentation Essential Genes – the ‘fermentome’ In most cases, deletion = slow fermentation About 1/3 rd of the genes are novel – rest have been highlighted through previous ‘single stressor’ screens (i.e. our screen works) Ethanol tolerance, heat sensitivity, oxidative stress, hyperosmotic stress, etc BY4743 Del1 Del2 Del3 For 9 of the deletants the fermentations were stuck Key genes/processes included ion homeostasis, NAD or ubiquitin recycling, signalling, transcription. Role of entire set of genes explored through gene ontology to highlight over- represented processes

9. Fermentation Essential Genes – the ‘fermentome’ Gene Ontology (GO) - bioinformatics annotated genes (products) description of functionality Importance of these processes? ■ Frequency in genome ■ Frequency in FEG gene set

10. The Fermentome and Directed Evolution Fermentome equals genes needed for high sugar fermentation Findings guide: Strain construction/isolation Design of selection strategies (eg. [ion] in future DE expts?) Optimisation of fermentation conditions using existing strains Insights into other high sugar fermentations (eg. bioethanol) Provides basis for screening of wine yeast deletion library (or other more suitable libraries) and interrogation of wine yeast data Directed evolution yields industry-ready strains and their characterisation guides further strain construction Collectively the work improves our understanding of wine yeast and the basis of their behaviour and will help optimise fermentation and tailor winemaking outcome

11. Acknowledgements Researchers Dr Jennie Gardner Dr Michelle Walker Dr Colin McBryde Dr Tommaso Liccioli Dr Frank Schmid Mr Trung Dzung Nguyen Dr Joanna Sundstrom Collaborators Dr Warwick Dunn – U Manchester Prof Steve Oliver – U Cambridge Dr Paul Chambers – AWRI Dr Miguel de Barros Lopes – UniSA