Evolutionary engineering and genome-wide screening to improve wine microorganisms

Professor Vladimir Jiranek

Wine Microbiology & Microbial Biotechnology Laboratory

Improving yeast and their management

• Overall goal is to provide superior yeast strains- Tolerant of various and multiple stresses (eg, high sugar, SO2, 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

Directed (Adaptive) Evolution applied in number of ways1. 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

More robust evolved yeast strains

11-Feb-13 18-Feb-13 25-Feb-13 04-Mar-13 11-Mar-13 18-Mar-13 25-Mar-130

50

100

150

200

FM strain added as rescue

Res

idua

l sug

ar (

g/L

)

2013 Barossa ShirazStuck after ~2 weeksWarming and pump-overs fail to restart Rescue strain added in week 6

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

Evolved yeast with improved fructose useVia 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 phenotype14C-fructose reveals a 50% rise in fructose uptake (nears known* ‘fructophilic’ strains)* see Guillaume et al AEM (2007)

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, O2, 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: Butfermentation not affected if add key nutrients to medium ►

• Conduct as micro-fermentations

Key aim: Find genes linked to fermentprogress – ie Fermentation Essential Genes

Fermentation Essential Genes – the ‘fermentome’ mutants

1st screen

4th screen

4835

336

101

72

93 21

Most not essential for fermentation

genes with related function ~80

Fermentation Essential Genes – the ‘fermentome’In most cases, deletion = slow fermentation

About 1/3rd 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

BY4743Del1Del2Del3

For 9 of the deletants thefermentations 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

Fermentation Essential Genes – the ‘fermentome’

vacuolar acidification/regulation of cellular pH

cell./chem./ion homeostasis

(micro)autophagy

membrane invagination

cellular response to glucose/carbohydrate stimulus

0 5 10 15 20 25 30

Gene frequency (%)

GO

te

rm

Gene Ontology (GO) - bioinformatics• annotated genes (products)• description of functionality

Importance of these processes?

■ Frequency in genome

■ Frequency in FEG gene set

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

Acknowledgements

ResearchersDr Jennie GardnerDr Michelle WalkerDr Colin McBrydeDr Tommaso LiccioliDr Frank SchmidMr Trung Dzung NguyenDr Joanna Sundstrom

CollaboratorsDr Warwick Dunn – U ManchesterProf Steve Oliver – U CambridgeDr Paul Chambers – AWRIDr Miguel de Barros Lopes – UniSA