directed evolution: limiting problematic fermentation
TRANSCRIPT
Contact: Vladimir JiranekPhone: +61 8 8313 6651Email: [email protected] www.agwine.adelaide.edu.au/wine/micro/
Directed evolution: limiting problematic fermentation through microbial improvement
Acknowledgements:This work is supported by Wine Australia (Project UA1302)
Michelle Walker, 1,2 Tommaso Watson1,2 Paul Grbin1,2 and Vladimir Jiranek1,2
University of Adelaide1 Dept. Wine and Food Science, Waite Campus, South Australia; Wine Innovation Cluster2 Adelaide, South Australia
Problem: Shortened, hot vintages, together with a trend towards more full-bodied wine can result in fruit with excessivelyhigh sugar content, leading to increased ethanol concentrations (>15%) with problematic fermentation due to microbial failure
How to solve microbial failure? Directed evolution; a paradigm to generate Saccharomyces cerevisiae wine yeast betterable to complete fermentation in problematic juice-like conditions
Targeting: Efficient nutrient usage and tolerance to stress-producing chemicals in grape must
Results and Discussion:• Strains which performed well in 2-3
conditions to be evaluated in a variety ofproblematic juices and stuck wines
• The genomes of best 2-3 strains to besequenced to identify genetic basis forimproved phenotype
High-throughput evaluation of isolates from DE
100 mL fermentations – ‘Teebot’ platform Micro-fermentations – 0.2 mL scale
C7HB (mix)
5
10
15
C7HB(mix)
5
10
15
1
6
11
16
1
6
11
16
2
7
12
Uva 43
2
7
12
Uva43
3
8
13
Q7
3
8
13
Q7
4
9
14
C7H
4
9
14
C7H
C7HB(mix)
5
10
15
C7HB (mix)
5
10
15
1
6
11
16
1
6
11
16
2
7
12
Uva43
2
7
12
Uva43
3
8
13
Q7
3
8
13
Q7
4
9
14
C7H
4
9
14
C7H
BLK
Sugar assays
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H
C7HB (mix)
21
26
31
C7HB (mix)
21
26
31
17
22
27
32
17
22
27
32
18
23
28
Uva 43
18
23
28
Uva43
19
24
29
Q7
19
24
29
Q7
20
25
30
C7H
20
25
30
C7H
C7HB(mix)
21
26
31
C7HB(mix)
21
26
31
17
22
27
32
17
22
27
32
18
23
28
Uva43
18
23
28
Uva43
19
24
29
Q7
19
24
29
Q7
20
25
30
C7H
20
25
30
C7H
BLK
Sugar assays
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H
C7HB (mix)
37
42
47
C7HB (mix)
37
42
47
33
38
43
48
33
38
43
48
34
39
44
Uva 43
34
39
44
Uva43
35
40
45
Q7
35
40
45
Q7
36
41
46
C7H
36
41
46
C7H
C7HB (mix)
37
42
47
C7HB(mix)
37
42
47
33
38
43
48
33
38
43
48
34
39
44
Uva43
34
39
44
Uva43
35
40
45
Q7
35
40
45
Q7
36
41
46
C7H
36
41
46
C7H
BLK
Sugar assays
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H
Fermentation progress – residual sugar
• Stress inducing conditions: ethanol, high sugar, low pH, medium chain fatty acids• Yeast: A and B (single strain), C (sporulated/mated populations, single hybrid, EMS mutant), D (yeast from A, B, C)• Generations: A (~150), B (~150), C (40-60), D (45)
DE – batch fermentation A B C
D
This study aims to provide new ‘industry ready’ wine yeast better suited for Australian winemaking
1: DE - C (Marsanne + 50 g/L sugar)
Examples of DE cultures where fermentation duration is reduced
B: DE - D (Marsanne + 4.5% ethanol)
RM3 MP 18.58 ± 7.78 93.313 1.54 ± 1.43 99.515 1.40 ± 1.80 99.531 0.02 ± 0.18 99.930 0.32 ± 0.40 99.825 0.14 ± 0.43 99.9
Uvaferm 43 0.23 ± 0.23 99.9Q7 23.12 ± 7.32 91.7
Sugar consumed as %
Residual sugar g/L ± SD
faster