Galactose utilization Metabolome analysis
62A - REF 62B - REF
Asn Galactose(int) Ala Aps, Glu, His Leu, Tyr
Glu 6P Fru 6P 0 Trehalose Dihydro- Glycogen ergosterol Galactose 1P, Ergosterol Glucose 1P NADP NADH NADPH ADP
AMP Tyr, His Phe, Lys
62C - REF
PNAS (2011) 108:12179-12184 Galactose utilization Integration of transcriptome and metabolome
Trehalose-6P TPS2 Trehalose NTH1 D-glucose Glycolysis
* Transcript increase 20 All mutants Trehalose 10 TPS1 Evolved mutants 0 More in 62A, 62C GSY1 UDP-Galactose UDP-Glucose Glycogen GDB1 D-glucose Glycolysis GSY2 60
40 Glycogen UGP1 20 0,4 Glucose 1P 0 Galactose int Galactose-1P Glucose-1P 0,2
1,0 Galactose 1P 0,0 PGM2 (umol/ 0,5 g DCW) 0,4 0,0 Glucose 6P
Glucose-6P 0,2
0,0 Glycolysis
PNAS (2011) 108:12179-12184 Galactose utilization
Genome analysis
No mutations in GAL genes, including PGM2 and genes associated with storage carbohydrate metabolism (both coding and non-coding regions)
Adaptation strategies different from rational engineering What are the “driving mutations” (beneficial mutations for galactose utilization)?
PNAS (2011) 108:12179-12184 Galactose utilization Hypothesis
Galactose
Gal 2
Galactose RAS2 62A:RAS2Lys77 62B:RAS2Tyr112
Galactose 1P CYR1 62C:CYR1Asn822
UDP-Galactose UDP-Glucose cAMP Pgm2p Ugp1p Glucose 1P PKA Trehalose 6P 62B:ERG5Pro370
Glucose 6P Glycogen Trehalose Different Ratio of Ergosterol & Dihydroergosterol Glycolysis 62A, 62B, 62C More in 62A, 62C More in 62B
PNAS (2011) 108:12179-12184 Galactose utilization
Inverse metabolic engineering Improvement of specific galactose uptake rate (%) Evaluation of two single nucleotide mutations 80 identified in RAS2 70 RAS2Lys77 and one single nucleotide mutation in ERG5 60 50 RAS2Tyr112 62C % 40 62B 30 ERG5Pro370 62A 20 10 0
Improvement of maximum specific growth rate (%)
40 RAS2Lys77 35
30 62B 62A 25 62C % 20 15 RAS2Tyr11 2ERG5Pro37 10 0 5 0
PNAS (2011) 108:12179-12184 Outline
Strain design for succinic acid production
ALE for growth in galactose
ALE for temperature tolerance
Temperature tolerance
Enzymes for biomass degradation often derived from thermophilic organisms -> activity at high temperature
⇒ Thermotolerant yeast strains needed for simultaneous saccharification and fermentation
Fermenter cooling is expensive Temperature tolerance
Evolution
Characterization
Caspeta et al. (2014) Science 346:75-78 Temperature tolerance
Characterization of thermotolerant strains
• higher growth rate at 40°C
• higher biomass yield
• higher ethanol production
• no growth on non-fermentable carbon sources
• higher tolerance towards osmotic stress
• reduced tolerance towards oxidative stress
Caspeta et al. (2014) Science 346:75-78 Temperature tolerance
Genome sequencing
⇒ partial duplications of Chr III
Caspeta et al. (2014) Science 346:75-78 Temperature tolerance
Dublication of several genes involved in the cell cycle (underlined) including transcription factors that regulate cell cycle genes
=> Chromosome duplication as fast adaptation mechanism
Caspeta et al. (2014) Science 346:75-78 Temperature tolerance
Genome sequencing
=> point mutations
Caspeta et al. (2014) Science 346:75-78 Temperature tolerance Evaluation of point mutations
A large fraction of the SNVs results in appearance of stop codons (26%), and 66% of these are in ATP3 and ERG3
Introduction of one of the stop codons identified in ERG3 results in 85% recovery of the temperature tolerance and results in the same remodeling of the sterol composition as found in the TT strains
Caspeta et al. (2014) Science 346:75-78 Temperature tolerance
Thermotolerant strains showed trade-off at 30°C
The M7 strain with re-introduced point mutation does not show any trade-off at 30°C and grows on non-fermentable carbon sources
Caspeta et al. (2014) Science 346:75-78 Conclusion
Systems biology can help in
• Predicting engineering targets
• Explaining adaptation mechanisms
Thank you