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Galactose Utilization Metabolome Analysis

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Galactose Utilization Metabolome Analysis 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 .
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