I NTACT – EI B.10.008 I NTegral engineering of ACetic acid Tolerance in yeast Ton van Maris Delft University of Technology Department of Biotechnology Section Industrial Microbiology Delft, the Netherlands W arsaw , February 26, 26, 2014 2014 1 1
… the Team: Isabel Sa-Correia Nuno Mira Margarida Palma Joana Guerreiro Joaquin Arino & students Boris Rodriguez Ton van Maris Elke Nevoigt Dani Gonzalez Ramos Steve Swinnen Erik de Hulster & students Bianca e.d. Bianca (Bra) & students 2 2
Desired feedstocks for I ndustrial Biotechnology cornstover bagasse wheatstraw 3
Acetic acid Structural component of lignocellulosic biomass Ac - HAc Synthetic Plant ↔ medium hydrolysate + H + - Weak organic acid, pKa = 4.75 4
Main mode of toxicity Low pH Neutral pH H + + Ac - H + + Ac - HAc HAc 100 Ac - 80 H + pK a = 4.75 % HAc 60 ATP 40 20 0 2.75 3.75 4.75 5.75 6.75 ADP pH H + Ac - 5
Growth of lab strain (CEN.PK) at various concentrations pH 4.5 defined media 0.0% 0.1% 0.2% 0.3% 0.9% 0.4% 0.5% 0.6% 0.7% 0.8% 1.0% 6
Exposure of exponentially growing cells to acetic acid decreases specific growth rate (µ max ) and lag (latency) phase 9 g/l Strain: CEN.PK, pH 4.5 9 g/l Swinnen et al ., submitted 7
Consortium Aim • Understand and rationally improve acetic acid tolerance of S. cerevisiae , through integrating: – Identification of tolerant natural isolates – Genetic mapping and comparative genomics – Transcription factor engineering – Evolutionary engineering – Physiological analysis including ion homeostasis – (Reverse) metabolic engineering 8
S. cerevisiae strains strongly differ in acetic acid tolerance (particularly lag phase) 9 g/l acetic acid, pH 4.5 9 Swinnen et al., submitted
S. cerevisiae strains strongly differ in acetic acid tolerance (particularly lag phase) 9 g/l acetic acid, pH 4.5 10 Swinnen et al., submitted
S. cerevisiae strains strongly differ in acetic acid tolerance (particularly lag phase) 9 g/l acetic acid, pH 4.5 11 Swinnen et al., submitted
The major challenge in reverse engineering: How to identify the causative genetic differences? All genetic differences (either causative or unimportant for phenotype) Strain with desirable phenotypic trait Reference strain without the desirable trait Genetic differences causative for phenotype 12 12
Acetic acid tolerance is a quantitative trait Acetic acid - strain Acetic acid + strain × 1n 1n 2n Sporulation Isolation of single segregants Quantification of acetic acid tolerance 1n 13
Identification of the crucial genetic determinants 1n Select only segregants with acetic acid + phenotype Pooled segregant whole genome analysis Significant genetic association? 14
Genome-wide genetic association analysis Acetic acid + strain Acetic acid - strain Mating Diploid hybrid strain Sporulation Isolating and phenotyping of segregants Selection of segregants with acetic acid + phenotype Segregants with acetic acid + phentotype Method reviewed by Swinnen et al. (2012) FEMS Yeast Res. … Position of a causative determinant 15
Genome-wide genetic association mapping of the two strains with different acetic acid tolerance 16 Analysis of determinant regions ongoing
Role of Haa1 and the Haa1-regulon in yeast response and resistance to acetic acid ( www.yeastract.com ) Haa1 Acetic acid-resistance genes indirectly regulated by Haa1 Mira et al. (2011) Nucleic Acids Res 39(16): 6896-907 16 acetic acid-resistance genes Teixeira et al. (2014) Nucleic Acids directly regulated by Haa1 Res 42(1): D161-6 TFs regulated by Haa1
Transcription Factor Engineering without acetic acid 0.95% acetic acid (*) addition of uracil to the medium Screening for growth on Synthetic medium with 0.95% acetic acid (pH 4.5) 18
Transcription factor engineering 1. Error prone PCR 2. Restriction of pRS416- HAA1 Homologous recombination in CEN.PK113-13D and CEN.PK113-13D haa1 ∆ Selection of plasmid-containing transformants Screening of library for acetic acid tolerance 19
Transcription factor engineering 1. A mutant HAA1 library has been enriched in acetic acid containing medium 1,4 1,2 1,0 Library Enrichment 1 & 2 OD 600 0,8 0,6 Wild-type HAA1 0,4 0,2 0,0 0 20 40 60 Time [h] 2. Several transformants expressing a mutated HAA1 gene showed an improved acetic acid tolerance (in terms of the duration of the latency phase) as compared to the strain expressing the wild type HAA1 gene 3. Focus on the HAA1 allele with the lowest number of mutations * * HAA1 PROMOTER ORF TERMINATOR 2 1 20 20
Transcription factor engineering • Introduction of the mutations in the genome of CEN.PK113-7D: Eliminate any possible effects of the plasmid and auxotrophic background Purpose Determine the individual effect of each mutation Strains 1 2 Strain 1 HAA1 1 Strain 2 HAA1 2 Strain 3 HAA1 Strain 4 + HAA1 HAA1 chr VII 21 21
Transcription factor engineering • Introduction of the mutations in the genome of CEN.PK113-7D: Screening of the mutant strains for acetic acid tolerance 160 mM – pH 4.5 22 22
Role of Haa1 and the Haa1-regulon in yeast response and resistance to acetic acid The protein kinase Hrk1 is among the ( www.yeastract.com ) Haa1p-targets exerting the strongest protective effect against acetic acid Haa1 Mira et al. (2011) Nucleic Acids Res 16 acetic acid-resistance genes 39(16): 6896-907 directly regulated by Haa1 Teixeira et al. (2014) Nucleic Acids Res TFs regulated by Haa1 42(1): D161-6
Tolerance test - VM-HAc 90 mM at 15 hours 40 35 30 25 Ratio 20 15 10 5 0 strains 24
K + Sky1 Hal4 Hal5 Ppz1 H + Pma1 Sap185 Hal3 Ptk2 Sap155 Hrk1 Sit4 H + Modified from Arino et al. 2010 Na + ,K + Na + ,K +
The role of the Haa1p regulon in yeast response and resistance to acetic acid stress Proteome-w ide yeast response to acetic acid stress: role of Hrk1 10 50 mM acetic acid OD 600nm -Hrk1p is the Haa1p-target exerting the strongest protective effect against acetic 1 acid wt hrk1 ∆ -Hrk1p belongs to a family of kinases involved in the 0,1 regulation of plasma 80 20 60 0 40 1h membrane transporters Time (h) Proteome extraction The phosphoproteome of a membrane-enriched fraction obtained from WT and hrk1 cells cultivated in the presence of acetic acid w as 26 compared (iTRAQ)
Proteome profiling of acetic acid stressed yeast strains – phosphoproteomic analysis to elucidate Hrk1 biological activity - Phosphate metabolism - Translation The phosphoproteomes of acetic acid - Cellular transport stressed and unstressed parental strain - Stress response S. cerevisiae BY4741 and hrk1 ∆ cells - (…) were compared using iTRAQ Acetic acid-responsive proteins I ncreased phosphorylation Decreased 108 221 phosphorylation ~20% of the proteins with an increased phosphorylation level in parental cells in response to acetic acid stress are Hrk1- dependent
Evolutionary Engineering in Sequential Batch Cultivation Start a batch culture Add fresh medium, End of the sometimes increasing exponential phase [acetic acid] Pump out 99% of the medium • Ability to grow at higher [acetic acid] • Faster growth at a given [acetic acid] 28
[Acetic acid] (g/L) 0 1 2 3 4 5 6 14 0.25 12 Batch Duration (days) Duration 0.20 Rate 10 u max (hr -1 ) 0.15 8 6 0.10 4 0.05 2 0 0.00 0 10 20 30 40 50 Batch Number However, acquired phenotype not constitutive, but hyper inducible 29 Wright et al. 2011 FEMS Yeast Res. 11: 299-306
Induction of acetic acid tolerance 0,34 0,29 CEN.PK113-7D 0,24 OD 660 exposed CEN.PK113-7D 0,19 0,14 0,09 0 10 20 30 40 50 60 Time (h) 30 30
Evolutionary ON/OFF approach for constitutive tolerance OFF OFF OFF OFF [Acetic acid] 31 31
Measurements of acetic acid tolerance CEN.PK113-7D Evolution mutant Dilute to the same OD 660 [Acetic acid] Measure OD 660 after 5 days 32 32
Aim of the study Why are the evolution strains more tolerant to acetic acid? Whole genome sequencing of 6 parallel evolution lines resulting in 10-30 mutations per strain Crossing and sporulation 33 33
Number of mutations responsible for tolerance? Dominant or recessive? HAT2A IMK439 Diploid HAT2A-D 34
Sporulation and screening priority targets identified & reverse engineering ongoing 144x 144 haploid segregants Inoculate without OD 660 measurement Measure OD 660 after 5 days
An alternative approach to deal with acetic acid? Can the inhibitor acetic acid be converted (reduced) to ethanol? 2 NADH • Attractive option (less acetic acid, more ethanol) • But where should the reducing equivalents come from?
An engineering strategy to eliminate glycerol production NADH NAD + NADH NAD + ATP AMP + PP i acetyl- acetic acetaldehyde ethanol Coenzyme A acid Acs2 Adh1-5 E. coli MhpF 1. Express heterologous acetylating acetaldehyde dehydrogenase ATP NAD + ADP NADH P i NADH NAD + dihydroxy- glycerol-3- 0.5 glycerol acetone- phosphate glucose Gpd1, Gpd2 Gpp1, Gpp2 phosphate 2. Eliminate glycerol production (delete GPD1, GPD2) Predicted benefits • less acetic acid, no glycerol, more ethanol 37 • 6% higher ethanol yield (industrial conditions)
Strain characterization in Batch GPD1 D1 GPD2 D2 38
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