I NTegral engineering of ACetic acid Tolerance in yeast Ton van - - PowerPoint PPT Presentation

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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

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… the Team:

Isabel Sa-Correia Nuno Mira Margarida Palma Joana Guerreiro & students Elke Nevoigt Steve Swinnen & students Ton van Maris Dani Gonzalez Ramos Erik de Hulster Bianca e.d. Bianca (Bra) & students Joaquin Arino Boris Rodriguez

Desired feedstocks for I ndustrial Biotechnology

3 cornstover bagasse wheatstraw

Acetic acid

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Synthetic medium Plant hydrolysate

+ H+

↔

HAc Ac-

• Weak organic acid, pKa = 4.75

Structural component of lignocellulosic biomass

HAc H+ + Ac- H+ Ac- H+ Ac- ATP ADP Neutral pH H+ + Ac- Main mode of toxicity

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HAc Low pH

20 40 60 80 100 2.75 3.75 4.75 5.75 6.75 pH % HAc pKa = 4.75

Growth of lab strain (CEN.PK) at various concentrations

pH 4.5 defined media 6

0.0% 0.1% 0.2% 0.3% 0.4% 0.5% 0.6% 0.7% 0.8% 0.9% 1.0%

Strain: CEN.PK, pH 4.5

9 g/l 9 g/l

Swinnen et al., submitted

Exposure of exponentially growing cells to acetic acid decreases specific growth rate (µmax) and lag (latency) phase

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• 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

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Consortium Aim

9 g/l acetic acid, pH 4.5 Swinnen et al., submitted

• S. cerevisiae strains strongly differ in acetic acid tolerance

(particularly lag phase)

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9 g/l acetic acid, pH 4.5

• S. cerevisiae strains strongly differ in acetic acid tolerance

(particularly lag phase)

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Swinnen et al., submitted

9 g/l acetic acid, pH 4.5

• S. cerevisiae strains strongly differ in acetic acid tolerance

(particularly lag phase)

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Swinnen et al., submitted

The major challenge in reverse engineering: How to identify the causative genetic differences?

Reference strain without the desirable trait Strain with desirable phenotypic trait All genetic differences (either causative or unimportant for phenotype) Genetic differences causative for phenotype

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Acetic acid- strain Acetic acid+ strain 1n 1n

×

2n Sporulation 1n Quantification of acetic acid tolerance Isolation of single segregants

Acetic acid tolerance is a quantitative trait

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1n

Select only segregants with acetic acid+ phenotype Pooled segregant whole genome analysis

Significant genetic association?

Identification of the crucial genetic determinants

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… Position of a causative determinant Acetic acid- strain Acetic acid+ strain Segregants with acetic acid+ phentotype

Mating Selection of segregants with acetic acid+ phenotype Sporulation Isolating and phenotyping of segregants

Diploid hybrid strain

Method reviewed by Swinnen et al. (2012) FEMS Yeast Res.

Genome-wide genetic association analysis

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 Analysis of determinant regions ongoing

Genome-wide genetic association mapping of the two strains with different acetic acid tolerance

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Haa1

Acetic acid-resistance genes indirectly regulated by Haa1 16 acetic acid-resistance genes directly regulated by Haa1 TFs regulated by Haa1

(www.yeastract.com)

Role of Haa1 and the Haa1-regulon in yeast response and resistance to acetic acid

Mira et al. (2011) Nucleic Acids Res 39(16): 6896-907 Teixeira et al. (2014) Nucleic Acids Res 42(1): D161-6

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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)

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

Transcription factor engineering

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Transcription factor engineering

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• 1. A mutant HAA1 library has been enriched in acetic acid containing medium
• 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

* * 1 2

PROMOTER ORF TERMINATOR

0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 20 40 60

OD600 Time [h]

Library Enrichment 1 & 2 Wild-type HAA1

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• Introduction of the mutations in the genome of CEN.PK113-7D:

Purpose  Eliminate any possible effects of the plasmid and auxotrophic background  Determine the individual effect of each mutation

HAA1

1 2

HAA1

1

HAA1

2

HAA1 HAA1

Strain 1 Strain 2 Strain 3 Strain 4

chr VII

Strains

+

Transcription factor engineering

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• 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

Transcription factor engineering

16 acetic acid-resistance genes directly regulated by Haa1

TFs regulated by Haa1

Role of Haa1 and the Haa1-regulon in yeast response and resistance to acetic acid

(www.yeastract.com)

Mira et al. (2011) Nucleic Acids Res 39(16): 6896-907 Teixeira et al. (2014) Nucleic Acids Res 42(1): D161-6 The protein kinase Hrk1 is among the Haa1p-targets exerting the strongest protective effect against acetic acid

Haa1

5 10 15 20 25 30 35 40 Ratio strains

Tolerance test - VM-HAc 90 mM at 15 hours

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Ppz1 Hal3 Pma1 Hal4 Hal5

K+ H+

Sky1

H+ Na+,K+

Sap185 Sap155 Sit4

Na+,K+

Ptk2

Hrk1

Modified from Arino et al. 2010

0,1 1 10 20 40 60 80

OD600nm Time (h)

wt

50 mM acetic acid

hrk1∆

1h

Proteome-w ide yeast response to acetic acid stress: role of Hrk1

Proteome extraction

The phosphoproteome of a membrane-enriched fraction obtained from WT and hrk1 cells cultivated in the presence of acetic acid w as compared (iTRAQ)

• Hrk1p is the Haa1p-target

exerting the strongest protective effect against acetic acid

• Hrk1p belongs to a family of

kinases involved in the regulation of plasma membrane transporters

The role of the Haa1p regulon in yeast response and resistance to acetic acid stress

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Proteome profiling of acetic acid stressed yeast strains – phosphoproteomic analysis to elucidate Hrk1 biological activity

The phosphoproteomes of acetic acid stressed and unstressed parental strain

• S. cerevisiae BY4741 and hrk1∆ cells

were compared using iTRAQ

• Phosphate metabolism
• Translation
• Cellular transport
• Stress response
• (…)

I ncreased phosphorylation

~20% of the proteins with an increased phosphorylation level in parental cells in response to acetic acid stress are Hrk1- dependent

Decreased phosphorylation

Acetic acid-responsive proteins

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Start a batch culture End of the exponential phase Pump out 99%

• f the medium

Add fresh medium, sometimes increasing [acetic acid]

• Ability to grow at higher [acetic acid]
• Faster growth at a given [acetic acid]

Evolutionary Engineering in Sequential Batch Cultivation

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2 4 6 8 10 12 14 10 20 30 40 50

Batch Number Batch Duration (days)

0.00 0.05 0.10 0.15 0.20 0.25

umax (hr-1)

Duration Rate

0 1 2 3 4 5 6

[Acetic acid] (g/L) However, acquired phenotype not constitutive, but hyper inducible

Wright et al. 2011

FEMS Yeast Res. 11: 299-306

Induction of acetic acid tolerance

0,09 0,14 0,19 0,24 0,29 0,34 10 20 30 40 50 60

OD660 Time (h)

CEN.PK113-7D exposed CEN.PK113-7D

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Evolutionary ON/OFF approach for constitutive tolerance

OFF OFF OFF OFF [Acetic acid]

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Measurements of acetic acid tolerance

[Acetic acid] Measure OD660 after 5 days Dilute to the same OD660

CEN.PK113-7D Evolution mutant

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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

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Number of mutations responsible for tolerance? Dominant or recessive?

HAT2A IMK439 Diploid HAT2A-D

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Sporulation and screening

144 haploid segregants Inoculate without OD660 measurement Measure OD660 after 5 days 144x

priority targets identified & reverse engineering ongoing

An alternative approach to deal with acetic acid?

Can the inhibitor acetic acid be converted (reduced) to ethanol?

• Attractive option (less acetic acid, more ethanol)
• But where should the reducing equivalents come from?

2 NADH

An engineering strategy to eliminate glycerol production Predicted benefits

• less acetic acid, no glycerol, more ethanol
• 6% higher ethanol yield (industrial conditions)

glycerol

NAD+ NADH dihydroxy- acetone- phosphate glycerol-3- phosphate Pi ADP ATP 0.5 glucose Gpd1, Gpd2 Gpp1, Gpp2

• 2. Eliminate glycerol production (delete GPD1, GPD2)

NAD+

NADH

ethanol

acetyl- Coenzyme A acetaldehyde AMP + PPi

ATP

• E. coli MhpF

Adh1-5

• 1. Express heterologous acetylating acetaldehyde dehydrogenase

Acs2 acetic acid NAD+

NADH

NAD+

NADH

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GPD1 D1 GPD2 D2

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Strain characterization in Batch

GPD1 D1 GPD2 D2 gpd1 gpd1 Δ gpd2 gpd2Δ + m hpF pF

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Strain characterization in Batch 13 % increased ethanol yield

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Integration and knowledge based engineering of tolerance

Activities Time-table

Please give a diagrammatical representation (block diagram) of the workpackage activities vs. time. Activity scheme S1 S2 S3 S4 S5 S6 WP1 Screening of natural and industrial isolates (Br & L) WP2 Evolutionary engineering improved acetic acid tolerance (D) WP3 Identification of relevant genetic loci in tolerant strains (Br & D) WP4 High-copy number screen for genes confering tolerance (Ba) WP 5 Generation of gTME library & screening transformants (Br&L) WP6 Proteome & Metabolome profiling (L) WP7 Characterization of Haa1 regulon (L & Ba) WP8 Characterization of Rim101p regulon (L & Ba) WP 9 Identification of acetate exporters (L & D) WP10 Potassium homeostasis in relation to tolerance (Ba & L) WP11 Knowledge-based metabolic engineering of tolerance (C) (Ba) Barcelona, (Br) Bremen, (D) Delft, (L) Lisbon (C) Consortium. S indicates Semester (half year)

completed completed

• ngoing

completed

• ngoing

deprioritized completed completed

• ngoing
• ngoing

continued collaboration

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Conclusions

• Improved understanding on how acetic acid affects

processes (single cells, genomics, induction)

• Strains with improved tolerance to acetic acid identified

 Synthetic biology tools rapidly developed the last 3 years

• Evolutionary engineering can dramatically improve

constitutive tolerance to acetic acid.

• Reverse metabolic engineering still ongoing

Tpo3

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I NTACT – EI B.10.008

I ntegral Engineering of Acetic Acid Tolerance in yeast