EP4065688A1 - Means and methods to modulate acetic acid tolerance in industrial fermentations - Google Patents

Abstract

The present invention relates to the field of fermentation, more particularly to ethanol production. Even more particularly the present invention relates mutant alleles and chimeric genes useful to engineer the acetic acid tolerance in yeast. These yeast strains are particularly useful in the production of bioethanol based on both first- and second-generation substrates.

Description

MEANS AND METHODS TO MODULATE ACETIC ACID TOLERANCE IN

INDUSTRIAL FERMENTATIONS

Field of the invention

The present invention relates to the field of fermentation, more particularly to ethanol production. Even more particularly the present invention relates to mutant alleles and chimeric genes useful to engineer the acetic acid tolerance in yeast. These yeast strains are particularly useful in the production of bioethanol based on both first- and second-generation substrates.

Background

Acetic acid is commonly used in the food industry as an antimicrobial preservative (Piper 2011 Adv Appl Microbiol 77). At low pH, the protonated form can easily diffuse through membranes and drastically lower the internal pH of cells and organelles, causing wide-spread inhibition of many cellular functions (Ullah et al 2012 Appl Environ Microbiol 78; Fernandez-Nino et al 2015 Appl Environ Microbiol 81). However, acetic acid is also a side-product in the microbial production of ethanol. Because of its antimicrobial activity, acetic acid accumulation may lead to fermentation arrests and reduced ethanol volumetric production. Acetic acid is a very efficient inhibitor of the microbial fermentation of so-called second-generation substrates, i.e. hydrolysates of lignocellulosic biomass derived from waste streams or bioenergy crops (Deparis et al 2017 FEMS Yeast Res 17; Jonsson et al 2013 Biotechnol Biofuels 6). (Ligno)cellulose fibrils contain large numbers of acetyl groups, which are released during pretreatment and enzymatic hydrolysis and accumulate to high levels in the medium. Together with the ethanol produced, the acetic acid inhibits the fermentation process, and especially the artificially engineered capacity of yeasts optimized for xylose fermentation (Bellissimi et al 2009 FEMS Yeast Res 9). But also in bioethanol production based on starch from food crops, sugar cane or molasses (so-called first- generation substrates), acetic acid produced by contaminating acetic acid bacteria can accumulate to high levels, especially by water recycling practices, and cause significant inhibition of yeast fermentation (Graves et al 2006 J Ind Microbiol Biotechnol 33). Acetic acid tolerance is therefore a trait of major importance in the field of industrial yeast fermentation.

Many approaches have been used to improve the tolerance of yeast in ethanol production processes, including tolerance to acetic acid (Caspeta et al 2015 Front Bioeng Biotechnol 3; W02008153890A1). A major component conferring acetic acid tolerance is the Haal transcription factor and its overexpression or modification is well known to enhance acetic acid tolerance (W02014030745A1). Further polygenic analysis of yeast strains with high acetic acid tolerance revealed GLOl, DOT5, CUP2 and VMA7 as additional causative factors, but also suggested the existence of many other factors involved in high acetic acid tolerance (WO2016083397A1; Meijnen et al 2016 Biotechnol Biofuels 9). There is however still a need for additional regulators of acetic acid tolerance in microorganisms, to improve the efficiency of acetic acid sensitive industrial processes.

Summary

Here, intra-species whole genome transformation (WGT) is used to idenitfy novel acetic acid tolerance mutations present in yeast strains with superior acetic acid tolerance. Surprisingly, it was found that mutations in the Sucrose non-fermenting 4 (Snf4) gene conferred increased acetic acid tolerance. Snf4 is an activating subunit of the Snfl protein kinase essential for sucrose utilization. Therefore, it was even more surprisingly found that mutating Snf4 has only a slight negative effect on growth in industrial strain backgrounds. Interestingly, the Snf4 mutation or deletion even further enhances acetic acid tolerance of industrial yeasts already engineered for increased acetic acid tolerance by the HAA1 allele. Current application therefore provides a novel tool to solve the problem of acetic acid accumulation in industrial microbiological processes.

In a first aspect, the application provides a mutant SNF4 yeast allele comprising a nonsense mutation on nucleic acid position 805. Other SNF4 mutations leading to a loss of Snf4 function can also increase acetic acid tolerance in yeast. Therefore, a chimeric gene construct is provided comprising a promoter which is active in a eukaryotic cell operably linked to a CRISPR guide RNA targeting the SNF4 allele.

In another aspect, a yeast strain is provided comprising a homozygous or hemizygous mutantSA/F4 allele, said allele being the above SNF4 allele or comprising the above chimeric gene construct. Since the art is completely silent about Snf4 and acetic acid tolerance in yeast, the invention extends to industrial yeast strains in which the SNF4 allele has been disrupted or deleted. In a particular embodiment, said yeast is a xylose fermenting yeast strain or is industrially optimized to ferment second-generation substrates. In other particular embodiments, an acetic acid tolerant yeast strain is provided devoid of a functional SNF4 allele. In more particular embodiments, said yeast strains comprises the above described chimeric gene construct. Surprisingly, the recessive SNF4 mutation can further enhance the acetic acid tolerance in yeasts which are already engineered to tolerate acetic acid. Therefore, the application also provides the herein disclosed yeast strains further comprising an allele of one or more of the GLOl, DOTS, CUP2 and HAA1 genes that confers increased tolerance to acetic acid as described in WO2015/181169 and WO2016/083397.

In another aspect, the use of any of the mutant SNF4 alleles herein disclosed or of any of the chimeric gene construct herein disclosed is provided to increase acetic acid tolerance in a culture of yeast cells. Also the use of any of the yeast strains herein disclosed is provided to increase the production of ethanol. In yet another aspect, a method of producing ethanol is provided comprising the step of fermenting a medium with any of the yeast strains of current application. Brief description of the Figures

Figure 1. Fermentation performance of the parent strain ER18, whole-genome transformant MS164 and the gDNA donor strain Kll in the presence of acetic acid. Fermentations were performed with constant stirring at 120 rpm, at 35°C and pH 4.7 in YPD medium with 40g/l glucose and supplemented with 8g/l (left) or lOg/l (right) acetic acid.

Figure 2. Fermentation performance of the original ER18 parent strain, ER18 derivatives engineered for SNP1 (A) or SNP5 (B), MS164 transformant and MS164 derivatives reverse engineered for SNP1 (A) or SNP5 (B) in the presence of acetic acid. The control strains are indicated with dashed lines, ER18 (blue) and MS164 (red). All SNP-engineered strains are indicated with full lines. Fermentations were performed at 35°C, constant stirring at 120 rpm, pH 4.7 in 50 ml YPD medium with 40g/l glucose and supplemented with lOg/l acetic acid. The downgraded derivatives of MS164 are shown with open symbols (red, □, D, V, 0), while the upgraded derivatives of ER18 are shown with closed symbols (blue, ■, A, □, □).

Figure 3. Fermentation performance of the original ER18 parent strain, ER18 derivatives engineered for SNP1 (A) or SNP5 (B), MS164 transformant and MS164 derivatives reverse engineered for SNP1 (A) orSNP5 (B) in the absence of acetic acid. The control strains are indicated with dashed lines, ER18 (blue) and MS164 (red). All SNP-engineered strains are indicated with full lines. Fermentations were performed at 35°C, constant stirring at 120 rpm, in 50 ml YPD medium with 40 g/l glucose (and no acetic acid addition). The downgraded derivatives of MS164 are shown with open symbols (red, □, D, V, 0), while the upgraded derivatives of ER18 are shown with closed symbols (blue, ■, A, □, □).

Figure 4. Fermentation performance of the strains ER18 and ER18 derivatives (A) or PE2 and PE2 derivatives (B) with engineered SNP1 (sn/4^E269*) or sn/421 in all SNF4 alleles present, in the presence of acetic acid. The strains are indicated with different colours, ER18 and PE2 (green, ·), ER18 and PE2 derivatives with engineered SNP1 (sn/4^E269*) in all SNF4 alleles present (blue, ■, A, □, □) and ER18 eh 4D and PE2 snf4AA strains (blue, □, D, V, 0). Fermentations were performed at 35°C, constant stirring at 120 rpm, pH 4.7 in 50 ml YPD medium with 40 g/l glucose and supplemented with 10 g/l (ER18) or 11 g/l (PE2) acetic acid.

Figure 5. Glucose consumption, ethanol production and acetic acid consumption during semi- anaerobic fermentation or aerobic growth of the strains ER18 and ER18 derivatives with engineered SNP1 (sn/4^E269*) in SNF4 or snf4l 1 in the presence of acetic acid. The strains are indicated with different colours, ER18 (green, ·), ER18 derivatives with engineered SNP1 (sn/4^E269*) in SNF4 (blue, ■, A, □, □) and ER18 snf4A (blue, □, D, V, 0). Semi-anaerobic fermentations were performed at 35°C, constant stirring at 120 rpm, pH 4.7 in 50 ml YPD medium with 40 g/l glucose and supplemented with 10 g/l acetic acid. Growth assays under aerobic conditions were performed at 30°C, constant shaking at 200 rpm, pH 4.7 in 50 ml YPD medium with 40 g/l glucose and supplemented with 10 g/l acetic acid.

Figure 6. Growth assays in microtiter plate format using different carbon sources of the strains ER18, PE2, MS488, and ER18, PE2 or MS488 derivatives with engineered SNP1 (sn/4^E269*) or snf4A in all SNF4 alleles present in the absence of acetic acid. The strains are indicated with different colours, ER18, PE2 and MS488 (green, ·), ER18, PE2 and MS488 derivatives with engineered SNP1 (sn/4^E269*) in SNF4 (blue, ■, A, □, □) and ER18 snf4A, PE2 snf4AA and MS488 snf4AA strains (blue, □, D, V, 0). Growth assays were performed in microtiter plates in a Multiskan apparatus at 30°C, with intermittent shaking, in 200 pL YP medium with 40g/l glucose, sucrose or maltose (and absence of acetic acid).

Figure 7. Growth of the strains MS488, MS488 with engineered SNP1 (sn/4^E269*) or snf4A in all SNF4 alleles present, in four different concentrations of sugar cane molasses. The strains are indicated with different colours, MS488 (green, ·) and MS488 derivatives with engineered SNP1 (sn/4^E269*) in all SNF4 alleles present (blue, ■, A, □, □) and MS488 snf4AA (blue, □, D). Growth assays were performed under aerobic conditions in shake flasks at 200 rpm, 30°C, in 25 ml in media with different concentrations of molasses (and absence of acetic acid).

Figure 8. Fermentation performance of the strains JT28541, MS488, MS488 derivatives with engineered SNP1 (sn/4^E269*) or snf4A in all SNF4 alleles, in the presence of acetic acid. The strains are indicated with different colours, JT28541 (black, □), MS488 (JT28541 HAA1^S506N ) (green, ·) and MS488 (JT28541 HAA1^S506N ) derivatives with engineered SNP1 (sn/4^E269*) (blue, ■, A, □, □) and MS488 (JT28541 HAA1^S506N ) snf4AA (blue, □, D). Fermentations were performed at 35°C, constant stirring at 120 rpm, pH 4.7 in 50 ml YPD medium with 40 g/l glucose and supplemented with lOg/l acetic acid.

Detailed description

Definitions

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein. The following terms or definitions are provided solely to aid in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Michael R. Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4^th ed., Cold Spring Harbor Laboratory Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999), for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.

The Applicants of current application aimed at increasing the acetic acid tolerance in yeast, more particularly in industrial yeasts. Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom and like all fungi, yeast may have asexual and sexual reproductive cycles. The most common mode of vegetative growth in yeast is asexual reproduction by budding. Here, a small bud or daughter cell, is formed on the parent cell. The nucleus of the parent cell splits into a daughter nucleus and migrates into the daughter cell. The bud continues to grow until it separates from the parent cell, forming a new cell. This reproduction cycle is independent of the yeast's ploidy, thus both haploid and diploid yeast cells can duplicate as described above. Haploid cells have in general a lower fitness and they often die under high-stress conditions such as nutrient starvation, while under the same conditions, diploid cells can undergo sporulation, entering sexual reproduction (meiosis) and producing a variety of haploid spores or haploid segregants, which can go on to mate (conjugate), reforming the diploid. The budding yeast Saccharomyces cerevisiae reproduces by mitosis as diploid cells when nutrients are abundant, but when starved, this yeast undergoes meiosis to form haploid spores. Haploid cells may then reproduce asexually by mitosis. Yeast, as used in current application, can be any yeast useful for industrial applications. Preferable, said yeast is useful for ethanol production, including, but not limited to Saccharomyces, Zygosaccharomyces, Brettanomyces and Kluyveromyces. Preferably, said yeast is a Saccharomyces sp., even more preferably it is a Saccharomyces cerevisiae sp. In current application, the Applicants report on a whole genome transformation experiment set up to identify acetic acid tolerance alleles in the sake yeast strain Kll. The most tolerant transformants showed an improved tolerance to lOg/l acetic acid compared to 6g/l of the sensitive host strain before transformation. Whole genome sequencing of the most promising transformants revealed 7 SNPs among which the recessive SNF4^G805T mutation. Interestingly and very surprisingly, the SNF4 mutation causative to the increased acetic acid tolerance of the transformants was induced by the transformation event itself and was not present in the donor strain Kll.

SNF4 or Sucrose Non-Fermenting 4 (also known as YGL115W, SGD:S000003083, CAT3 or SCI 1) is the activating gamma subunit of the AMP-activated Snfl protein kinase complex, that plays a central role in the response to glucose starvation in the yeast Saccharomyces cerevisiae (Jiang and Carlson 1997 Mol Cell Biol 17). The SNF4 gene is required for expression of glucose-repressible genes in response to glucose deprivation in S. cerevisiae. The mutant SNF4 allele as herein disclosed is provided in SEQ ID No. 1. The G805T mutation is underlined, indicate in bold and highlighted in bigger font. The truncated Snf4 protein encoded by SEQ ID No. 1 is shown in SEQ ID No. 2. The corresponding wild-type sequences are shown in SEQ ID No. 3 and 4.

SEQ ID No. 1

ATGAAACCGACACAGGATTCACAAGAAAAGGTTTCTATTGAACAGCAGTTAGCTGTAGAATCGATAAGGAAGTT

TTTGAACTCGAAAACATCTTATGACGTGTTGCCTGTTTCTTACCGTTTAATTGTCTTGGACACCTCGTTGTTAGTGA

AGAAATCACTGAATGTTCTTTTGCAAAATAGCATTGTCTCCGCGCCATTATGGGACTCCAAGACTTCCAGGTTCGC

TGGACTTCTAACTACTACAGATTTTATTAATGTCATCCAGTATTACTTCTCCAATCCAGATAAGTTCGAATTAGTAG

ACAAATTACAGTTAGATGGATTAAAAGATATAGAGCGGGCTCTCGGTGTTGATCAACTAGATACAGCTTCAATTC

ATCCTTCTAGACCCTTATTTGAGGCGTGTCTTAAGATGTTAGAATCAAGAAGTGGTAGAATACCACTGATCGATC

AAG ATG AAG AG AC AC AT AG AG AAATT GTCGTT AGT GTT CTT ACGCAAT AT AG AATT CTG AAGTTCGTTGCTTT AA

ATTGCAGGGAAACACATTTTCTAAAGATTCCAATTGGGGACTTGAACATTATTACGCAAGATAACATGAAAAGCT

GTCAAATGACCACTCCGGTCATAGACGTCATTCAGATGCTTACCCAAGGTCGGGTTTCTTCCGTCCCTATTATTGA

CGAAAACGGCTACTTAATCAACGTATATGAAGCATACGATGTCCTAGGCTTGATAAAAGGAGGCATCTACAACG

ACCTGTCATTGAGCGTCGGAGAAGCCCTTATGAGGAGAAGTGATGATTTTTAAGGTGTTTATACATGCACTAAG

AATGAT AAATT ATCT ACT ATT ATGGATAACATCAGAAAAGCAAGGGTGCATAGATTCTTTGTAGTTGATGACGTC GGACGGTTGGTTGGTGTCTTGACGTTAAGCGATATTCTCAAATATATCCTTCTAGGTAGCAACTGA

SEQ ID No. 2

MKPTQDSQEKVSIEQQLAVESIRKFLNSKTSYDVLPVSYRLIVLDTSLLVKKSLNVLLQNSIVSAPLWDSKTSRFAGLLTTT

DFINVIQYYFSNPDKFELVDKLQLDGLKDIERALGVDQLDTASIHPSRPLFEACLKMLESRSGRIPLIDQDEETHREIVVSV

LTQYRILKFVALNCRETHFLKIPIGDLNIITQDNMKSCQMTTPVIDVIQMLTQGRVSSVPIIDENGYLINVYEAYDVLGLI

KGGIYNDLSLSVGEALMRRSDDF* SEQ ID No. 3

ATGAAACCGACACAGGATTCACAAGAAAAGGTTTCTATTGAACAGCAGTTAGCTGTAGAATCGATAAGGAAGTT

TTTGAACTCGAAAACATCTTATGACGTGTTGCCTGTTTCTTACCGTTTAATTGTCTTGGACACCTCGTTGTTAGTGA

AGAAATCACTGAATGTTCTTTTGCAAAATAGCATTGTCTCCGCGCCATTATGGGACTCCAAGACTTCCAGGTTCGC

TGGACTTCTAACTACTACAGATTTTATTAATGTCATCCAGTATTACTTCTCCAATCCAGATAAGTTCGAATTAGTAG

ACAAATTACAGTTAGATGGATTAAAAGATATAGAGCGGGCTCTCGGTGTTGATCAACTAGATACAGCTTCAATTC

ATCCTTCTAGACCCTTATTTGAGGCGTGTCTTAAGATGTTAGAATCAAGAAGTGGTAGAATACCACTGATCGATC

AAG ATG AAG AG AC AC AT AG AG AAATT GTCGTT AGT GTT CTT ACGCAAT AT AG AATT CTG AAGTTCGTTGCTTT AA

ATTGCAGGGAAACACATTTTCTAAAGATTCCAATTGGGGACTTGAACATTATTACGCAAGATAACATGAAAAGCT

GTCAAATGACCACTCCGGTCATAGACGTCATTCAGATGCTTACCCAAGGTCGGGTTTCTTCCGTCCCTATTATTGA

CGAAAACGGCTACTTAATCAACGTATATGAAGCATACGATGTCCTAGGCTTGATAAAAGGAGGCATCTACAACG

ACCTGTCATTGAGCGTCGGAGAAGCCCTTATGAGGAGAAGTGATGATTTTGAAGGTGTTTATACATGCACTAAG

AATGAT AAATT ATCT ACT ATT ATGGATAACATCAGAAAAGCAAGGGTGCATAGATTCTTTGTAGTTGATGACGTC

GGACGGTTGGTTGGTGTCTTGACGTTAAGCGATATTCTCAAATATATCCTTCTAGGTAGCAACTGA

SEQ ID No. 4

MKPTQDSQEKVSIEQQLAVESIRKFLNSKTSYDVLPVSYRLIVLDTSLLVKKSLNVLLQNSIVSAPLWDSKTSRFAGLLTTT

DFINVIQYYFSNPDKFELVDKLQLDGLKDIERALGVDQLDTASIHPSRPLFEACLKMLESRSGRIPLIDQDEETHREIVVSV

LTQYRILKFVALNCRETHFLKIPIGDLNIITQDNMKSCQMTTPVIDVIQMLTQGRVSSVPIIDENGYLINVYEAYDVLGLI

KGGIYNDLSLSVGEALMRRSDDFEGVYTCTKNDKLSTIMDNIRKARVHRFFVVDDVGRLVGVLTLSDILKYILLGSN*

In a first aspect, the application provides a mutant SNF4 yeast allele comprising a mutation on nucleic acid position 805. In one embodiment, said mutant SNF4 allele is an isolated mutant SNF4 yeast allele. In particular embodiments, said mutation is a nonsense or missense mutation, more particularly on nucleic acid position 805. In more particular embodiments, said mutant allele is a snf4E269* allele or encodes a Snf4 protein comprising a E269* mutation. In even more particular embodiments said mutant allele comprises a G805T mutation. In most particular embodiments, said mutant allele is the allele as depicted in SEQ ID No. 1. In the rest of this document, the above described mutant SNF4 yeast alleles will be referred to as "one of the mutant SNF4 alleles of the application". The allele depicted in SEQ ID No. 1 encodes the truncated Snf4 yeast protein of which the amino acid sequence is depicted in SEQ ID No 2.

A "nonsense mutation" as used herein refers to a point mutation in a sequence of DNA that results in a premature stop codon (herein illustrated as or a nonsense codon in the transcribed mRNA, and hence in a truncated (more particularly a C-terminal truncated), incomplete, and nonfunctional protein product. A "missense mutation" means a point mutation where a single nucleotide is changed to cause substitution of a different amino acid.

A "mutation on nucleic acid position 805" is equivalent as saying that the nucleobase on position 805 is mutated. With "mutation on nucleic acid position 805" as used herein, it is thus meant that nucleobase 805 from the wild-type SNF4 gene as depicted in SEQ ID No. 3 is mutated. "Position 805" or "nucleobase 805" as used herein refers to the nucleobase that is 804 positions removed downstream from the first nucleobase (i.e. adenosine) from the start codon. This nucleobase 805 is a guanine (G), see SEQ ID No. 3. In the specific mutant SNF4 allele disclosed in the application said G is replaced by a thymine (T). As such the mutation can also be referred to as a G805T mutation. Given that said mutation changes a glutamic acid (E) codon into a premature stop codon (*), said SNF4 allele can also be referred to as a snf4E269* allele or an allele that encodes a E269* mutation.

The mutant SNF4 alleles of the application as well as the mutant Snf4 proteins which they encode may have additional mutations (besides G805T or E269*) and are also envisaged in this application as long as the additional Snf4 mutations do not restore Snf4 function. Thus, in one embodiment the mutant SNF4 allele or mutant Snf4 yeast protein which are provided herein comprise at least a G805T or at least an E269* mutation.

"Nucleobases" are nitrogen-containing biological compounds that form nucleosides, which in turn are components of nucleotides; all which are monomers that are the basic building blocks of nucleic acids. Often simply called bases, as in the field of genetics, the ability of nucleobases to form base-pairs and to stack one upon another leads directly to long-chain helical structures such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). There are four so-called DNA-bases: adenine (A), cytosine (C), guanine (G) and thymine (T).

As used herein, "nucleic acid" includes reference to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g. peptide nucleic acids).

By "encoding" or "encodes" or "encoded", with respect to a specified nucleic acid, is meant comprising the information for transcription into an RNA molecule and in some embodiments, translation into the specified protein or amino acid sequence. A nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid, or may lack such intervening non- translated sequences (e.g., as in cDNA). The information by which a protein is encoded is specified by the use of codons. Typically, the amino acid sequence is encoded by the nucleic acid using the "universal" genetic code.

The disclosed SNF4 mutant alleles have a loss-of-function effect and are recessive, meaning that the mutation has to be homozygous (for a diploid organism) or hemizygous to lead to the mutant phenotype, i.e. increased acetic acid tolerance. "Homozygous" refers to having identical alleles for a single trait. An "allele" represents one particular form of a gene. Alleles can exist in different forms and diploid organisms typically have two alleles for a given trait. A homozygous mutant SNF4 allele thus means that all SNF4 alleles are identical. "Hemizygous" refers to having only one allele for a single trait or gene. In case of a diploid organism thus only one allele of its pairs is present, while all other genes are represented by two alleles. This can for example be achieved by deleting one allele of a gene or by introducing one allele of a gene that is not present in an organism. Therefore, in another aspect, a yeast strain is provided comprising a homozygous or hemizygous mutant SNF4 allele, said allele being on of the SNF4 alleles of the application as described above. In one embodiment, a haploid yeast segregant comprising a disrupted, partially deleted or completely deleted SNF4 allele is provided. In a particular embodiment, said haploid segregant comprises a mutant SNF4 yeast allele according to the application. Haploid cells contain one set of chromosomes, while diploid cells contain two. A haploid segregant as used herein is equivalent as a haploid spore, the result of sporulation.

In another aspect, an industrial yeast strain is provided comprising a homozygous or hemizygous mutant SNF4 allele compromising, partially abolishing or completely abolishing Snf4 function. An allele that compromises, partially abolishes or completely abolishes Snf4 function is equivalent to a disrupted, partially deleted or completely deleted SNF4 allele. In a particular embodiment, a yeast strain or more particularly an industrial yeast strain is provided comprising a homozygous or hemizygous mutant SNF4 allele, wherein said mutant allele comprises a nonsense or missense mutation on nucleic acid position 805. In more particular embodiments, said mutant SNF4 allele is a snf4E269* allele or encodes a Snf4 protein comprising a E269* mutation. In even more particular embodiments said mutant SNF4 allele comprises a G805T mutation. In most particular embodiments, said mutant SNF4 allele is the allele as depicted in SEQ ID No. 1.

"Disrupted, partially deleted or completely deleted function" or "disrupting, partially deleting or completely deleting the functional expression" is equivalent as saying partially or completely inhibiting the formation of a functional mRNA molecule encoding Snf4. Means and methods to disrupt, partially delete or completely delete a gene or protein are well known in the art. In order to disrupt the SNF4 allele, any site may be disrupted, for example, a promoter site of SNF4, an open reading frame (ORF) site, and a terminator site, or combination thereof may be disrupted. The skilled person can select from a plethora of techniques to affect the expression or function of Snf4. The particular method used to reduce or abolish the expression of SNF4 is not critical to the invention.

In one embodiment, disruption can be accomplished by homologous recombination (as was demonstrated in the Experimental part of current application), whereby the gene to be disrupted is interrupted (e.g., by the insertion of a selectable marker gene) or made inoperative (e.g., "gene knockout"). A "knock-out" can be a gene knockdown (leading to reduced gene expression) or the gene can be knocked out by a mutation such as, a point mutation, an insertion, a deletion, a frameshift, or a missense mutation by techniques known in the art. The lack of transcription can e.g. be caused by epigenetic changes (e.g. DNA methylation) or by loss-of-function mutations. A "loss-of-function" or "LOF" mutation as used herein is a mutation that prevents, reduces or abolishes the function of a gene product as opposed to a gain-of-function mutation that confers enhanced or new activity on a protein. Disruption, partial deletion or complete deletion at the DNA level can also be achieved by inducing mutations in the promoter of a gene encoding Snf4. Also gain-of-function mutations can inhibit the formation of a functional mRNA molecule for example through gene silencing, whereas dominant negative mutations can disrupt the functional expression of a gene. Both dominant negative or LOF mutations can be caused by a wide range of mutation types, including, but not limited to, a deletion of the entire gene or part of the gene, splice site mutations, frame-shift mutations caused by small insertions and deletions, nonsense mutations, missense mutations replacing an essential amino acid and mutations preventing correct cellular localization of the product.

Methods for gene knockout and multiple gene knockout are well known. See, e.g. Rothstein, 2004, "Targeting, Disruption, Replacement, and Allele Rescue: Integrative DNA Transformation in Yeast" In: Guthrie et al., Eds. Guide to Yeast Genetics and Molecular and Cell Biology, Part A, p. 281-301; Wach et al., 1994, "New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae" Yeast 10:1793-1808. Methods for insertional mutagenesis are also well known. See, e.g., Amberg et al., eds., 2005, Methods in Yeast Genetics, p. 95-100; Fickers et al., 2003, "New disruption cassettes for rapid gene disruption and marker rescue in the yeast Yarrowia lipolytica" Journal of Microbiological Methods 55:727-737; Akada et al., 2006, "PCR-mediated seamless gene deletion and marker recycling in Saccharomyces cerevisiae" Yeast 23:399-405; Fonzi et al., 1993, "Isogenic strain construction and gene mapping in Candida albicans" Genetics 134:717-728. Other methods to disrupt a gene in a microorganism include the use of nucleases, such as zinc-finger nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), meganucleases but especially the CRISPR-Cas system. "Nucleases" as used herein are enzymes that cut nucleotide sequences. These nucleotide sequences can be DNA or RNA. If the nuclease cleaves DNA, the nuclease is also called a DNase. If the nuclease cuts RNA, the nuclease is also called an RNase. Upon cleavage of a DNA sequence by nuclease activity, the DNA repair system of the cell will be activated. Yet, in most cases the targeted DNA sequence will not be repaired as it originally was and small deletions, insertions or replacements of nucleic acids will occur, mostly resulting in a mutant DNA sequence. ZFN are artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA cleavage domain. Zinc finger domains can be engineered to target desired DNA sequences, which enables zinc-finger nucleases to target a unique sequence within a complex genome. By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of simple and higher organisms. Other technologies for genome customization that can be used to knock out genes are meganucleases and TAL effector nucleases (TALENs, Cellectis bioresearch). A TALEN is composed of a TALE DNA binding domain for sequence- specific recognition fused to the catalytic domain of an endonuclease that introduces double strand breaks (DSB). The DNA binding domain of a TALEN is capable of targeting with high precision a large recognition site (for instance 17bp). Meganucleases are sequence-specific endonucleases, naturally occurring "DNA scissors", originating from a variety of single-celled organisms such as bacteria, yeast, algae and some plant organelles. Meganucleases have long recognition sites of between 12 and 30 base pairs. The recognition site of natural meganucleases can be modified in order to target native genomic DNA sequences (such as endogenous genes).

One very efficient technique is the CRISPR-Cas technology which has also been used in the Examples of this application. CRISPR interference is a genetic technique which allows for sequence-specific control of gene expression in prokaryotic and eukaryotic cells. It is based on the bacterial immune system-derived CRISPR (clustered regularly interspaced palindromic repeats) pathway and has been modified to edit basically any genome. By delivering the Cas nuclease (in many cases Cas9) complexed with a synthetic guide RNA (gRNA) in a cell, the cell's genome can be cut at a desired location depending on the sequence of the gRNA, allowing existing genes to be removed and/or new one added and/or more subtly removing, replacing or inserting single nucleotides (e.g. DiCarlo et al 2013 Nucl Acids Res 41:4336-4343; Sander & Joung 2014 Nat Biotech 32:347-355). Therefore, also a yeast strain is provided in which the SNF4 allele has been disrupted or deleted by using nuclease technology, more particularly by means of the CRISPR- Cas technology. In order to achieve a SNF4 mutant via CRISPR-Cas, a synthetic guide RNA targeting the SNF4 allele should be expressed in the industrial yeast strains of the application. Therefore, the application provides also a chimeric gene construct comprising a promoter which is active in a eukaryotic cell, more particularly in a yeast cell, operably linked to a CRISPR guide RNA targeting the SNF4 allele. "Targeting" as used herein is equivalent to "annealing" or "binding" to the SNF4 allele. As a result, the CRISPR gRNA is complementary to a part of the SNF4 allele. In one embodiment, said promoter of said chimeric gene construct is also operably linked to a nucleic acid molecule encoding a Cas endonuclease, more particularly a Cas9 endonuclease. In another embodiment, the chimeric gene comprising the SNF4 targeting gRNA is expressed together with the chimeric gene comprising the Cas encoding nucleic acid molecule. Said chimeric gene construct(s) is/are expressed in yeast according to standard methods known to the skilled person. In one embodiment, the chimeric gene constructs from current application additionally comprise a 3' end region involved in transcription termination and/or polyadenylation.

The skilled person is familiar with how CRISPR gRNA molecules are designed. The most commonly used gRNA is about 100 base pairs in length. By altering the 20 base pairs towards the 5' end of the gRNA, the CRISPR-Cas9 system can be targeted towards any genomic region complementary to that sequence, e.g. towards the yeast SNF4 allele. Upon annealing to the complementary region, the accompanying Cas endonuclease induces DSB which upon incorrect repair leads to a mutant SNF4. Alternatively, a specific mutation in theSNF4 allele can be introduced via the CRISPR-Cas system as explained in the Experimental details herein.

A "chimericgene" or "chimeric construct" as used herein is a recombinant nucleic acid sequence in which a promoter or regulatory nucleic acid sequence is operably linked to, or associated with, a nucleic acid sequence that codes for a mRNA and encodes an amino acid sequence, such that the regulatory nucleic acid sequence is able to regulate transcription or expression of the associated nucleic acid coding sequence. The regulatory nucleic acid sequence of the chimeric gene is not operably linked to the associated nucleic acid sequence as found in nature.

In the present application a "promoter" comprises regulatory elements, which mediate the expression of a nucleic acid molecule. For expression, the nucleic acid molecule must be linked operably to or comprise a suitable promoter which expresses the gene at the right point in time and with the required spatial expression pattern. The term "operably linked" as used herein refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest. A promoter that enables the initiation of gene transcription in a eukaryotic cell is referred to as being "active". To identify a promoter which is active in a eukaryotic cell, the promoter can be operably linked to a reporter gene after which the expression level and pattern of the reporter gene can be assayed. Suitable well-known reporter genes include for example beta- glucuronidase, beta-galactosidase or any fluorescent or luminescent protein. The promoter activity is assayed by measuring the enzymatic activity of the beta-glucuronidase or beta-galactosidase. Alternatively, promoter strength may also be assayed by quantifying mRNA levels or by comparing mRNA levels of the nucleic acid, with mRNA levels of housekeeping genes such as 18S rRNA, using methods known in the art, such as Northern blotting with densitometric analysis of autoradiograms, quantitative real-time PCR or RT-PCR (Heid et al., 1996 Genome Methods 6: 986-994).

The term "a 3' end region involved in transcription termination or polyadenylation" encompasses a control sequence which is a DNA sequence at the end of a transcriptional unit which signals 3' processing or polyadenylation of a primary transcript and is involved in termination of transcription. The control sequence for transcription termination or terminator can be derived from a natural gene or from a variety of genes. For expression in yeast the terminator to be added may be derived from, for example, the TEF or CYC1 genes or alternatively from another yeast gene or less preferably from any other eukaryotic or viral gene. In a particular embodiment the promoter in the chimeric gene of the invention is active in yeast. In a preferred embodiment, said promoter is selected from the list comprising pTEFl (Translation Elongation Factor 1); pTEF2; pFIXTl (Flexose Transporter 1); pHXT2; pHXT3; pHXT4; pTDH3 (Triose-phosphate Dehydrogenase) also known in the art as pGADPH (Glyceraldehyde-3-phosphate dehydrogenase) or pGDP or pGLDl or pHSP35 or pHSP36 or pSSS2; pTDH2 also known in the art as pGLD2; pTDHl also known in the art as pGLD3; pADHl (Alcohol Dehydrogenase) also know in the art as pADCl; pADH2 also known in the art as pADR2; pADH3; pADH4 also known in the art as pZRG5 or pNRC465; pADH5; pADH6 also known in the art as pADHVI; pPGKl (3-Phosphoglycerate Kinase); pGALl (Galactose metabolism); pGAL2; pGAL3; pGAL4; pGAL5 also known in the art as pPGM2 (Phosphoglucomutase); pGAL6 also known in the art as pLAP3 (Leucine Aminopeptidase) or pBLHl or pYCPl; pGAL7; pGALlO; pGALll also known in the art as pMED15 or pRAR3 or pSDS4 or SPT13 or ABE1; pGAL80; pGAL81; pGAL83 also know in the art as pSPMl; pSIP2 (SNFl-interacting Protein) also know in the art as pSPM2; pMET (Methionine requiring); pPMAl (Plasma Membrane ATPase) also known in the art as pKTHO; pPMA2; pPYKl (Pyruvate Kinase) also known in the art as pCDC19; pPYK2; pENOl (Enolase) also known in the art as pHSP48; pEN02; pPHO (Phosphate metabolism); pCUPl (Cuprum); pCUP2 also known in the art as pACEl; pPET56 also known in the art as pMRMl (Mitochondrial rRNA Methyltransferase); pNMTl (N-Myristoyl Transferase) also known in the art as pCDC72; pGREl (Genes de Respuesta a Estres); pGRE2; GRE3; pSI P18 (Salt Induced Protein); pSV40 (Simian Vacuolating virus) and pCaMV (Cauliflower Mosaic Virus). These promoters are widely used in the art. The skilled person will have no difficulty identifying them in databases. For example, the skilled person will consult the Saccharomyces genome database website (http://www.yeastgenome.org/) or the Promoter Database of Saccharomyces cerevisiae (http://rulai.cshl.edu/SCPD/) for retrieving the yeast promoters' sequences.

In another embodiment, a vector is provided comprising one of the chimeric genes of the application. In yet another embodiment, a yeast or an industrial yeast strain is provided comprising said vector or one of the chimeric gene constructs described herein.

The term "vector" refers to any linear or circular DNA construct comprising one of the above described chimeric gene constructs. The vector can refer to an expression cassette or any recombinant expression system for the purpose of expressing one of the chimeric gene constructs of the application in vitro or in vivo, constitutively or inducibly, in any cell, including, in addition to plant cells, prokaryotic, yeast, fungal, insect or mammalian cells. The vector can remain episomal or integrate into the host cell genome. The vector can have the ability to self-replicate or not (i.e., drive only transient expression in a cell). The term includes recombinant expression cassettes that contain only the minimum elements needed for transcription of the recombinant nucleic acid. The vector of the application is a "recombinant vector" which is by definition a man-made vector.

The yeasts or industrial yeast strains envisaged in current application are yeasts useful for ethanol production, including, but not limited to Saccharomyces, Zygosaccharomyces, Brettanomyces and Kluyveromyces. Preferably, said yeast is a Saccharomyces sp., even more preferably it is a Saccharomyces cerevisiae sp. In most particular embodiments, said yeast or said industrial yeast is not S288C, CEN.PK, MCY1830, MCY1853 or MCY2634.

Industrial yeast strains are often diploid, polyploid or aneuploid and have proven capabilities for application in large scale industrial fermentation. Suitable industrial yeast strains include but are not limited to e.g. the commercial strains Gert Strand Turbo yeasts, Alltech SuperStart TM, Fermiol Super HATM, ThermosaccTM and Ethanol RedTM. Also suitable are yeast cells derived from any of these strain by modifications as described herein.

In a particular embodiment, the yeast or industrial yeast herein disclosed is a xylose fermenting yeast. In another particular embodiment, the yeasts or industrial yeasts herein disclosed are acetic acid tolerant yeasts. Therefore, also yeast or industrial yeast strains are provided that are metabolically active, actively dividing, actively fermenting first- and/or second generation substrates, and/or producing ethanol at a concentration of at least 8 g/l, 9 g/l or 10 g/l acetic acid or between 7 g/l and 10.5 g/l or between 8.5 g/l and 11 g/l acetic acid. More particularly, yeasts are provided that are actively fermenting in the presence of at least 8, 9 or 10 g/l acetic acid and pH between 4.3 and 4.8, or 4.4 and 4.9 or between 4.5 and 4.7 or a pH of 4.6 or 4.7. Even more particularly, yeasts are provided that actively ferment in the presence of 8 g/l and pH 4.7 or in the presence of 10 g/l acetic acid and pH 4.7 Said yeasts are actively fermenting at a rate of at least 90%, at least 92% or at least 95% of the rate of fermentation in the absence of acetic acid. "Actively fermenting" as used herein refers to a situation wherein the yeast are metabolically active, more particularly producing metabolites such as carbon dioxide and ethanol.

In other particular embodiment, said yeast strains are devoid of or deficient in a functional Snf4 protein or functional Snf4 protein production. In most particular embodiments, said yeast strains comprise any of the chimeric gene constructs disclosed herein or a homozygous or hemizygous SNF4 mutation, the SNF4 mutation leading to a loss-of-Snf4 function.

In another embodiment, the yeast strains as disclosed herein are industrial yeast strains engineered for increased acetic acid tolerance. Besides the disrupted or deleted Snf4 function said yeasts can comprise additional genetic modifications that independent of Snf4 result in increased tolerance to acetic acid. More particularly said modifications are for example those as described in WO2015/181169 and WO2016/083397, even more particularly modifications that introduces an allele of one or more of the GLOl, DOT5, CUP2 and HAA1 genes that confers increased tolerance to acetic acid.

In another aspect the use of any of the mutant SNF4 alleles or proteins herein described or of any of the chimeric gene constructs herein described is provided to increase acetic acid tolerance in a culture of yeast cells. Said use is also provided to increase the production of ethanol in a culture of yeast cells.

In yet another aspect, the use of any of the yeast strains or industrial yeast strains herein described is provided to increase the production of ethanol.

"Increasing" or "increase" as used herein means an at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90, at least 95% or 100% increase or a 2-fold, 3-fold, 5-fold or 10-fold increase in the tolerance of acetic acid or in the production of ethanol compared to the same situation where the mutant SNF4 allele or protein or the chimeric gene constructs of the application are not present, or alternatively phrased compared to a situation wherein the yeasts have at least one functional SNF4 allele.

The envisaged increase in acetic acid tolerance or production of ethanol can also be between 10% and 50%, between 20% and 60%, between 30% and 70%, between 40% and 80% or between 60% and 150% more compared to a situation wherein the yeasts comprise at least one functional Snf4 protein.

In yet another aspect, a method of producing ethanol is provided comprising the step of fermenting a medium with one of the yeast strains herein disclosed. In one embodiment, said medium comprises first- generation and/or second-generation carbon substrates. "First generation substrates" refer to carbon sources like starch, sugar, animal fats and vegetable oil. "Second generation substrates" refer to lignocellulosic biomass for example the residual non-food parts of current crops, such as stems, leaves and husks that are left behind once the food crop has been extracted, as well as other crops that are not used for food purposes, such as switch grass, cereals that bear little grain and more fibre, and also industry waste such as wood chips, skins and pulp from fruit pressing etc. Fermentation of the first- and second-generation substrates by the yeasts of current application leads respectively to first- and second generation bioethanol. Therefore, in yet another aspect a fermented solution, more particularly bio ethanol, is provided comprising any of the yeast described in current application.

It is to be understood that although particular embodiments, specific configurations as well as materials and/or molecules, have been discussed herein for cells and methods according to the present invention, various changes or modifications in form and detail may be made without departing from the scope and spirit of this invention. The following examples are provided to better illustrate particular embodiments, and they should not be considered limiting the application. The application is limited only by the claims. Examples

Example 1. Isolation of whole-genome transformants

We have identified in our Saccharomyces cerevisiae strain collection a sake strain, Kll, with high acetic acid tolerance during fermentation, in spite of the fact that it comprises the inferior alleles of all five genes previously identified as causative elements for high acetic acid tolerance in strain JT22689 (PYCC 4542) (Meijnen et al 2016 Biotechnol Biofuels 9). Hence, Kll should contain novel genetic elements determining high acetic acid tolerance. We decided to use whole-genome transformation to identify novel causative alleles in strain Kll. As host strain, we used strain ER18, a haploid derivative of the industrial strain Ethanol Red, widely used for first-generation bioethanol production and displaying relatively low acetic acid tolerance. After LiAc/SS-DNA/PEG transformation of the recipient strain ER18 with gDNA of Kll and selection on YPD plates with different levels of acetic acid from 5g/l to 8g/l and pH 4.7, about 60 independent transformants were obtained. They were tested for acetic acid tolerance in fermentations and showed a wide range of acetic acid tolerance. The most tolerant transformant, MS164, was selected for further analysis. Strain MS164 showed clear improvement in growth on solid YPD medium supplemented with 0.9% acetic acid at pH 4.7, whereas ER18 only grew up to 0.6% acetic acid. Furthermore, MS164 also displayed better performance compared to ER18 in small-scale fermentations in the presence of different concentrations of acetic acid. Its performance was comparable to that of strain Kll from which the gDNA was derived (Fig. 1).

Example 2. Whole-genome sequence analysis of SNPs in transformant MS164 Transformant MS164 was submitted to whole-genome sequence analysis to identify the genetic changes introduced by transformation of the host strain ER18. Unexpectedly, MS164 and ER18 only differed in 12 single nucleotide polymorphisms (SNPs), of which five were synonymous while the other seven were non-synonymous mutations (Table 1). Surprisingly, none of the seven non-synonymous SNPs was present in the genome of donor strain Kll. Also none of these SNPs was present in 1011 whole-genome sequenced S. cerevisiae strains (Peter et al 2018 Nature 556), except for SNP3, which was present in 7 strains (Table 1).

Table 1. The seven non-synonymous and intergenic SNPs introduced during whole genome transformation in the ER18 host strain. Chr, chromosome; ER18, ER18 parent; MS164, MS164 transformant; Kll, Kll gDNA donor

Example 3. Identification of the causative SNP by allele exchange

We assessed the relevance of all seven non-synonymous SNPs individually for high acetic acid tolerance by allele exchange between the ER18 and MS164 strains using the CRISPR/Cas9 methodology. The nucleotide from the inferior ER18 host strain was exchanged into the corresponding position in the MS164 strain to assess whether it downgraded acetic acid tolerance and the nucleotide from the superior transformant MS164 was exchanged into the inferior ER18 strain to assess whether it upgraded acetic acid tolerance. The resulting strains were evaluated for fermentation performance in YPD medium containing lOg/l acetic acid with at least three biological replicates. The results for SNP2 and SNP5 are shown in Fig. 2. Probably due to the harsh conditions caused by the high acetic acid levels, we observed large differences in the length of the lag phase between technical as well as biological replicates. Hence, all replicates are shown as individual fermentations rather than an average from different replicates with standard deviation.

For an SNP to be considered causative for high acetic acid tolerance, all replicates of the upgraded ER18 strain should ferment better than the inferior parent ER18, while all the replicates of the downgraded MS164 strain should ferment worse than the superior parent MS164. After assessing all 7 SNPs individually, we can conclude that only SNP1 and SNP5 had a major effect on fermentation performance in the presence of acetic acid (Fig. 2). To distinguish whether the effect of the SNP was truly due to improvement of acetic acid tolerance or whether it affected fermentation capacity itself, we tested the strains also under the same conditions in YPD medium in the absence of acetic acid (Fig. 3). The results showed that SNP1 exchange had no effect in the absence of acetic acid. On the other hand, the downgrading of MS164 for SNP5 caused a reduction in the fermentation rate in the absence of acetic acid. The upgrading of ER18 for SNP5 did not have a significant effect on the fermentation rate. Upgrading SNP5 in other genetic backgrounds gave variable effects on fermentation performance in the presence of acetic acid, ranging from worsening, little or no significant effect, to improvement (data not shown). For instance, upgrading the two copies of YJR120W in the diploidized ER18 strain did not improve performance. Because of the apparent side-effect on the growth rate of MS164 with the engineered SNP5 and the variable effects of SNP5 upgrading in other genetic backgrounds, we did not explore SNP5 further for improvement of acetic acid tolerance. SNP1 is located in theSA/F40RF at position 805 for which the host strain ER18, as well as the gDNA donor strain Kll contain guanine (G) while the transformant MS164 has thymine (T). The modification from G to T leads to a change from glutamic acid at position 269 of the encoded protein into a stop codon resulting in a truncated SNF4 gene product. The wild type Snf4 protein (SEQ ID No. 4) contains 323 amino acids and the early stop codon results in a 54 amino acids shorter protein.

Example 4. Comparison of snf4^E269* and snf4A for acetic acid tolerance

We also compared the effect of the snf4^E2m* nonsense mutation with that of snf4A (knock-out) on fermentation performance of ER18 in the presence of acetic acid (Fig. 4). The ER18 strain with the SNF4^e269* mutation showed the same improvement of fermentation performance in the presence of acetic acid as the snf4A strain, suggesting that the truncated Snf4* protein is inactive. To evaluate the effect of SNF4 modification in another industrial strain genetic background, we introduced SNF4^E2m* or snf4A each in two copies in the diploid Brazilian bioethanol strain PE2. In both cases we observed a strong improvement of the fermentation rate in the presence of acetic acid, indicating that inactivation of Snf4 also improves acetic acid tolerance in other genetic backgrounds (Fig. 4). Single deletion of SNF4 had no effect indicating that the mutation is recessive.

Next, we have evaluated whether the snf4^E2m* and snf4A genetic modifications affected other properties of the ER18 strain in the presence of acetic acid: glucose consumption and ethanol production during semi-anaerobic fermentations and during aerobic growth in shake flasks. YP medium with 40g/l glucose, lOg/l acetic acid at pH 4.7 and 30°C was used. Samples were taken every few hours, both from fermentation tubes and growth flasks and analysed by H PLC. The snf4^E2m* and snf4A engineered ER18 strains showed much faster glucose consumption and ethanol production both during semi-anaerobic fermentation and during aerobic growth (Fig. 5). We also tested whether the strains consumed the added acetic acid during semi-anaerobic fermentation and aerobic growth. Acetic acid was only consumed during aerobic growth and not during semi-anaerobic fermentation. The snf4^E2m* and snf4A engineered ER18 strains initially showed faster acetic acid consumption than the ER18 strain possibly due to their higher acetic acid tolerance, whereas later the difference with the control strain disappeared when the acetic acid had dropped to lower levels (Fig. 5). The results confirmed superior and similar performance of the snf4^E2m* and snf4A engineered ER18 strains compared to the control ER18 strain.

Example 5. Effect of snf4^E269* and snf4A on growth in different conditions

Snf4 is an activating subunit of the Snfl protein kinase ('sucrose nonfermenting') which is essential for sucrose utilization. Flowever, mutations in SNF1 were found to have pleiotropic effects also on utilization of other sugars like galactose, raffinose and maltose (Carlson et al 1981 Genetics 98). For the SNF4-319 mutant pleiotropic defects are reported on the utilization of carbon sources controlled by glucose repression. The SNF4-139 mutant was also compromised in producing secreted invertase (Neigeborn and Carlson 1984 Genetics 108). To test for additional effects of the snf4^E269* and snf4A mutations, we measured growth on different carbon sources of the snf4^E269* and snf4A (in all SNF4 alleles present) strains in the ER18, PE2 and MS488 genetic backgrounds (Fig. 6). The utilization of glucose was not significantly affected by snf4^E2m* or snf4A in the three genetic backgrounds ER18, PE2 and MS488. On the other hand, growth on sucrose and maltose was delayed to variable extents in the three backgrounds (Fig. 6). This appears to be consistent with the role of Snf4 in glucose derepression.

Previous work has shown that the requirement for Snf4 is less stringent at lower temperature (23°C, 30°C) than at higher temperature (37°C), possibly due to lower levels of Snfl protein. While in the wild type strain the level of Snfl protein was constant at 23°C, 30°C and 37°C, snf4A mutants showed similar levels at 23°C and 30°C, yet strongly reduced levels at 37°C (Celenza et al 1989 Mol Cell Biol 9). Flence, we evaluated growth of the snf4^E2m* and snf4A strains in the three genetic backgrounds ER18, PE2 and MS488 with glucose, sucrose and maltose as carbon sources. Flowever, there was no significant difference for growth rate on any of the three carbon sources and in any of the three genetic backgrounds between the two temperatures (data not shown).

We also tested whether the snf4^E2m* and snf4A modifications affect the proliferation rate of the industrial strain MS488 in molasses medium using four different concentrations of sugar cane molasses. In all cases there was a delay in the second phase of growth and a slightly lower final OD (Fig. 7).

Example 6. Cumulative effect of snf4^E269* or snf4A and HAA1^S506N on acetic acid tolerance

Finally, we tested whether the snf4^E2m* or snf4A modifications were able to further enhance acetic acid tolerance in an industrial yeast strain engineered with the superior HAA1^S506N allele, which in itself already causes a strong increase in acetic acid tolerance (Meijnen et al 2016 Biotechnol Biofuels 9). Surprisingly the fermentation performance of yeast strains comprising the superior HAA1 allele could even further be increased in the absence of Snf4 or in the homozygous presence of the Snf4E269* mutation (Fig. 8).

Materials and methods

Yeast strains and media

The following Saccharomyces cerevisiae strains are used and constructed in this work: Ethanol Red (ER) (first generation bioethanol production strain; source: Fermentis), ER18 (segregant of ER; Meijnen et al 2016 Biotechnol Biofuels 9), Kll (sake production strain; lab strain collection), JT28541 (molasses bioethanol production strain; lab strain collection), PE2 (Brazilian bioethanol production strain; lab strain collection), MS488 (JT28541 HAA1^S506N; Meijnen et al 2016 Biotechnol Biofuels 9), MS164 (ER18), MS 488 (JT28541 HAA1*), ER18-, MS164-, PE- and MS488-derivatives are shown in Table 2. In most experiments YP medium (10 g/l yeast extract, 20 g/l bacteriological peptone) was used, supplemented with different concentrations of glucose. In addition, media with different concentrations of molasses were used, as indicated. Yeast propagation was done in YP with 20 g/l glucose, while fermentation and growth assays were done in YP supplemented with 40 g/l glucose. Propagation was performed in a shaking incubator at 30°C with constant shaking of 200 rpm. For selection of transformants, solid YP medium was used, containing 20g/l glucose and 15 g/l bacto agar and supplemented with different concentrations of acetic acid without or with pH correction to 4.7 using 4M KOH. The pH was corrected to 4.7, which is just below the pKa of acetic acid (4.76) to assure stringent conditions.

As inferior host strain for WGT, we used ER18, a haploid segregant derived from Ethanol Red, an industrial yeast strain used in commercial first-generation bioethanol production. The strain has a high robustness and fermentation capacity, yet relatively low tolerance to acetic acid. As a gDNA donor, we have used Kll, a sake strain with high acetic acid tolerance. To evaluate the beneficial effect of the newly discovered genetic modifications in different strain backgrounds, we have used PE2, an industrial strain used in Brazil for bioethanol production with sugar cane and MS488, an industrial strain developed for bioethanol production with molasses.

Screening of the yeast strain collection

To identify the strain most suitable as donor of the gDNA, we screened all s cerevisiae strains with known genome sequence for high acetic acid tolerance and selected from the strains with the highest tolerance the Kll sake strain. This strain lacked all the superior alleles for high acetic acid tolerance we identified in our previous study (Meijen et al 2016 Biotechnol Biofuels 9) and thus was expected to contain novel causative alleles for high acetic acid tolerance. The screening was done on solid YPD medium supplemented with different concentrations of acetic acid ranging from 4 g/l to 6.5 g/l, without pH correction. The Kll strain was able to grow up to 6 g/l acetic acid (without pH correction).

Genomic DNA extraction and whole genome transformation

High-quality gDNA was extracted using the MasterPure™ Yeast DNA purification kit from Epicentre according to the manufacturer's instructions. The gDNA fragments obtained were not cut further into smaller pieces for whole-genome transformation. An overnight culture of the host strain ER18 was transformed with 2 pg of donor gDNA using the LiAc/SS-DNA/PEG method (Gietz and Schiestl 2007 Nat Protoc 2; Gietz et al 1995 Yeast 11). The transformed culture was plated on solid YPD medium containing a range of acetic acid concentrations from 5 g/l to 8 g/l, with pH corrected to 4.7 using 4M KOH, and grown for two days at 30°C. All colonies obtained on the medium with the highest concentration of acetic acid (8 g/l) were plated again in serial dilutions on YPD solid medium with 8 g/l to 10 g/l acetic acid, pH 4.7, and grown overnight at 30°C, to select the most acetic acid tolerant strain, which was named MS164.

Whole genome sequence analysis and identification ofSNPs

The gDNA was extracted and sent to the Beijing Genomics Institute (BGI, Hong Kong) for whole-genome sequence analysis with the lllumina (HiSeq2500) platform. The libraries contained 500bp inserts and sequencing was done with pair-end reads of 125bp. The genomic sequence of the transformant MS164 was compared with that of the host strain ER18 to identify all genetic changes introduced by whole genome transformation. The gDNA of MS164 and ER18 was extracted with the MasterPure Yeast DNA Purification Kit (Epicentre) and sent to the Beijing Genomics Institute (BGI, Hong Kong) for whole- genome sequence analysis. A library of 125 pair-end reads with an average insert length of 500bp was generated with the lllumina HiSeq2500 platform. Reads of strain Kll were obtained from NCBI (SRR1568238). All reads were mapped against the S288C reference genome (version R64-2-1; SGD) with bowtie2 using parameters -I 0 -X 600 -a -t. Variant detection was performed using NGSEP (Duitama et al 2014 Nucleic Acids Res 42) using parameters -maxBaseQS 30 -minQuality 40 -maxAlnsPerStartPos 2. Repetitive regions were masked using Tandem Repeats Finder (Benson 1999 Nucleic Acids Res 27). The final VCF file was filtered with parameter -q 40 and annotated. Custom in-house scripts were used to extract variants introduced in MS164 that did not occur in ER18. These variants were then compared to the corresponding sequence in Kll to determine whether these variants could have been derived from Kll or were novel mutations introduced during the transformation of ER18 with the gDNA from Kll. Non-mapped reads were collected and de novo assembled using CLC Genomics Workbench (QIAGEN Bioinformatics) with default parameters. These contigs were used as a reference and a similar mapping strategy was followed after which the variants of MS164 and ER18 were compared again. All contigs of MS164 were blasted against the Kll genome to identify possible large-scale insertions. Additionally, copy number variations were analysed with in-house scripts to identify possible duplications/deletions or large-scale genome rearrangements.

CRiSPR/Cas9 technology

We have used clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology (Bao et al 2015 ACS Synth Biol 4; DiCarlo et al 2013 Nucleic Acids Research 41; Horwith et al 2015 Cell Syst 1; Mans et al 2015 FEMS Yeast Res 15) to transfer SNP1, into strains with different genetic backgrounds using the following steps:

(i) Cas9 plasmid transformation was done with the P51 (pTEF-Cas9-KANMX) single copy plasmid derived from the p414-TEFlp-Cas9-CYClt plasmid (DiCarlo et al). Transformants were selected on YPD plates containing geneticin. Transformation was done with 1 pg of Cas9 plasmid using the LiAc/SS-DNA/PEG method.

(ii) For gRNA plasmid transformation, a specific gRNA targeting SNP1 was designed. gRNA was flanked by GCAGTGAAAGATAAATGATC (promoter) and GTTTTAGAGCTAGAAATAG (terminator) and without a protospacer adjacent motif (PAM) site. Both, forward and reverse oligomers were ordered, duplexed and assembled by Gibson Assembly kit in the Xhol-EcoRV-digested P58 vector. P58 contains the HPH marker in the p426-SNR52p-gRNA.CANl.Y-SUP4t backbone (DiCarlo et al). Forward and reverse oligomers at 500 mM dissolved in STE buffer (10 m M Tris pH 8.0, 50 mM NaCI, I mM EDTA) were duplexed by incubating equimolar concentrations of the primers for 3 min at 94°C and slowly cooling down by turning off the heat block.

(iii) For the SNP1 replacement we used 59-bp oligo donor DNA containing the desired SNP1: forward ( AG AAGCCCTT ATG AGG AG AAGT GAT G ATTTTT AAG AT GTTT AT AC ATGCACTAAG AAT G ) and reverse

( C ATT CTT AGTG C ATGTAT AA AC AT CTT AA AA AT CAT C ACTT CT CCTC AT A AG G G CTT CT) oligos were duplexed using the same protocol as described for gRNAs.

A successful Cas9 transformant was afterwards transformed with 1 pg of gRNA plasmid and 2 pg of donor DNA using LiAc/SS-DNA/PEG method. Transformation mix was plated on solid YP medium with 20 g/l glucose supplemented with 200 pg/ml geneticin and 300 pg/ml hygromycin B. The gRNA plasmids for application of the CRISPR-Cas9 technology were amplified in Escherichia coli cells grown overnight at 37°C on solid Luria broth (LB) medium with 15 g/l bacto agar and the respective antibiotic.

Targeted gene deletion

Depending on the ploidy of the strain, the SNF4 gene was deleted in one or two copies. Deletion cassettes were amplified using the plasmids with NATMX and KANMX antibiotic markers with 60bp flanking regions for homologous recombination. The amplification was done by PCR using Q5 enzyme. Two different PCRs were performed to verify whether the SNF4 gene deletion was correct, the first PCR comprised primers binding inside the antibiotic marker and outside the gene, while the second PCR comprised primers inside and outside the gene. If we got a positive result for the first and a negative for the second PCR reaction, we concluded that deletion of the gene was correct.

Allele-specific PCR and Sanger sequencing

To verify correct SNP1 replacement we first performed allele-specific PCR. The forward primer differed in a desired SNP at the 3' end, one being specific for ER18 (ATGAGGAGAAGTGATGATTATG) and the other for MS164 SNP (ATGAGGAGAAGTGATGATTATT). Both primers contained an extra mismatch at the third nucleotide position from the 3' end to increase specificity. The reverse primer (GCCTGTACCTTTTTGATG) was common for both PCR reactions and was designed to be at a distance of about 500bp. Depending on whether SNP1 was present, only one of the two PCRs gives a positive result. When a transformant with the correct mutation was identified, it was grown with several transfers in liquid YPD in order to lose the Cas9 and gRNA plasmids. Single cells were picked using a micromanipulator and the strains sent for final verification by Sanger sequencing.

Small-scale fermentations

Whole-genome transformants, SNP1 replacement strains and gene deletion strains were evaluated for acetic acid tolerance under semi-anaerobic fermentation conditions. The medium used was YP containing 40 g/l glucose and various concentrations of acetic acid, with initial pH adjusted to 4.7 using 4M KOH. Yeast cells used were pre-grown in YP with 20 g/l glucose for 48h at 30°C, until stationary phase. After measuring the optical density (OD) of each culture, the correct volume needed was calculated and the cells harvested by centrifugation at 3000 rpm for 5 min at 4°C. Starting OD of the fermentations was 2, corresponding to approximately 0.5 g/l cell density. Fermentations were performed in 50 mL volume at 35°C with continuous stirring at 120 rpm. Fermentation performance was assessed by measuring the weight loss which corresponds to CO2 release. Due to the differences in strain sensitivity the ER18 and MS488 strains were tested at 10 g/l acetic acid, while for PE2 we used 11 g/l acetic acid, both at pH 4.7. Under our conditions (50ml, 40 g/l glucose) the maximum weight loss was lg.

Growth assays

Growth assays of the strains were done in shaking flasks under aerobic conditions. The medium used was YP containing 40 g/l glucose and 10 g/l acetic acid, with pH adjusted to 4.7 using 4M KOH. Growth tests were done with starting OD 2 in flasks containing 50 ml of the culture, in a shaking incubator at 30°C and constant stirring of 200 rpm. To evaluate the utilization of different carbon sources, we performed screening using a Multiskan FC microplate photometer (Thermofisher), with starting OD 0.1 in 200 pL YP medium containing 40 g/l glucose, maltose or sucrose. The growth was done at 30°C and 37°C with pulsed stirring of 1 min every 15 min. Additionally, we tested the aerobic growth in flasks containing 25 ml of the culture in 100 g/l and 200 g/l molasses with the starting OD 0.5, in shaking incubator at 30°C and constant stirring of 200 rpm.

High performance liquid chromatography (HPLC)

During the growth assays in shaking flasks, as well as during the fermentations in YP with 40 g/l glucose, samples were taken every few hours to assess glucose utilization, ethanol production and the level of acetic acid. Samples were analysed using a Shimadzu Nexera X2 HPLC system. 5mM H2SO4 was used as mobile phase with a flow rate of 0.7 mL/min over an Agilent MetaCarb 87H column (300 x 7.8 mm) at 70°C. The compounds were detected using refractive index detection and analysed using LabSolutions software (version 5.86, Shimadzu Corporation). Table 2. Overview of ER18-, MS164-, MS488- and PE2-derivatives constructed herein.

Claims

Claims (clean version)

1. A mutant SNF4 yeast allele comprising a nonsense mutation on nucleic acid position 805.

2. A chimeric gene construct comprising a promoter which is active in a eukaryotic cell operably linked to a CRISPR guide RNA targeting the SNF4 allele.

3. A yeast strain comprising a homozygous or hemizygous mutant SNF4 allele, said allele being the SNF4 allele of claim 1 or comprising the chimeric gene construct of claim 2.

4. An industrial yeast strain in which the SNF4 allele has been disrupted or deleted.

5. The yeast strain according to claim 4 comprising the chimeric gene construct of claim 2.

6. The yeast according to any of claims 3-5 actively fermenting in the presence of at least 8 g/l acetic acid.

7. The yeast according to any of claims 3-5 actively fermenting in the presence of 10 g/l acetic acid and pH 4.7 at a rate of at least 90% of the fermentation rate of the same yeast in the absence of acetic acid.

8. The yeast strain according to any of claims 3-7 further comprising an allele of one or more of the GLOl, DOT5, CUP2 and HAA1 genes that confers increased tolerance to acetic acid as described in W02015/181169 and WO2016/083397.

9. The yeast strain according to any of claims 3-8, wherein the yeast strain is a xylose fermenting yeast strain.

10.A fermented solution comprising the yeast according to any of claims 3-9.

11. Use of the mutant SNF4 allele of claim 1 or the chimeric gene construct of claim 2 to increase acetic acid tolerance in a culture of yeast cells.

12. Use of the yeast strain of any of claims 3-9 to increase the production of ethanol.

13. A method of producing ethanol comprising the step of fermenting a medium with a yeast strain according to any of claims 3-9.