WO2017129607A1

WO2017129607A1 - Improvement of salt tolerance in yeast

Info

Publication number: WO2017129607A1
Application number: PCT/EP2017/051521
Authority: WO
Inventors: Johan Thevelein; Maria Remedios FOULQUIÉ MORENO; Yingying Li
Original assignee: Vib Vzw; Katholieke Universiteit Leuven, K.U.Leuven R&D
Priority date: 2016-01-26
Filing date: 2017-01-25
Publication date: 2017-08-03

Abstract

The present invention provides a chimeric gene useful to develop yeast strains for industrial fermentation at salt level conditions that reduce the fermentation efficiency. The invention also provides recombinant yeast strains for fermentation of second-generation substrates including cellulosic or lignocellulosic hydrolysates or of feedstocks with high salt levels. In addition, the invention relates to the use of such yeast strains for the production of second-generation biofuels.

Description

IMPROVEMENT OF SALT TOLERANCE IN YEAST

Field of the invention

The present invention relates to the field of abiotic stress tolerance, more particularly to salt tolerance. Even more particularly the present infection relates to a Zygosaccharomyces rouxii gene encoding a malate dehydrogenase for the generation of eukaryotic cells with increased salt tolerance. The present invention provides chimeric genes useful to develop yeast strains for industrial fermentation at salt level conditions that reduce the fermentation efficiency. The invention also provides recombinant yeast strains for fermentation of second-generation substrates including cellulosic or lignocellulosic hydrolysates or of feedstocks with high salt levels. In addition, the invention also relates to the use of such yeast strains for the production of second-generation biofuels.

Background

Because of the conflict between increasing energy consumption and environmental concerns due to climate change, bioethanol has become a major alternative transport fuel for petroleum-based fossil fuels in the short-term [1]. Bioethanol has been produced on a large scale from foodstuffs, like sugar cane, corn and wheat, so-called first-generation substrates, which has led to a food versus fuel discussion. Although the latter remains highly contentious, it has stimulated interest in the production of bioethanol with non-foodstuffs, especially lignocellulosic waste streams (cellulosic biofuels). These materials are available in prolific amounts, are often an environmental nuisance and expensive to dispose of. The first commercial-scale second-generation bioethanol production units have now been constructed, yet the industrial process still needs plenty of optimization. One of the major challenges is the development of an industrial microorganism able to produce bioethanol by anaerobic fermentation with high efficiency in concentrated, undetoxified lignocellulose hydrolysates. The composition of the latter is highly variable depending on the feedstock and usually contains large amounts of inhibitors derived either from the feedstock itself or generated during the feedstock pretreatment process [2-4]. It would thus be advantageous to develop microorganisms, including yeast, with an increased tolerance to the high levels of various fermentation inhibitors present in the feedstocks or generated during the pre-treatment. Salts for instance are present in all fermentation media but are toxic to wild-type strains of Saccharomyces cerevisiae, leading to inhibition of cell growth, glucose consumption and ethanol production [5-6].

Because of its inherent robustness, its high ethanol tolerance and the availability of potent first- generation industrial bioethanol strains, the yeast Saccharomyces cerevisiae has been a preferred microorganism for production of second-generation bioethanol. Multiple studies have been devoted to further increasing its tolerance to fermentation inhibitors commonly present in the hydrolysates, like acetic acid, furfural, HMF and phenolic compounds [7-12]. However, tolerance to salts present in the hydrolysates has received little attention up to now. Salts that are present in bioethanol fermentations may originate from different sources, either from the biomass itself, from chemicals added during pretreatment, detoxification and/or neutralization [11, 13- 15], from the recirculation of processing water [5, 16], also possibly from the cleaning and corrosion of the process equipment and from stabilization of the pH during the fermentation process. In addition, certain process configurations, such as combined fermentation of lignocellulosic hydrolysates and molasses, may also result in elevated salt levels. Sodium hydroxide (NaOH), slaked lime (Ca(OH)2), ammonium hydroxide (NH₄OH) and other chemicals can be used to remove lignin and part of the hemicellulose, which significantly increases the enzymatic hydrolysis efficiency but also increases the salt content [17-19]. Marine algae as an alternative substrate for biofuel production have recently become a focus of intensive research, with one of the challenges being the high salt content or salt level of the feedstock [19-21]. Moreover, utilising seawater, instead of fresh water, as a water source for succinic acid fermentation has been successfully accomplished [22], which indicates the potential of seawater utilization instead of scarce freshwater resources also for bioethanol production. This will obviously increase the salt concentration in the fermentation process. In seaweed hydrolysate, a salt concentration as high as 11.25 % was present [23]. Furthermore, sodium chloride (NaCI) has been used for the pretreatment of seaweed, resulting in increased efficiency of the enzymatic saccharification [19]. However, the accumulation of salt placed an additional burden on the fermentation process, while desalting requires too much energy to be economically feasible in industrial practice [23]. Because of these reasons it would be advantageous to develop efficient approaches to enhance salt tolerance in industrial yeast strains used for second-generation bioethanol production. An additional advantage of using high-salt-tolerant industrial yeast in a fermentation medium with high salinity is that it may significantly reduce the risk of bacterial contamination, a persistent problem in all non-sterile, large-scale industrial fermentations [24].

High salts are toxic to most living cells because of specific ion toxicity and osmotic stress. Both cations and anions, as present in salts, have significant inhibitory effect on microbial conversion of various sugar substrates to ethanol. For S. cerevisiae the inhibition increased in the following order: KCI < MgS0₄ < MgCI₂ < KH2PO4 < NaCI, N H4CI < CaCI₂, (NH₄)2S0₄ [5]. Six pairs of anions (CI^" and S0₄ ^2") and cations (Na⁺, K⁺, and NH ⁺) all displayed an inhibitory effect on glucose and xylose co-fermentation by a genetically engineered strain of S. cerevisiae, with xylose fermentation being most sensitive [25]. Furthermore, CI^" was more inhibitory than S0₄ ^2~, which is in agreement with results of previous research on the effect of salts on bioethanol production with Zymomonas mobilis [26].

Many efforts have been devoted to the search of genes responsible for salt tolerance with the expectation that they might be useful for development of industrial yeast strains with improved salt tolerance. The transport systems relevant to salt tolerance have been studied in great detail in S. cerevisiae and also in other yeast species, especially those with very high halotolerance [27-29]. More recently, two genes, MDJ1 and VPS74, were identified as being required for growth under salt stress in two S. cerevisiae strains tolerant to 10 % (w/v) NaCI and isolated by screening a transposon-mediated mutant library [30]. Improvement of salt tolerance in yeast by overexpression of specific genes has been achieved, such as with S. cerevisiae HAL2 [31], a plant Arabidopsis thaliana gene AtNHXl [32] and the Zygosaccharomyces rouxii gene ZrSOD2 [33]. Z. rouxii is a halotolerant and osmotolerant yeast species phylogenetically closely related to S. cerevisiae [34]. Z. rouxii has been isolated from a variety of salty environments and can grow in the presence of up to 18% NaCI [35]. Z. rouxii thus represents a promising potential source of salt tolerance conferring genes. Though some genes important for salt tolerance have already been isolated from Z. rouxii and functionally expressed in S. cerevisiae, this was first done in osmosensitive mutant strains defective in the S. cerevisiae homolog and/or other genes, and did not lead to substantial improvement in S. cerevisiae general salt tolerance [36-37]. The ZrSOD2 (encoding the plasma membrane Na⁺/H⁺-antiporter) and ZrPMAl (encoding the plasma membrane H⁺-ATPase) genes are important for the high salt tolerance of Z. rouxii. Single expression of ZrSOD2 was effective in conferring salt-tolerance up to 1.5 M and although a slight synergic effect was observed with co- expression of ZrSOD2 and ZrPMAl, the usefulness of this co-expression is likely to be minimal with regard to salt-tolerance [33]. Also a hybrid was obtained by protoplast fusion of Z. rouxii TIST 1750 and S. cerevisiae M30, which showed high ethanol production efficiency in a medium containing 3% NaCI [38].

In all the above described cases, however, the improvement in salt tolerance was limited or the research was performed with laboratory yeast strains. This raises the question of this approach's applicability to industrial yeast strains, especially the pentose-utilizing strains developed for second-generation bioethanol production. Indeed, improvement of salt tolerance in yeast for fermentation of lignocellulosic hydrolysates to ethanol has not been reported yet.

Summary

It is an object of the invention to identify heterologous genes from the halophilic yeast Z. rouxii that could improve salt tolerance in S. cerevisiae for fermentation of lignocellulosic hydrolysates into ethanol. A Z. rouxii cDNA library was transformed in the industrial yeast strain GSE16 [10, 39] (Example 1). Three transformants with higher salt tolerance were selected (Example 2). This led to identification of three Z. rouxii genes that improved salt tolerance in the industrial yeast strain GSE16 (Example 3 and 4). Furthermore, the transformant with the most potent Z. rouxii gene, a homolog of S. cerevisiae MDH3, also displayed improved fermentation performance in whole corn cob hydrolysates, both for glucose and xylose (Example 5). Surprisingly, the improved fermentation performance was observed both in the absence and presence of added NaCI.

Brief description of the Figures

Figure 1. Evaluation of salt tolerance in transformants and control strains.

(A) Assessment of salt tolerance in the strains S288c, E and GSE16 by spot assay. The media used were solid SCD without and with addition of 1.6, 1.8 and 2.0 M NaCI. (B) Assessment of salt tolerance by growth in liquid medium in 30 GSE16 transformants obtained with the Z. rouxii cDNA library. The media used were liquid SCD with addition of 1.8 or 2.0 M NaCI. (C) Evaluation of salt tolerance by spot assay in the 16 most promising GSE16 transformants with expression of Z. rouxii cDNA. The media used were solid SCD without and with addition of 1.8, 2.0 or 2.2 M NaCI. All experiments were performed in duplicate/triplicate with independent cultures.

Figure 2. Evaluation of salt tolerance in the most promising transformants.

(A) Fermentation performance of 16 candidate salt tolerant transformants in YPD5% medium supplemented with 1.7 M NaCI in comparison with parent strain GSE16. (B) Salt tolerance of the three most promising original transformants and the corresponding plasmid-loss strains as determined by spot assay. The media used were solid SCD without and with addition of 1.8 or 1.9 M NaCI. The parent strain GSE16 and lab strain S288c were included as controls. Each experiment was performed in duplicate with independent cultures. Figure 3. Evaluation of salt tolerance in liquid medium for the three most promising transformants after plasmid retransformation.

Growth performance of the three most promising transformants after plasmid retransformation and the corresponding plasmid-loss strains in liquid SCD with 1.9 or 2.0 M NaCI. Each experiment was performed in triplicate with independent cultures.

Figure 4. Evaluation of salt tolerance upon overexpression or co-expression of the most potent Z. rouxii genes. Growth performance of the strains overexpressing or co-expressing the three Z. rouxii inserts on solid SCD medium without or with addition of 1.6 M NaCI. Each experiment was performed in duplicate with independent cultures. Figure 5. Assessment of salt tolerance of cell proliferation in the strains GSE16-p26 and GSE16 in corn cob hydrolysate without and with addition of 0.6 M NaCI. The experiment was repeated twice with two biological duplicates for each strain. Error bars represent standard deviation from the average of the two biological duplicates. Figure 6. Fermentation performance of strains in whole corn cob hydrolysate without or with addition of 0.6 M NaCI.

Glucose, xylose and ethanol levels were determined as a function of time during fermentation with GSE16-p26 (filled markers), and the control strain GSE16 (open markers). The experiment was repeated twice with two biological replicates for each strain. Error bars represent standard deviation from the average of the two biological duplicates.

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 . 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.

Because of its natural capability of converting hexose sugars very efficiently to ethanol and because of its high ethanol tolerance, S. cerevisiae has been the most popular microorganism for industrial bioethanol production. However, one of the bottlenecks in the conversion of lignocellulosic materials to biofuels is the low tolerance of all microorganisms, including yeast, to the high levels of various fermentation inhibitors present in the feedstocks or generated during the pretreatment. Salts for instance are present in all fermentation media and are toxic to wild-type strains of S. cerevisiae, leading to inhibition of cell growth, glucose consumption and ethanol production [5, 25]. To overcome the inhibition, two main options are available. First, a detoxification process can be used to remove most of the inhibitors although this leads to additional expenses for the industrial process [15, 41]. Chemical detoxification by alkali treatment is one of the most effective approaches. It improves the efficiency of the subsequent enzymatic hydrolysis and ethanol fermentation by S. cerevisiae [11, 42-46]. Sodium chlorite, for instance, has been used for pretreatment of some types of biomass, such as Ceylon moss [19]. However, large amounts of salts are accumulated in the hydrolysates during the detoxification processes, which are also inhibitory for the fermentation process. The second option to overcome the inhibition of fermentation by the chemicals present or generated during the industrial process is to develop more inhibitor-tolerant yeast strains.

We have identified and isolated 3 genes (ZYRO0D06204g, ZYRO0G17556g and ZYRO0A08470g) from Zygosaccharomyces rouxii that when expressed in industrial S. cerevisiae strains are separately responsible for increased salt tolerance. Surprisingly, the gene with the strongest positive effect on salt tolerance (i.e. ZYRO0A08470g) is a homologue of S. cerevisiae malate dehydrogenase MDH3. Adjusting the expression levels or production levels of malate dehydrogenase in a eukaryotic organism to obtain or increase salt tolerance has never been shown or anticipated. Expression of the Z. rouxii ZYRO0A08470g gene in an industrial S. cerevisiae strain improved the fermentation efficiency in the presence of added salt. Surprisingly, expression of the Z. rouxii ZYRO0A08470g gene in an industrial S. cerevisiae strain improved glucose and xylose fermentation on second-generation substrate even in the absence of added salt.

In a first embodiment the invention provides a chimeric gene comprising a promoter which is active in a eukaryotic cell, a nucleic acid sequence encoding an amino acid sequence depicted in SEQ ID N° 2 and a 3' end region involved in transcription termination or polyadenylation. In a particular embodiment the nucleic acid sequence of the chimeric gene of the invention encodes an amino acid sequence with a sequence identity to SEQ ID N° 2 preferably of at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% over a range of at least 300 amino acids. Throughout this application, the said nucleic acid sequence will also be referred to as the nucleic acid sequence of the invention. The nucleic acid described is designated in the art as ZYRO0A08470g. The nucleic acid sequence of the invention originates from Zygosaccharomyces rouxii and is annotated as a malate dehydrogenase (MDH) gene, encoding for the enzyme malate dehydrogenase. The "sequence identity" of two related nucleotide or amino acid sequences, expressed as a percentage, refers to the number of positions in the two optimally aligned sequences which have identical residues (xlOO) divided by the number of positions compared. A gap, i.e., a position in an alignment where a residue is present in one sequence but not in the other is regarded as a position with non-identical residues. The alignment of the two sequences is performed by the Needleman and Wunsch algorithm (Needleman and Wunsch (1970) J Mol Biol. 48: 443-453). The computer-assisted sequence alignment above, can be conveniently performed using standard software program such as GAP which is part of the Wisconsin Package Version 10.1 (Genetics Computer Group, Madison, Wisconsin, USA) using the default scoring matrix with a gap creation penalty of 50 and a gap extension penalty of 3. Sequences are indicated as "essentially similar" when such sequences have a sequence identity of at least about 75%, particularly at least about 80 %, more particularly at least about 85%, quite particularly about 90%, especially about 95%, more especially about 100%, quite especially are identical.

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; pHXTl (Hexose 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 pAD 2; 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; pSIP18 (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. Yeast, as used here, 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 the present invention 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 (here the nucleic acid sequence encoding an amino acid sequence with a sequence identity to SEQ ID N° 2 preferably of at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), 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 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 PC or RT-PCR (Heid et al., 1996 Genome Methods 6: 986-994).

A "chimeric gene" or "chimeric construct" 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.

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 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 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 CYCl genes or alternatively from another yeast gene or less preferably from any other eukaryotic or viral gene. For expression in plants the terminator to be added may be derived from, for example the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic or viral gene. For expression in mammalian cells the terminator to be added may be derived from, for example the human gastrin gene, or alternatively from another mammalian gene, or less preferably from any other eukaryotic or viral gene. In yet another embodiment the invention provides a vector comprising the chimeric gene of the invention. The said chimeric gene of the invention comprises a promoter which is active in a eukaryotic cell, a nucleic acid sequence encoding an amino acid sequence depicted in SEQ ID N° 2 and a 3' end region involved in transcription termination or polyadenylation. In a particular embodiment the nucleic acid sequence of the chimeric gene of the invention encodes an amino acid sequence with a sequence identity to SEQ ID N° 2 preferably of at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. The term "vector" refers to any linear or circular DNA construct containing the above described chimeric gene of the invention. The vector can refer to an expression cassette or any recombinant expression system for the purpose of expressing a nucleic acid sequence of the invention 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 invention is a "recombinant vector" which is by definition a man-made vector.

In yet another embodiment the invention, the chimeric gene of the invention comprising a nucleic acid sequence encoding an amino acid sequence with a sequence identity to SEQ ID N° 2 preferably of at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% is used for obtaining salt tolerance or increasing salt tolerance in a eukaryotic organism. Another embodiment is the use of the vector comprising the chimeric gene of the invention for obtaining salt tolerance or increasing salt tolerance in a eukaryotic organism. This is equivalent as saying that a method is provided to obtain salt tolerance in a eukaryotic organism, said method comprising expressing the chimeric gene of the invention or the vector of the invention in said eukaryotic organism. In a particular embodiment, said eukaryotic organism is a yeast, more particularly a yeast strain different from Zygosaccharomyces, even more particularly said yeast strain is a Saccharomyces yeast. In a preferred embodiment said chimeric gene of the invention or said vector comprising the chimeric gene of the invention is used to obtain salt tolerance or to increase salt tolerance in yeast, in plant cells or mammalian cells. "Obtaining salt tolerance" or "increasing salt tolerance" as used herein means that the yeast, plant cell or mammalian cell that comprises the nucleic acid sequence, chimeric gene or vector of the invention shows less of an effect, or no effect, compared to a corresponding reference yeast, plant cell or mammalian cell lacking the nucleic acid sequence, chimeric gene or vector of the invention in response to salt levels that have an inhibitory effect on the said reference yeast, plant cell or mammalian cell. This effect can be related to growth, proliferation or metabolic activity of the organism. Preferably, for yeast, increasing salt tolerance or obtaining salt tolerance is achieved when a yeast strain comprising the nucleic acid sequence, chimeric gene or the vector of the invention is still actively dividing or metabolically active in the fermentation process in contrast to the control strain lacking the nucleic acid, chimeric gene or vector of the invention. This effect can be convincingly measured by using the optical density or absorbance of a sample of the yeast culture at a wavelength of 600 nm also referred to in the art as OD600. More preferably, the OD600 of the salt tolerant yeast strain comprising the nucleic acid sequence of the invention would preferably at least be 20%, preferably at least be 30%, more preferably at least be 40%, more preferably at least be 50%, even more preferably at least be 60%, even more preferably at least be 70%, even more preferably at least be 80%, even more preferably at least be 90%, and most preferably at least be 100% higher compared to a control strain lacking the nucleic acid sequence of the invention at growth limiting salt levels for the said control strain.

The term "salt" is not restricted to common salt (NaCI), but may be any one or more of: NaCI, KCI, LiCI, MgC , CaC , amongst others.

In another embodiment the invention provides a recombinant yeast strain, comprising the chimeric gene of the invention thus comprising the nucleic acid sequence of the invention encoding an amino acid sequence with a sequence identity to SEQ ID N° 2 preferably of at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or comprising the vector of the invention thus comprising the chimeric gene of the invention. Preferably, said yeast is any yeast useful for industrial applications. More preferable, said yeast is useful for ethanol production, including, but not limited to Saccharomyces, Zygosaccharomyces, Brettanomyces and Kluyveromyces. Even more preferably, said yeast is a Saccharomyces sp., most preferably it is a Saccharomyces cerevisiae sp.

In yet another embodiment the invention provides a recombinant yeast strain different from Zygosaccharomyces rouxii comprising a nucleic acid sequence encoding an amino acid sequence depicted in SEQ ID N° 2 or that has a sequence identity to SEQ ID N° 2 preferably of at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. Preferably, said yeast is any yeast different from Zygosaccharomyces rouxii useful for industrial applications. More preferable, said yeast different from Zygosaccharomyces rouxii is useful for ethanol production. Even more preferably, said yeast is a Saccharomyces sp., most preferably it is a Saccharomyces cerevisiae sp. In yet another embodiment, the above described recombinant yeast strains according to the invention is used for industrial fermentation at salt level conditions that reduce the fermentation efficiency. Salt level conditions that reduce the fermentation efficiency can be defined as those salt levels of the yeast substrate that inhibit or at least negatively influence the growth, proliferation or metabolic activity of yeast cells with at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% compared to the growth, proliferation or metabolic activity of yeast cells on a substrate optimized for fermentation, preferably industrial fermentation. The production of metabolites as output of "metabolic activity" can be convincingly measured by high performance liquid chromatography (HPLC). In another embodiment, a method is provided to produce a recombinant yeast strain suitable for fermentation of second-generation substrates or for production of second-generation biofuels, the method comprising the step of expressing the chimeric gene of the invention or the vector of the invention in a yeast strain to obtain a recombinant yeast strain suitable for fermentation of second- generation substrates or for production of second-generation biofuels. In a particular embodiment, said method further comprises the step of identifying a recombinant yeast strain with increased salt tolerance. In a particular embodiment, said yeast is a yeast strain different from Zygosaccharomyces, even more particularly said yeast strain is a Saccharomyces yeast.

In yet another embodiment, the above described recombinant yeast strain according to the invention is used for fermentation of second-generation substrates or for the production of second-generation biofuels. "Second-generation substrates" as used herein are lignocellulosic biomass or woody crops, agricultural residues, non-foodstuffs or waste, especially lignocellulosic waste streams. Lignocellulosic refers to plant biomass composed of carbohydrate polymers (cellulose, hemicellulose) and an aromatic polymer (lignin). These carbohydrate polymers contain different sugar monomers (six and five carbon sugars) and they are tightly bound to lignin. Lignocellulosic biomass can be broadly classified into virgin biomass, waste biomass and energy crops. Virgin biomass includes all naturally occurring terrestrial plants such as trees, bushes and grass. Waste biomass is produced as a low value byproduct of various industrial sectors such as agricultural (corn stover, sugarcane bagasse, straw etc.), forestry (saw mill and paper mill discards). Energy crops are crops with high yield of lignocellulosic biomass produced to serve as a raw material for production of second generation biofuel, not limiting examples are poplar trees, willow trees, switch grass (Panicum virgatum) and Elephant grass. "Second-generation biofuels" are biofuels produced from second-generation substrates. Fermentation of second-generation substrates can be convincingly evaluated by analysis of the substrate content and metabolites by high performance liquid chromatography (HPLC) as described in the materials and methods section of the present application. Fermentation is then defined as a process during which the level of one or more substrate components (e.g. glucose, xylose) is decreased and the level of one or more metabolites (e.g. ethanol, glycerol) is increased. In yet another embodiment the invention provides a method for obtaining salt tolerance or for increasing salt tolerance during yeast fermentation, comprising at least the expression of the chimeric gene of the invention or at least the vector according to the invention in a yeast strain and using the resulting yeast strain in a fermentation process. Preferably, said yeast is a Saccharomyces sp., even more preferably it is a Saccharomyces cerevisiae sp.

In yet another embodiment, the invention provides a method for industrial fermentation at salt level conditions that reduce the fermentation efficiency, said method comprising expressing the chimeric gene of the invention or the vector of the invention in a yeast strain. In a particular embodiment, said method further comprises the step of identifying a recombinant yeast strain with increased salt tolerance. In a particular embodiment, said yeast is a yeast strain different from Zygosaccharomyces, even more particularly said yeast strain is a Saccharomyces yeast.

In yet another embodiment the invention provides a method for fermentation using second-generation substrates, comprising at least the expression of the chimeric gene of the invention or at least the vector according to the invention in a yeast strain and using the resulting yeast strain in a fermentation process using second-generation substrates. Preferably, said yeast is a Saccharomyces sp., even more preferably it is a Saccharomyces cerevisiae sp.

Besides the negative impact of excessive amounts of salt on yeast growth and fermentation, salt also affects plant growth. Plant salt stress is a condition where excessive salts in soil solution cause plant death or at least inhibition of plant growth. On a world scale, no toxic substance restricts plant growth more than does salt [53]. Salt stress presents an increasing threat to plant agriculture and an ever- increasing problem in arid and semi-arid regions [54]. It was estimate that arid and semi-arid lands represent around 40% of the earth's area [55]. Among the various sources of soil salinity, irrigation combined with poor drainage is the most serious, because it represents losses of once productive agricultural land. General symptoms of damage by salt stress are growth inhibition, accelerated development and senescence and death during prolonged exposure. It is thus advantageous to develop plants with increased salt tolerance. In yet another embodiment the invention provides a chimeric gene comprising a promoter which is active in a plant or plant cell, a nucleotide sequence encoding an amino acid sequence with a sequence identity to SEQ ID N° 2 preferably of at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and a 3' end region involved in transcription termination or polyadenylation. In a preferred embodiment, said promoter is a constitutive promoter, a promoter that is active in the root system of a plant, a promoter that is active in the growth zone of the root system of a plant, a promoter that is active in the leaves of a plant, a promoter that is active in the shoot apical meristem of the plant or a promoter that is active in the plant embryo. In another embodiment the said promoter is an inducible promoter, i.e. a promoter which gets activated in the presence or absence of biotic or abiotic stress. "Biotic stresses" are typically those stresses caused by pathogens, such as bacteria, viruses, fungi, nematodes and insects. The "abiotic stress" may be an osmotic stress caused by a water stress (e.g. due to drought), salt stress, or freezing stress.

The term "plant" as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, wherein each of the aforementioned comprise the chimeric gene or vector according to the invention. The term "plant" also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the chimeric gene or vector according to the invention. In yet another embodiment the chimeric gene comprising a promoter which is active in a plant or plant cell, a nucleotide sequence encoding an amino acid sequence with a sequence identity to SEQ ID N° 2 preferably of at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and a 3' end region involved in transcription termination or the vector comprising said chimeric gene for use in obtaining salt tolerance or for increasing salt tolerance in a plant or a plant cell. "Obtaining salt tolerance" or "increasing salt tolerance" as used herein means that the plant or plant cell that comprises the said chimeric gene of the invention or the vector of the invention has the ability to tolerate salt levels that have an inhibitory effect on growth, metabolism or proliferation of the control. The choice of suitable control plants is a routine part of an experimental setup by which a person skilled in the art is very familiar with and may include corresponding wild type plants or corresponding plants or plant cells without the chimeric gene or vector of the invention. The control plant is typically of the same plant species or even of the same variety as the plant to be assessed. The control plant may also be a nullizygote of the plant to be assessed. Nullizygotes are individuals missing the transgene by segregation. A "control plant" as used herein refers not only to whole plants, but also to plant parts, including seeds and seed parts. Multiple protocols for evaluating salt tolerance in plant are present in the art. Most of them will use growth analyses of one or different parts (e.g. root, shoot) of the plant at a series of salt levels in the substrate. The salt levels that will inhibit plant growth depend first and foremost on the plant species itself. Most plants are glycophytes that cannot tolerate salt stress. Glycophytes are severely inhibited in their growth or even killed by 100-200 millimoles per liter of NaCI [53]. There are however also very salt-sensitive glycophytes, for example fruit trees such as citrus and avocado, which are sensitive to a few millimoles per litre of NaCI [53]. The term "salt" is not restricted to common salt (NaCI), but may be any one or more of: NaCI, KCI, LiCI, MgC , CaC , amongst others. The term "tolerant" when used in reference to a stress condition of a plant, means that the particular plant, when exposed to a stress condition, shows less of an effect, or no effect, in response to the condition as compared to a corresponding reference plant (naturally occurring wild-type plant or a plant not containing the chimeric gene or vector of the present invention). As a consequence, a plant encompassed within the present invention shows improved agronomic performance as a result of enhanced abiotic stress tolerance and grows better under more widely varying conditions, such as increased biomass and/or higher yields and/or produces more seeds. Preferably, the transgenic plant is capable of substantially normal growth under environmental conditions where the corresponding reference plant shows reduced growth, yield, metabolism or viability, or increased male or female sterility.

The terms "increase", "obtain", "improve" or "enhance" are interchangeable and shall mean in the sense of increasing salt tolerance in a plant or plant cell at least a 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% more yield and/or growth in comparison to control plants as defined herein at growth limiting salt levels for the said control plants.

For the purposes of the invention, "transgenic", "transgene" or "recombinant" means with regard to, for example, a nucleic acid sequence, a chimeric gene, an expression cassette, gene construct or a vector comprising the nucleic acid sequence or an organism transformed with the nucleic acid sequences, expression cassettes or vectors according to the invention.

A transgenic plant for the purposes of the invention is thus understood as meaning, as above, that the nucleic acids used in the method of the invention are not present in, or originating from, the genome of said plant, or are present in the genome of said plant but not at their natural locus in the genome of said plant, it being possible for the nucleic acids to be expressed homologously or heterologously.

In yet another embodiment the invention provides the use of the chimeric gene of the invention or the vector according to the invention to preserve the yield of a plant at salt levels in the soil that limit the optimal and intrinsic yield of a plant. "Optimal and intrinsic yield of a plant" is defined as the yield of the plant at non-limiting growth conditions. The term "yield" as used herein generally refers to a measurable product from a plant, particularly a crop. Yield, yield preservation and yield increase (in comparison to a non-transformed starting or wild-type plant) can be measured in a number of ways, and it is understood that a skilled person will be able to apply the correct meaning in view of the particular embodiments, the particular crop concerned and the specific purpose or application concerned. The terms "yield preservation" or "yield increase" shall mean in the sense of the increasing salt tolerance in a plant or plant cell at least a 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% more yield and/or growth in comparison to control plants as defined herein at yield limiting salt levels for the said control plants. "Yield limiting" salt levels are levels of salt which decrease the yield with at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to conditions optimized for yield.

In yet another embodiment the invention provides the use of the chimeric gene of the invention or the vector according to the invention to increase the seedling vigor of plants at salt levels in the soil that limit optimal and intrinsic seedling vigor of a plant. "Optimal and intrinsic seedling vigor of a plant" is defined as the seedling vigor of the plant at non-limiting growth and germinating conditions. The terms "increased seedling vigor" shall mean in the sense of the increasing seedling vigor of a plant at least a 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 50%, 60%, 70%, 80%, 90%, 100% more germination or seedling growth in comparison to control plants as defined herein at germination or seedling vigor limiting salt levels for the said control plants.

In yet another embodiment the invention provides the use of the chimeric gene of the invention or the vector according to the invention to increase the drought tolerance of plants. "Increasing drought tolerance" as used herein means that the plant or plant cell that comprises the said chimeric gene of the invention or the vector of the invention has the ability to tolerate drought levels that have an inhibitory effect on growth, metabolism or proliferation of the control. The term "increasing drought tolerance " shall mean in the sense of increasing drought tolerance in a plant at least a 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 50%, 60%, 70%, 80%, 90%, 100% more yield and/or growth in comparison to control plants as defined herein at drought levels that inhibit yield and/or growth for the said control plants.

In yet another embodiment the invention provides a plant or plant cell comprising the chimeric gene comprising a promoter which is active in a eukaryotic cell, a nucleotide sequence encoding an amino acid sequence with a sequence identity to SEQ ID N° 2 preferably of at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and a 3' end region involved in transcription termination. In a preferred embodiment, the said promoter is active in a plant or plant cell. In another preferred embodiment the said plant is a crop. In another specific embodiment the said crop is a cereal. In yet another specific embodiment the said crop is a grass. In yet another specific embodiment said plant of plant cell is selected from the list comprising the genera Solanum, Saccharum, Zea, Triticum, Secale, Hordeum, Glycine, Oryza, Sorghum, Lolium, Vitis, Medicago, Miscanthus, Panicum, Phalaris, Cannabis, Salix, Populus, and Eucalyptus.

In another embodiment the invention provides a plant or plant cell comprising a vector comprising the chimeric gene, wherein said chimeric gene comprises a promoter which is active in a eukaryotic cell, a nucleotide sequence encoding an amino acid sequence with a sequence identity to SEQ ID N° 2 preferably of at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and a 3' end region involved in transcription termination. In a preferred embodiment, the said promoter is active in a plant or plant cell. In another preferred embodiment the said plant is a crop. In another specific embodiment the said crop is a cereal. In yet another specific embodiment the said crop is a grass. In yet another specific embodiment said plant of plant cell is selected from the list comprising the genera Solanum, Saccharum, Zea, Triticum, Secale, Hordeum, Glycine, Oryza, Sorghum, Lolium, Vitis, Medicago, Miscanthus, Panicum, Phalaris, Cannabis, Salix, Populus, and Eucalyptus.

In yet another embodiment the invention provides a method to produce a plant with increased salt tolerance comprising the following steps:

a. providing plant cells with the chimeric gene according to the invention to create transgenic plant cells,

b. regenerating a population of transgenic plant lines from said transgenic plant cell; and c. identifying a plant with increased salt tolerance.

Still another aspect of the invention is a method to produces a plant with increased salt tolerance as compared to a corresponding control plant, whereby the method comprises introducing or transforming the chimeric gene or vector according to the invention to created transgenic plant cell, regenerating a population of transgenic plant lines from said transgenic plant cell and identifying a plant with increased salt tolerance. In another embodiment the invention provides a chimeric gene comprising a promoter which is active in a mammalian cells, a nucleotide sequence encoding an amino acid sequence with a sequence identity to SEQ ID N° 2 preferably of at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and a 3' end region involved in transcription termination or polyadenylation.

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: Screening for high salt tolerant transformants using a Z. rouxii cDNA library

First, we have assessed the salt tolerance of S. cerevisiae GSE16, in comparison with Ethanol Red (ER), a widely-used commercial first-generation bioethanol strain, and S288c, a laboratory strain, on solid SCD nutrient plates containing different concentrations of NaCI (Fig. 1A). In present application "salt tolerance" and "halotolerance" are used interchangeably. Also "salt tolerant" and "halotolerant" are used interchangeably. Spot assays showed that the strain GSE16 was the most salt-sensitive, growing in the presence of maximally 1.6 M NaCI, followed by ER (1.8 M NaCI) and S288c (2.0 M NaCI). To improve the halotolerance of strain GSE16 and identify genes sustaining high salt tolerance, strain GSE16 was transformed with a cDNA library constructed from RNA isolated from the halotolerant strain Z. rouxii CBS732. Around 10,000 transformants were selected on hygromycin plates and then screened on plates containing different concentrations of NaCI, from 1.6 M to 2.4 M. A total of 216 transformants formed visible colonies on 2.0 M NaCI plates. In order to confirm the improvement in salt tolerance and select the transformants with the highest salt tolerance, the 216 transformants were replica plated onto plates containing higher concentrations of NaCI (2.2 M and 2.4 M). No transformants grew on the 2.4 M salt plates while 92 transformants were obtained from the 2.2 M NaCI plates. They were replica plated on plates with the same concentration of 2.2 M NaCI and 50 transformants reproducibly formed visible colonies on such plates. Next, they were selected for growth in liquid medium with 1.8 M or 2.0 M NaCI. All the 50 transformants showed improved salt tolerance under those conditions compared to the control GSE16 strain (Fig. IB). In order to identify the most halotolerant transformants, 25 transformants behaving reproducibly better than the control under all tested conditions were selected for confirmation of the improved salt tolerance on plates by spot test. The lab strain S288c, which has a high intrinsic salt tolerance, and three transformants were able to form colonies on plates with 2.2 M NaCI. Another 13 transformants could grow in the presence of 2.0 M NaCI, while the control strains GSE16 and GSE16-EP (harboring the empty plasmid) could only grow in the presence of 1.8 M NaCI (Fig. 1C). The 16 transformants that showed higher salt tolerance both in liquid medium and on solid nutrient plates were selected for further analysis.

Example 2: Evaluation in small-scale fermentations and confirmation of plasmid-based high salt tolerance

The 16 selected transformants were subsequently evaluated in small-scale semi-anaerobic static fermentations with YPD5% in the presence of 1.5 M, 1.6 M (results not shown) and 1.7 M NaCI (Fig. 2A). In this case, only two transformants, GSE16-p26 and GSE16-p20, displayed a higher and more sustained fermentation rate than the control GSE16 strain. The largest difference between these transformants and the control was observed in the fermentations with 1.7 M NaCI (Fig. 2A). The fermentation performance of the other transformants was similar or worse than that of the control strain GSE16. In the absence of added salt, all transformants showed the same fermentation performance as the control strain GSE16. Based on the results of the salt tolerance assays in liquid medium, on solid nutrient plates and in the small-scale fermentations, we selected the transformants GSE16-pl8, GSE16-p20 and GSE16- p26 for further characterization. In principle, the improvement in salt tolerance should be due to the plasmids with the Z. rouxii cDNA insert, but mutations induced by the transformation process cannot be excluded at this point. To assess this possibility, the three superior transformants, GSE16-pl8, GSE16-p20 and GSE16-p26, were grown until they lost their plasmid and the resulting strains were named GSE16-[pl8 lost], GSE16-[p20 lost] and GSE16-[p26 lost]. Their salt tolerance was compared with the original transformants and the controls by spot assay (Fig. 2B). The original transformants GSE16-pl8, GSE16-p20 and GSE16-p26 grew on 1.8 M and 1.9 M NaCI, while the corresponding plasmid-loss strains GSE16-[pl8 lost], GSE16-[p20 lost] and GSE16-[p26 lost] did not. This result was consistent with the growth test in liquid salt media (data not shown). Transformant GSE16-p26 always performed better than GSE16-pl8 and GSE16-p20 under all tested conditions. Our results indicate that the improvement in salt tolerance of the selected transformants was due to the insert present in the plasmid.

To further assess whether the improvement in salt tolerance was due to the Z. rouxii genes expressed, the 3 superior plasmids were isolated and transformed into the starting strain GSE16 separately, resulting in new transformants GSE16-pl8, GSE16-p20 and GSE16-p26. The plasmids of these new transformants were lost resulting in new strains GSE16-[pl8 lost], GSE16-[p20 lost] and GSE16-[p26 lost]. The growth of these strains was compared with the parent strain GSE16 in liquid salt media. The novel transformants GSE16-pl8, GSE16-p20 and GSE16-p26 showed better growth than the starting strain GSE16 and the corresponding plasmid-lost strains in 1.9 M and 2.0 M NaCI (Fig. 3). These results were consistent with those of a growth test of these strains on solid salt media (data not shown). Hence, this further confirmed that the improvement in salt tolerance of these strains was due to the Z. rouxii genes expressed in GSE16.

Example 3: Identification of the Z. rouxii genes conferring higher halotolerance

To identify the Z. rouxii genes conferring the higher halotolerance to the GSE16 strain, the plasmids from the superior transformants were first isolated and named pFL39-hph-18, pFL39-hph-20 and pFL39-hph- 26, respectively. After sequencing the cDNA inserts, the sequences obtained were blasted against the genome sequence of Z. rouxii type strain CBS732 as present in SGD (http://www.yeastgenome.org/). This identified the genes ZYRO0D06204g (insert 18), ZYRO0G17556g (insert 20) and ZYRO0A08470g (insert 26). Further analysis at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) showed that ZYRO0D06204g (insert 18) encodes a protein with 228 amino acid residues (aa) and 65 % sequence identity in a 63 aa overlap with the product of S. cerevisiae YLR110C (Ccwl2, 133 aa), ZYRO0G17556g (insert 20) encodes a protein with 92 aa and 92 % sequence identity in a 92 aa overlap with the product of S. cerevisiae YPR043W ( pl43A, 92 aa) and ZYRO0A08470g (insert 26) encodes a protein with 328 aa and 60 % sequence identity in a 297 aa overlap with the product of S. cerevisiae YDL078C (Mdh3 or malate dehydrogenases, 343 aa).

Example 4: Effect on salt tolerance of overexpression and co-expression of the Z. rouxii genes

The pFL39-hph plasmid used for the cDNA library construction was a single copy plasmid, while halotolerance is a polygenic trait. Hence, we next tested whether overexpression and co-expression of the three genes had the potential to further improve the salt tolerance in S. cerevisiae. In order to evaluate these possibilities, multicopy plasmids pBEVY-hph (pBh) and pBEVY-nat (pBn) (with dominant antibiotic marker hph and nat, respectively) were used to express the three cDNAs ZYRO0D06204g (insert 18), ZYRO0G17556g (insert 20) and ZYRO0A08470g (insert 26) from Z. rouxii alone and in different combinations together in S. cerevisiae. Hence, four novel multicopy plasmids pBhl8, pBn20, pBh26 and pBhl8+26 were constructed and were used for single expression in yeast together with four combinations: pBhl8-pBn20, pBn20-pBh26, pBhl8+26 and pBhl8+26-pBn20. They, as well as the empty plasmids (pBh and pBn), were transferred into the strain GSE16. The resulting strains were GSE16-pBh, GSE16-pBn, GSE16-pBhl8, GSE16-pBn20, GSE16-pBh26, for single overexpression, and GSE16-pBhl8- pBn20, GSE16-pBn20-pBh26, GSE16-pBhl8+26, GSE16-pBhl8+26-pBn20, for combined expression. The growth of these strains was compared with the original transformants GSE16-pl8, GSE16-p20, GSE16- p26 and the starting strain GSE16 on solid nutrient plates containing 1.6 M NaCI (Fig. 4). The original transformants again displayed higher salt tolerance than the control strain GSE16. However, the strains harboring multi-copy plasmids for overexpression of the genes showed lower salt tolerance than the original transformants and the control GSE16 strains with the empty plasmid (Fig. 4). Hence, the increased copy number of the three inserts from Z. rouxii did not enhance but rather decreased salt tolerance, which indicated that overexpression of these genes influenced salt tolerance negatively. In addition, the co-expression strains also showed lower salt tolerance than the starting strain GSE16 (Fig. 4). Combined expression of the three cDNAs could also not further improve salt tolerance. Since halotolerance is a polygenic trait, it is very surprising that the individual expression of the three cDNAs from Z. rouxii increased salt tolerance in S. cerevisiae.

Example 5: Fermentation performance of salt tolerant transformant GSE16-p26 in corn cob hydrolysate

We next investigated whether the transformant GSE16-p26, which displayed the best salt tolerance in synthetic medium, also displayed higher salt tolerance in an industrially relevant medium compared to the GSE16 parent strain. For that purpose, we first evaluated the growth of the GSE16-p26 and GSE16 strains in corn cob hydrolysate without and with addition of 0.6 M NaCI. The added NaCI caused strong inhibition of growth, while the GSE16-p26 strain displayed better growth than the GSE16 strain under all conditions (Fig. 7).

We next performed fermentation experiments with corn cob hydrolysate in the absence and in the presence of 0.6 M NaCI. The composition of the medium was measured during the course of the fermentation by HPLC, to determine the glucose and xylose consumption rates, the glycerol and ethanol production rates and the ethanol yield for the two strains in the two media. They are listed in table 1. Also the fermentation performance of the two strains was negatively influenced by the presence of 0.6 M NaCI. The glucose and xylose consumption rates were decreased causing the fermentation time to be prolonged, the ethanol yield was reduced and the glycerol yield increased (Fig 6). Hence, high salt clearly inhibited ethanol production in corn cob hydrolysate. The GSE16-p26 transformant showed a better fermentation performance than the GSE16 strain not only in the presence of 0.6 M NaCI but surprisingly also in corn cob hydrolysate without added NaCI. The improvement was observed both for glucose and xylose fermentation. This shows that the ZYRO0A08470g gene in insert 26, a homolog of S. cerevisiae MDH3, can be employed to improve salt tolerance in industrial yeast strains used for second-generation bioethanol production. In addition, it suggests that the salt level present in corn cob hydrolysates already exerts an inhibitory effect on the fermentation and that the expression of ZYRO0A08470g could improve fermentation performance in various other types of lignocellulose hydrolysates. Table 1 Glucose and xylose consumption rates, glycerol and ethanol production rates and ethanol yield in fermentations with the transformant GSE16-p26 and parent strain GSE16 of whole corn cob hydrolysate in the absence and presence of 0.6 M NaCI.

Example 6: Expression of the Z. rouxii ZYRO0A08470g gene in Arabidopsis thaliana

A chimeric gene is constructed containing the following DNA elements:

a promoter region which causes transcription of the 35S m NA of cauliflower mosaic virus (CaMV 35S) active in Arabidopsis thaliana (i.e. the promoter in pMON81 in U.S. Pat. No. 5,352,605)

a nucleic acid sequence encoding an amino acid sequence depicted in SEQ ID N° 2 and with optimized codon usage for expression in plants

- A CaMV 35S terminator

This chimeric gene is introduced into a T-DNA vector (pK7m24GW) in combination with a selectable GFP marker. The T-DNA vector is introduced into Agrobacterium tumefaciens and used to produce transgenic Arabidopsis.

Shoot and root growth of the transgenic is plants is analysed under optimal and salt stress conditions. Wild-type and transgenic seeds are grown in vitro with Murashige and Skoog (MS) medium containing 0.5% sucrose under a 16-h/8-h photoperiod. For salt stress, wild-type and transgenic seeds are allowed to germinate for 5-7 days and transferred to a series of agar plates to which 0 mM, 50 mM, 100 mM, 150 mM or 200 mM was added . Shoot fresh and dry weight, leaf area, root length and mass are measured at 14 days after germination. Under soil conditions, a high-throughput, fully automated water monitoring system, named WIWAM, implemented at the host institute is used [56]. This system enables to keep stable water levels and is capable of taking digital images of individual plants that can be used to determine rosette growth, leaf area and leaf shape. Plants are grown under control watering regime until stage 1.04 (approximately 12-13 days old), after which control (0 mM NaCI) or salt stress (50 mM, 100 mM, 150 mM or 200 mM NaCI) watering are applied for additional 10-12 days. At the end of the experiment, plants are harvested and the shoot production is recorded as a measurement of yield.

Example 7: Expression of the Z. rouxii ZYRO0A08470g gene in corn

A chimeric gene is constructed comprising the following DNA elements:

UBI1 promoter (SEQ ID NO: 3) active in monocots including corn

a CaMV 35S terminator

This chimeric gene is introduced into the destination vectors (pBbm42GW7), containing the BASTA herbicide under control of 35S CaMV promoter and followed by a nos terminator as a selectable marker. This construct is introduced into Agrobacterium tumefaciens (EHAlOl) and used to transform immature maize embryos of B104 which are regenerated by tissue culture to produce transgenic plants expressing the Z. rouxii ZYRO0A08470g gene. Maize transformation was performed according to Coussens et al [57].The transgenic plants are backcrossed to B104, resulting in a working population segregating in 50% sensitive and thus control plants and 50% transgenic plants. Leaf growth of the segregating population is analyzed under optimal and salt stress conditions. The plants are grown in soil and watered daily: the salt treated plants receive the water that is added to the control plants but to which 50 mM, 100 mM, 150 mM or 200 mM NaCI was added. The leaf growth is monitored by daily measuring the leaf length of the fourth leaf upon its appearance, providing data on the leaf elongation rate and the final leaf length. In addition final plant height, fresh weight and dry weight plants is determined as a measure for plant biomass.

Materials and methods

Strains, media and culture conditions

The strains utilized in this study are listed in Table 2. Yeast cells were propagated in yeast extract peptone (YP) medium (10 g/L yeast extract, 20 g/L bacteriological peptone) supplemented with 20 g/L D-glucose (YPD) or synthetic complete medium (1.7 g/L Difco yeast nitrogen base without amino acid and without ammonium sulfate, 5 g/L ammonium sulfate, 740 mg/L CSM) supplemented with 20 g/L D-glucose (SCD). For solid plates, 15 g/L Bacto agar was added (20 g/L agar for salt plates) after adjusting the pH to 6.5 using N H₄OH. For selection of strains expressing plasmids containing the hygromycin or/and nourseothricin resistance marker, 300 mg/L hygromycin or/and 100 mg/L nourseothricin was added to the medium. Escherichia coli DH5a was used for plasmid construction and grown in Luria-Bertani medium (LB) supplemented with 100 mg/L of ampicillin at 37 °C. Different concentrations (1.6 M, 1.7 M, 1.8 M, 1.9 M, 2.0 M, 2.1 M, 2.2 M and 2.4 M) of NaCI were added to the liquid media or solid plates. Media YP + 5% D-glucose (YP5%D) supplemented with 1.5 M, 1.6 M, 1.7 M and 1.8 M NaCI were used for small-scale fermentations. Whole corn cob hydrolysate with different concentrations (0 M, 0.5 M, 0.6 M and 0.7 M) of added NaCI was used to test the improvement of the growth and fermentation performance of the superior transformants.

Table 2 Yeast strains used in this study

Name Main characteristics Source/reference

S288c Lab strain; MATa/a [47]

Ethanol Red (ER) Industrial bioethanol production strain, MATa/a Fermentis

GSE16 ER background; obtained from GS1.11-26 by

[10]

backcrossing with a segregant of ER; ΜΑΤα/α

Z. rouxii CBS732 Haploid; MATa/homothallic [48]

GSE16-EP GSE16 transformed with pFL39-hph This work

GSE16-pl8 GSE16 transformed with pFL39-hph-18 This work

GSE16-p20 GSE16 transformed with pFL39-hph-20 This work

GSE16-p26 GSE16 transformed with pFL39-hph-26 This work

GSE16-pBh GSE16 transformed with pBEVY-hph This work

GSE16-pBhl8 GSE16 transformed with pBEVY-hph-18 This work

GSE16-pBn GSE16 transformed with pBEVY-nat This work

GSE16-pBn20 GSE16 transformed with pBEVY-nat-20 This work

GSE16-pBh26 GSE16 transformed with pBEV-hph-26 This work

GSE16 transformed with pBEVY-hph-18 and pBEVY- This work

GSE16-pBhl8-pBn20

nat-20

GSE16 transformed with pBEVY-hph-26 and pBEVY- This work

GSE16-pBh26-pBn20

nat-20

GSE16-pBhl8+26 GSE16 transformed with pBEVY-hph-18+26 This work

GSE16-pBhl8+26- GSE16 transformed with pBEVY-hph-18+26 and This work

pBn20 pBEVY-nat-20 cDNA library construction

The cDNA library was custom-made by Invitrogen and transferred into a modified single copy pFL39 shuttle vector. Construction of the plasmid included introduction of specific restriction sides (Pme\ and Not\) flanked by TEF promoter and terminator, where the cDNA was later introduced. A dominant restriction marker, hph, that confers resistance to hygromycin B was also inserted. Cells of the Z. rouxii type strain, CBS732, were grown at 30°C under high osmotic pressure in the presence of 70% glucose added to YP medium. Cells were then harvested in various growth stages and rapidly frozen in liquid nitrogen. NA isolation was performed by the company Invitrogen. The library was delivered in E. coli and isolated according to the company guidelines, by using QIAGEN Plasmid Mega Kit. cDNA library transformation and selection of transformants

The Z. rouxii cDNA library was transferred to S. cerevisiae strain GSE16 by an optimized electroporation protocol [49], with 2 to 3 μg cDNA library plasmids, 200 μΙ competent cells, 200 V voltage, 200 Ω with the 25 μί¹ capacitor giving a 5 msec time constant. The cultures were recovered in YPD for 4 hours and placed on YPD-hph plates. All the plates were sealed with parafilm and incubated at 30°C for 12 - 26 days. Around 10000 transformants were selected and replica plated on YPD plates supplemented with 1.6 M, 1.8 M, 2.0 M, 2.2 M and 2.4 M of NaCI. A total of 216 transformants formed macroscopic colonies on 2.0 M NaCI plates, while the GSE16 strain only grew on 1.8 M and no colonies grew on 2.2 M NaCI plates. In order to confirm the improvement in salt tolerance and select transformants with reproducibly better salt tolerance, the 216 transformants were replica plated onto higher salt concentration plates (YPD plates supplemented with 2.2 M or 2.4 M NaCI). Next, 92 transformants were selected from the 2.2 M NaCI plates and replica plated on the same concentration salt plates (2.2 M) for the second round replica plating. The best 50 transformants that formed robust colonies on 2.2 M NaCI plates were selected as salt tolerant transformants for further evaluation.

Growth on solid salt plates

The growth of the salt tolerant transformants on solid salt plates was compared by spot assay [50]. The starting strain GSE16, strain GSE16-EP (strain GSE16 with empty plasmid) and the lab strain S288c (a/alpha) were used as controls. Cells were harvested by centrifugation after overnight culture, re- suspended in Milli-Q water and the cell density was normalized to an initial Οϋεοο value of 1.0. A 10-fold serial dilution of the culture was prepared, and 5 μΙ of each dilution was spotted onto YPD/SCD salt plates and incubated at 30°C. The growth was recorded over a period of 2 - 12 days. Each experiment was performed in duplicate with independent cultures. Growth in liquid salt media

Growth determination of the salt tolerant transformants was performed in tubes or 24-well plates in liquid salt media. Cells (starting Οϋεοο value of 0.2) were inoculated in 3 mL (in tubes) or in 1 mL (in 24- well plates) YPD/SCD salt media supplemented with 0 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, 2.0 M and 2.1 M NaCI at 30°C with shaking (200 rpm). OD measurements were manually taken at 600 nm on a Novaspec II spectropho-tometer (Pharmacia Biotech) every few hours for 72 hours. Three replicates were conducted for each treatment.

Small-scale fermentations

Small-scale fermentations were performed with the three best salt tolerant transformants and the control in YPD5% medium supplemented with different concentrations of NaCI (0 M, 1.5 M, 1.6 M, 1.7 M and 1.8 M). The pH value was adjusted to 5.5 using N H₄OH or HCI. Cells were pregrown in 5 mL YPD medium overnight, and then grown in 50 mL YPD medium for 2 days till stationary phase and used for inoculation. The initial inoculum density was 2.0 g DW/L (starting Οϋεοο value of 7). The fermentations were conducted in 300 mL fermentation bottles with a working volume of 100 mL with cotton plugged rubber stopper and glass tubing. Cultures were continuously stirred magnetically at 200 rpm and incubated at 32°C. The weight of the tubes was measured till the end of the fermentation. Each experiment was performed in duplicate with independent cultures. Plasmid loss

The strains harboring plasmids were cultured in YPD medium without selection pressure (salt or antibiotic) and transferred to a new culture each day continuously for 5 days. At the end, the cultures were diluted and plated on YPD plates. Single colonies were obtained after cell isolation with a micromanipulator and tested for loss of the plasmid by PC and by absence of growth in medium based on the selection marker (hph) in the plasmid.

Molecular Biology methods and plasmid construction

All cloning procedures followed standard molecular biology methods [51]. Yeast cells were transformed by an optimized electroporation protocol based on a previous report [49]. Genomic DNA from yeast was extracted with PCI [phenol/chloroform/isoamyl-alcohol (25:24:1)] [52]. Polymerase chain reaction (PCR) was performed with Q5 DNA polymerase (New England Biolabs) for construction of vectors and for sequencing purposes and ExTaq (Takara) or Taq (NEB) for diagnostic purposes. Sanger sequencing was performed by the Genetic Service Facility of the VIB, Belgium. The plasmids and the primers used in this study are listed in Table 3. Plasmids were propagated in E. coli strain DH5a (NEB), and the cells were grown in LB medium containing 100 μg/mL ampicillin at 37°C. The E. coli cells were transformed using the CaC method [51]. The genes from inserts 18, 20 and 26 were amplified with the corresponding primers from the plasmids pFL39-hph-18/20/26, digested with corresponding enzymes, and linked to the vectors pBEVY-hph and pBEVY-nat, which were flanked with the proper restriction sites, resulting in the plasmids pBEVY-hph-18, pBEVY-nat-20 and pBEVY-hph-26. The gene from insert 18 was inserted into plasmid pBEVY-hph-26, to form the plasmid pBEVY-hph-18- 26. The primers used are listed in Table 3.

Table 3 Plasmids and primers used in this study

Plasmid or primer Relevant features Origin

pFL39-hph Single copy, hph under TEFp This work pBEVY-hph Multicopy, hph under TEFp [7]

pBEVY-nat Multicopy, nat under TEFp [7]

pBEVY-hph-18 Multicopy, hph under TEFp, with Z. rouxii ORF 18 This work pBEVY-nat-20 Multicopy, hph under TEFp, with Z. rouxii ORF 20 This work pBEVY-hph-26 Multicopy, hph under TEFp, with Z. rouxii ORF 26 This work pBEVY-hph-18-26 Multicopy, hph under TEFp, with Z. rouxii ORF 18 and 26 This work

Primers for gene expression

Fw-eamHI-18 ttttttggatccGTCGCTCGCTTATCACTA This work

Rv-Xbal-18 tttttttctagaTAAAGTACTTCTATTAATTTATTAGTAC This work

Fw-eamHI-20 ttttttggatccGGCTAAGAGAACAAAGAAGG This work

Fw-Xbal-20 ttttttctagaGGCTAAGAGAACAAAGAAGG This work

Rv-Xbal-20 tttttttctagaTTTGgAGTTCAGAACCAATGG This work

Fw-/ p/il-26 ttttttggt a cc ATCG CTTCTTG C AGTATAT This work v- pnl-26 ttttttggtaccTAACTAATTATTCATCAATTAATTCATATAAAG This work

Fermentations in whole corn cob hydrolysate without and with added salt

Whole corn cob hydrolysate with and without added salt was used to evaluate the fermentability of the best salt tolerant transformant GSE16-p26 in comparison with the control strain GSE16. Urea (3 g/L) was added as supplement and 50 mg/L chloramphenicol was used to prevent bacterial contamination. The initial pH value of the medium was adjusted to 5.2 using NH₄OH. The fermentations were performed in specially designed 250 mL bottles with an opening for sampling at the side and a working volume of 100 mL. The fermentations were started with an initial cell density of 2.86 g DW/L (starting Οϋεοο value of 10) and incubated at 32°C with continuous stirring with a magnetic rod at 200 rpm. Samples were taken every few hours through plastic tubing fitted to the opening on the side of the flasks, centrifuged and the supernatant stored at 4°C until HPLC analysis. The fermentations were repeated twice under the same conditions and each experiment was performed in duplicate with independent cultures.

Analysis of substrates and metabolites

The substrate content and metabolites were analysed by high performance liquid chromatography (HPLC). Metabolites and substrates in fermentation experiments with SCD or hydrolysate medium were analyzed by Waters Isocratic Breeze HPLC system using ion-exchange column WAT010290 and a refractive index detection system (Waters 2414 l detector). The injection volume was 10 μΙ and column temperature was maintained at 75°C. The eluent used was 5 mM H2SO4 at a flow rate of 1 ml/min, the syringe was washed by 10% methanol and the running time for each sample was 16 min.

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Claims

1. A chimeric gene comprising:

a. a promoter which is active in a eukaryotic cell

b. a nucleic acid sequence encoding an amino acid sequence depicted in SEQ ID N° 2 or that has at least 80% sequence identity to SEQ ID N° 2

c. a 3' end region involved in transcription termination or polyadenylation.

2. A vector comprising the chimeric gene of claim 1.

3. The use of the chimeric gene according to claim 1 or the vector of claim 2 for obtaining salt tolerance in a eukaryotic organism.

4. The use according to claim 3, wherein said eukaryotic organism is a yeast.

5. A recombinant yeast strain comprising the chimeric gene according to claim 1 or the vector of claim 2.

6. A recombinant yeast strain according to claim 5 wherein said yeast strain belongs to Saccharomyces, Zygosaccharomyces, Brettanomyces or Kluyveromyces.

7. A recombinant yeast strain different from Zygosaccharomyces comprising a nucleic acid sequence encoding an amino acid sequence depicted in SEQ ID N° 2 or that has at least 95% sequence identity to SEQ ID N° 2.

8. The use of the recombinant yeast strain according to claims 5-7 for industrial fermentation at salt level conditions that reduce the fermentation efficiency.

9. The use of the recombinant yeast strain according to claims 5-7 for fermentation of second- generation substrates or for the production of second-generation biofuels.

10. A chimeric gene according to claim 1 wherein said promoter is active in a plant or plant cell.

11. The use of the chimeric gene according to claims 1 or 10 or the vector of claim 2 for obtaining salt tolerance in a plant.

12. A plant or plant cell comprising the chimeric gene according to claims 1 or 10 or the vector of claim 2. A method to produce a plant with increased salt tolerance comprising the following steps: a. providing plant cells with the chimeric gene according to claims 1 or 10 to create transgen plant cells,