# Patent

## Title
Ectoine-producing yeast

## Abstract

The present invention relates to the field of bio-production of ectoine. There is a need in the art for ectoine production methods allowing its highly efficient synthesis and secretion. The solution proposed in the present invention is the use of a genetically modified yeast comprising many modifications as described in the present text.

## Background
SEQUENCE LISTING
This application includes a sequence listing which has been submitted in ST.25 format and is hereby incorporated by reference in its entirety. Said sequence listing, created on Dec. 21, 2020, is in a file named 2021-02-11_CorrectedSequenceListing.txt and is 159,266 bytes in size.
FIELD OF THE INVENTION
The present invention relates to the field of bio-production of ectoine.
BACKGROUND OF THE INVENTION
Ectoine (1, 4, 5, 6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is an heterocyclic amino acid naturally produced by halophilic organisms in nature. Indeed, in order to survive in salty environments, these organisms produce ectoine as compatible solute which serves as osmotic counterweights.
Ectoine is indeed also capable of protecting nucleic acids, proteins, cell membranes as well as whole cells against denaturation caused by numerous aggressions from the external environment, such as UV radiations, heating, freezing or chemical agents, but also against denaturation due to drying (see for example Lentzen G et al. Appl Microbiol Biot. 2006; 72:623-34, and Graf R et al. Clin Dermatol. 2008; 26:326-33). As such it is used in cosmetic for skin-care.
Ectoine has moreover been found to be interesting as proteins stabilizer, cosmetic additive, PCR enhancer and drying protective agent for microorganisms.
Due to these advantageous properties, ectoine is increasingly produced through bacterial processes using in particular halophilic bacteria such as Halomonas elongata. However, these methods necessitate a high salt concentration which complexifies the process and leads to an increase of the costs involved considering the important corrosion of the equipment.
Furthermore, the production of essential amino acids such as ectoine through the biosynthetic pathways of bacteria and yeasts requires an important amount of reducing power in the form of NADPH. However, the main pathway for the metabolisation of glucose in these microorganisms, and in particular in yeasts, is glycolysis followed by fermentation which only produces NADH. Maintaining an acceptable NADPH/NADH balance within the microorganism, albeit complex, is therefore essential to optimize bio-production of ectoine while maintaining a viable recombinant microorganism.
Accordingly, there is still a need in the art for further ectoine production methods allowing its highly efficient synthesis and secretion.
SUMMARY OF THE INVENTION
The present invention accordingly relates to an ectoine-producing recombinant yeast, in the genome of which:

    
    
        (A) (i) at least one nucleic acid encoding an aspartokinase is overexpressed and/or is under the control of an inducible or repressible promoter; and/or
        
            (ii) at least one nucleic acid encoding an aspartate kinase is overexpressed and/or is under the control of an inducible or repressible promoter;
        
        
        (B) at least one nucleic acid encoding an aspartate semi-aldehyde dehydrogenase and/or at least one nucleic acid encoding an aspartate semi-aldehyde dehydrogenase that can use as coenzyme both NAD and NADP is overexpressed and/or is under the control of an inducible or repressible promoter;
        (C) at least one nucleic acid encoding a diaminobutyrate aminotransferase is overexpressed and/or is under the control of an inducible or repressible promoter;
        (D) (i) at least one nucleic acid encoding an homoserine-O-acetyltransferase MET2 is overexpressed and/or is under the control of an inducible or repressible promoter;
        
            (ii) at least one nucleic acid encoding an homoserine-O-acetyltransferase METX is overexpressed and/or is under the control of an inducible or repressible promoter, and/or
            (iii) at least one nucleic acid encoding a diaminobutyric acid acetyltransferase is overexpressed and/or is under the control of an inducible or repressible promoter;
        
        
        (E) at least one nucleic acid encoding an ectoine synthase is overexpressed and/or is under the control of an inducible or repressible promoter;
        (F) (i) at least one, preferably all, endogenous nucleic acid encoding an homoserine dehydrogenase has been deleted and/or interrupted, and/or
        
            (ii) at least one, preferably all, nucleic acid encoding an homoserine dehydrogenase is independently:
        
        
        under the control of an inducible or repressible promoter;
        under the control of a weak promoter; and/or
        in a destabilized form.
    
    


As illustrated in the enclosed examples, the recombinant yeasts of the invention have an increased ectoine production.
Said advantageous property can be further increased by also recombining the yeast with additional modifications described here-after.
An ectoine-producing recombinant yeast can consequently advantageously be used in a method for producing ectoine as described here-after or be used for the production of ectoine.
The present invention further relates to a method for producing ectoine, said method comprising the steps of:

    
    
        (a) culturing a recombinant yeast of the invention in a culture medium; and
        (b) recovering the ectoine from said culture medium.
    
    


Preferably, the culture medium comprises at least a carbon source, preferably a carbon source selected from the group consisting of glucose and sucrose.
The invention also relates to the use of a recombinant yeast according to the invention for the production of ectoine.


DETAILED DESCRIPTION OF THE INVENTION
The inventors have conceived genetically modified microorganisms, and especially genetically modified yeasts, having an increased ability to produce ectoine, as compared to the parent microorganisms, and especially as compared to the parent yeasts.
These genetically modified microorganisms, including these genetically modified yeasts, are described throughout the present specification.
Definitions
The term “microorganism”, as used herein, refers to a yeast which is not modified artificially. The microorganism may be “donor” if it provides genetic element to be integrated in the microorganism “acceptor” which will express this foreign genetic element or if it used as tool for genetic constructions or protein expressions. The microorganism of the invention is chosen among yeast which expresses genes for the biosynthesis of ectoine.
The term “recombinant microorganism” or “genetically modified microorganism” or “recombinant yeast” or “genetically modified yeast”, as used herein, refers to a yeast genetically modified or genetically engineered. It means, according to the usual meaning of these terms, that the microorganism of the invention is not found in nature and is modified either by introduction or by deletion or by modification of genetic elements from equivalent microorganism found in nature. It can also be modified by forcing the development and evolution of new metabolic pathways by combining directed mutagenesis and evolution under specific selection pressure (see for instance WO 2004/076659).
A microorganism may be modified to express exogenous genes if these genes are introduced into the microorganism with all the elements allowing their expression in the host microorganism. A microorganism may be modified to modulate the expression level of an endogenous gene. The modification or “transformation” of microorganism, like yeast, with exogenous DNA is a routine task for those skilled in the art. In particular, a genetic modification of a microorganism according to the invention, more particularly the genetic modification(s) herein defined, may be carried out by using CRISPR-Cas systems, as described in DiCarlo et al. (Nucl. Acids Res., vol. 41, No. 7, 2013: 4336-4343).
The term “endogenous gene” means that the gene was present in the microorganism before any genetic modification, in the wild-type strain. Endogenous genes may be overexpressed by introducing heterologous sequences in addition to, or to replace endogenous regulatory elements, or by introducing one or more supplementary copies of the gene into the chromosome or a plasmid. Endogenous genes may also be modified to modulate their expression and/or activity. For example, mutations may be introduced into the coding sequence to modify the gene product or heterologous sequences may be introduced in addition to or to replace endogenous regulatory elements. Modulation of an endogenous gene may result in the up-regulation and/or enhancement of the activity of the gene product, or alternatively, in the down-regulation and/or attenuation of the activity of the endogenous gene product. Another way to enhance expression of endogenous genes is to introduce one or more supplementary copies of the gene onto the chromosome or a plasmid.
The term “exogenous gene” means that the gene was introduced into a microorganism, by means well known by the man skilled in the art, whereas this gene is not naturally occurring in the wild-type microorganism. Microorganism can express exogenous genes if these genes are introduced into the microorganism with all the elements allowing their expression in the host microorganism. Transforming microorganisms with exogenous DNA is a routine task for the man skilled in the art. Exogenous genes may be integrated into the host chromosome, or be expressed extra-chromosomally from plasmids or vectors. A variety of plasmids, which differ with respect to their origin of replication and their copy number in the cell, are all known in the art. The sequence of exogenous genes may be adapted for its expression in the host microorganism. Indeed, the man skilled in the art knows the notion of codon usage bias and how to adapt nucleic sequences for a particular codon usage bias without modifying the deduced protein.
The term “heterologous gene” means that the gene is derived from a species of microorganism different from the recipient microorganism that expresses it. It refers to a gene which is not naturally occurring in the microorganism.
In the present application, all genes are referenced with their common names and with references to their nucleotide sequences and, the case arising, to their amino acid sequences. Using the references given in accession number for known genes, those skilled in the art are able to determine the equivalent genes in other organisms, bacterial strains, yeast, fungi, mammals, plants, etc. This routine work is advantageously done using consensus sequences that can be determined by carrying out sequence alignments with genes derived from other microorganisms and designing degenerated probes to clone the corresponding gene in another organism.
The man skilled in the art knows different means to modulate, and in particular up-regulate or down-regulate, the expression of endogenous genes. For example, a way to enhance expression of, or over express, endogenous genes is to introduce one or more supplementary copies of the gene onto the chromosome or a plasmid.
Another way is to replace the endogenous promoter of a gene with a stronger promoter. These promoters may be homologous or heterologous. Promoters particularly interesting in the present invention are described in more detail elsewhere in the present specification.
The nucleic acid expression construct may further comprise 5′ and/or 3′ recognition sequences and/or selection markers.
The term “overexpression” means that the expression of a gene or of an enzyme is increased as compared to the non-modified microorganism. Increasing the expression of an enzyme is obtained by increasing the expression of a gene encoding said enzyme. Increasing the expression of a gene may be carried out by all techniques known by the one skilled in the art. In this regard, it may be notably cited the implementation of a strong promoter upstream the nucleic acid intended to be overexpressed or the introduction of a plurality of copies of the said nucleic acid between a promoter, especially a strong promoter, and a terminator.
The term “underexpression” means that the expression of a gene or of an enzyme is decreased as compared to the non-modified microorganism. Decreasing the expression of an enzyme is obtained by decreasing the expression of a gene encoding said enzyme. Decreasing the expression of a gene may be carried out by all techniques known by the one skilled in the art. In this regard, it may be notably cited the implementation of a weak promoter upstream the nucleic acid intended to be underexpressed. It may be also cited the implementation of a nucleic acid encoding a variant of the said enzyme that is less active than the parent enzyme or a variant of the said enzyme that is more rapidly degraded in the cell than the parent enzyme. Variants of a parent enzyme that is more rapidly degraded that the said parent enzyme encompass degron-tagged enzymes. It may also be cited the decrease of the expression of a transcription activator of the gene of interest.
The term “inducible promoter” is used to qualify a promoter whose activity is induced, i.e. increased:

    
    
        in the presence of one or more particular metabolite(s). The higher the metabolite concentration in the medium, the stronger the promoter activity; or
        in the presence of a low concentration, or in the absence, of one or more metabolite(s). These metabolites are different from those whose increasing presence induces the activity of the promoter. The lower the metabolite concentration in the medium, the stronger the promoter activity.
    
    


The term “repressible promoter” is used to qualify a promoter whose activity is repressed, i.e. reduced:

    
    
        in the presence of one or more particular metabolite(s). The higher the metabolite concentration in the medium, the weaker the promoter activity; or
        in the presence of a low concentration, or in the absence, of one or more metabolite(s). These metabolites are different from those whose increasing presence represses the activity of the promoter. The lower the metabolite concentration in the medium, the weaker the promoter activity.
    
    


A used herein, a “degron-tagged” enzyme means an enzyme comprising an added protein-degradation signal amino acid sequence that serves as a destruction signal that will cause the said enzyme to be the subject of a degradation, which may be either (i) a ubiquitin-independent degradation or (ii) an ubiquitin-dependent degradation. The said added protein-degradation signal, that is also termed “degron” in the art, encompasses an amino acid sequence that serves as a destruction signal, the said amino acid sequence consisting of a transferrable degradation signal causing a targeted protein degradation. Degrons encompass “N-degrons”, which are transferrable N-terminal amino acids that cause the target protein degradation following the well-known N-end rule (Bachmair et al., 1986, Science, Vol. 234 (4773): 179-186). The unstable nature of the N-degron is attributed to its first amino acids, which are prone to acetylation or arginylation modifications and ultimately lead to ubiquitination and degradation. Generally, a degron requires at least two components to ensure targeted protein degradation: (i) a target degradation recognition tag, such as a poly-ubiquitin tag and (ii) an unstructured amino acid sequence in close proximity to the degradation recognition tag. For degron-tagging a protein, and especially herein for degron-tagging an enzyme, the one skilled in the art may refer to Yu et al. (2015, Current Opinion in Biotechnology, Vol. 36: 199-204), Cho et al. (2010, Genes & Development, Vol. 24: 438-442), or to Fortmann et al. (2015, J Mol Biol, Vol. 427 (17): 2748-2756), Ravid et al. (2008, Nat Rev Mol Cell Biol, Vol. 9(9): 679-690) and Hochstrasser (1996, Annu Rev Genet, Vol. 30: 405-439).
The “activity” of an enzyme is used interchangeably with the term “function” and designates, in the context of the invention, the capacity of an enzyme to catalyze a desired reaction.
The terms “reduced activity” or “attenuated activity” of an enzyme mean either a reduced specific catalytic activity of the protein obtained by mutation in the amino acids sequence and/or decreased concentrations of the protein in the cell obtained by mutation of the nucleotide sequence or by deletion of the cognate corresponding gene or also by degron-tagging of the protein.
The term “enhanced activity” of an enzyme designates either an increased specific catalytic activity of the enzyme, and/or an increased quantity/availability of the enzyme in the cell, obtained for example by overexpression of the gene encoding the enzyme.
The terms “encoding” or “coding” refer to the process by which a polynucleotide, through the mechanisms of transcription and translation, produces an amino-acid sequence.
The gene(s) encoding the enzyme(s) considered in the present invention can be exogenous or endogenous.
“Attenuation” of genes means that genes are expressed at an inferior rate than in the non-modified microorganism. The attenuation may be achieved by means and methods known to the man skilled in the art and contains gene deletion obtained by homologous recombination, gene attenuation by insertion of an external element into the gene or gene expression under a weak promoter. The man skilled in the art knows a variety of promoters which exhibit different strengths and which promoter to use for a weak genetic expression.
The methods implemented in the present invention preferably require the use of one or more chromosomal integration constructs for the stable introduction of a heterologous nucleotide sequence into a specific location on a chromosome or for the functional disruption of one or more target genes in a genetically modified microbial cell. In some embodiments, disruption of the target gene prevents the expression of the related functional protein. In some embodiments, disruption of the target gene results in the expression of a non-functional protein from the disrupted gene.
Parameters of chromosomal integration constructs that may be varied in the practice of the present invention include, but are not limited to, the lengths of the homologous sequences; the nucleotide sequence of the homologous sequences; the length of the integrating sequence; the nucleotide sequence of the integrating sequence; and the nucleotide sequence of the target locus. In some embodiments, an effective range for the length of each homologous sequence is 20 to 5,000 base pairs, preferentially 50 to 100 base pairs. In particular embodiments, the length of each homologous sequence is about 50 base pairs. For more information on the length of homology required for gene targeting, see D. Burke et al., Methods in yeast Genetics— A cold spring harbor laboratory course Manual (2000).
In some embodiments, (a) disrupted gene(s) in which the above-mentioned DNA construct(s) is/are intended to be inserted may advantageously comprises one or more selectable markers useful for the selection of transformed microbial cells. Preferably, said selectable marker(s) are comprised in the DNA construct(s) according to the present invention.
In some embodiments, the selectable marker is an antibiotic resistance marker. Illustrative examples of antibiotic resistance markers include, but are not limited to the, NAT1, AUR1-C, HPH, DSDA, KAN<R>, and SH BLE gene products. The NAT 1 gene product from S. noursei confers resistance to nourseothricin; the AUR1-C gene product from Saccharomyces cerevisiae confers resistance to Auerobasidin A (AbA); the HPH gene product of Klebsiella pneumonia confers resistance to Hygromycin B; the DSDA gene product of E. coli allows cells to grow on plates with D-serine as the sole nitrogen source; the KAN<R> gene of the Tn903 transposon confers resistance to G418; and the SH BLE gene product from Streptoalloteichus hindustanus confers resistance to Zeocin (bleomycin).
In some embodiments, the antibiotic resistance marker is deleted after the genetically modified microbial cell of the invention is isolated. The man skilled in the art is able to choose suitable marker in specific genetic context.
In some embodiments, the selectable marker rescues an auxotrophy (e.g., a nutritional auxotrophy) in the genetically modified microbial cell. In such embodiments, a parent microbial cell comprises a functional disruption in one or more gene products that function in an amino acid or nucleotide biosynthetic pathway, such as, for example, the HIS3, LEU2, LYS1, LYS2, MET 15, TRP1, ADE2, and URA3 gene products in yeast, which renders the parent microbial cell incapable of growing in media without supplementation with one or more nutrients (auxotrophic phenotype). The auxotrophic phenotype can then be rescued by transforming the parent microbial cell with a chromosomal integration encoding a functional copy of the disrupted gene product (NB: the functional copy of the gene can originate from close species, such as Kluveromyces, Candida etc.), and the genetically modified microbial cell generated can be selected for based on the loss of the auxotrophic phenotype of the parent microbial cell.
For each of the nucleic acid sequences comprising a promoter sequence, a coding sequence (e.g. an enzyme coding sequence), or a terminator sequence, reference sequences are described herein. The present description also encompasses nucleic acid sequences having specific percentages of nucleic acid identity, with a reference nucleic acid sequence.
For each or the amino acid sequences of interest, reference sequences are described herein. The present description also encompasses amino acid sequences (e.g. enzyme amino acid sequences), having specific percentages of amino acid identity, with a reference amino acid sequence.
For obvious reasons, in all the present description, a specific nucleic acid sequence or a specific amino acid sequence which complies with, respectively, the considered nucleotide or amino acid identity, should further lead to obtaining a protein (or enzyme) which displays the desired biological activity. As used herein, the “percentage of identity” between two nucleic acid sequences or between two amino acid sequences is determined by comparing both optimally aligned sequences through a comparison window.
The portion of the nucleotide or amino-acid sequence in the comparison window may thus include additions or deletions (for example “gaps”) as compared to the reference sequence (which does not include these additions or these deletions) so as to obtain an optimal alignment between both sequences.
The identity percentage is calculated by determining the number of positions at which an identical nucleic base, or an identical amino-acid residue, can be noted for both compared sequences, then by dividing the number of positions at which identity can be observed between both nucleic bases, or between both amino-acid residues, by the total number of positions in the comparison window, then by multiplying the result by hundred to obtain the percentage of nucleotide identity between the two sequences or the percentage of amino acid identity between the two sequences.
The comparison of the sequence optimal alignment may be performed by a computer using known algorithms.
Most preferably, the sequence identity percentage is determined using the CLUSTAL W software (version 1.82) the parameters being set as follows: (1) CPU MODE=ClustalW mp; (2) ALIGNMENT=“full”; (3) OUTPUT FORMAT=“aln w/numbers”; (4) OUTPUT ORDER=“aligned”; (5) COLOR ALIGNMENT=“no”; (6) KTUP (word size)=“default”; (7) WINDOW LENGTH=“default”; (8) SCORE TYPE=“percent”; (9) TOPDIAG=“default”; (10) PAIRGAP=“default”; (11) PHYLOGENETIC TREE/TREE TYPE=“none”; (12) MATRIX=“default”; (13) GAP OPEN=“default”; (14) END GAPS=“default”; (15) GAP EXTENSION=“default”; (16) GAP DISTANCES=“default”; (17) TREE TYPE=“cladogram” and (18) TREE GRAP DISTANCES=“hide”.
The “fermentation” or “culture” is generally conducted in fermenters with an appropriate culture medium adapted to the microorganism being cultivated, containing at least one simple carbon source, and if necessary co-substrates.
Microorganisms disclosed herein may be grown in fermentation media for the production of a product from oxaloacetate. For maximal production of ectoine, the microorganism strains used as production hosts preferably have a high rate of carbohydrate utilization. These characteristics may be conferred by mutagenesis and selection, genetic engineering, or may be natural. Fermentation media, or “culture medium”, for the present cells may contain at least about 10 g/L of glucose. Additional carbon substrates may include but are not limited to monosaccharides such as fructose, mannose, xylose and arabinose; oligosaccharides such as lactose maltose, galactose, or sucrose; polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate cornsteep liquor, sugar beet molasses, and barley malt. Other carbon substrates may include glycerol.
Hence, it is contemplated that the source of carbon utilized in the present invention may encompass a wide variety of carbon containing substrates and will only be limited by the choice of organism.
Although it is contemplated that all of the above-mentioned carbon substrates and mixtures thereof are suitable in the present invention, preferred carbon substrates are glucose, fructose, and sucrose, or mixtures of these with C5 sugars such as xylose and/or arabinose for microorganisms modified to use C5 sugars, and more particularly glucose.
A preferred carbon substrate is glucose.
In addition to an appropriate carbon source, fermentation media may contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for the production of the desired product.
Besides, additional genetic modifications suitable for the growth of recombinant microorganisms according to the invention may be considered.
The terms “Aerobic conditions” refers to concentrations of oxygen in the culture medium that are sufficient for an aerobic or facultative anaerobic microorganism to use di-oxygene as a terminal electron acceptor.
“Microaerobic condition” refers to a culture medium in which the concentration of oxygen is less than that in air, i.e. oxygen concentration up to 6% 02.
An “appropriate culture medium” designates a medium (e.g. a sterile, liquid medium) comprising nutrients essential or beneficial to the maintenance and/or growth of the cell such as carbon sources or carbon substrate, nitrogen sources, for example, peptone, yeast extracts, meat extracts, malt extracts, urea, ammonium sulfate, ammonium chloride, ammonium nitrate and ammonium phosphate; phosphorus sources, for example, monopotassium phosphate or dipotassium phosphate; trace elements (e.g., metal salts), for example magnesium salts, cobalt salts and/or manganese salts; as well as growth factors such as amino acids, vitamins, growth promoters, and the like. The term “carbon source” or “carbon substrate” or “source of carbon” according to the present invention denotes any source of carbon that can be used by those skilled in the art to support the normal growth of a microorganism, including hexoses (such as glucose, galactose or lactose), pentoses, monosaccharides, oligosaccharides, disaccharides (such as sucrose, cellobiose or maltose), molasses, starch or its derivatives, cellulose, hemicelluloses and combinations thereof.
General features of genetic modifications introduced according to the invention


    
    
        Genes are over expressed by two kinds of non mutually exclusive modifications:
        
            Placing them under the control of a strong promoter; and/or
            Inserting a plurality of copies of the considered gene.
        
        
        All the genome modifications are inserted in yeast according to known genetic engineering techniques:
        The successive genes included in a gene construct that is introduced in the yeast genome according to the invention are of the following structure:
    
    


Prom1-ORF1-term1-ORF2-gene2-term2- . . . / . . . -Promn-ORFn-termn, wherein:

    
    
        Prom1 is a sequence regulating the expression of the coding sequence ORF1,
        ORF1 is a nucleic acid sequence encoding a desired protein PROT1, and especially a desired enzyme PROT1,
        Term1 is a transcription terminator sequence that mediates transcriptional termination by providing signals in the newly synthesized mRNA that trigger processes which release the mRNA from the transcriptional complex, and
        “1”, “2”, . . . / . . . “n” may or may not describe the same ORF (Open Reading Frame), promoter or terminator. The order of the genes does not matter. “n” is an integer usually ranging from 5 and 20. These constructs are inserted in one of the yeast chromosome at a controlled location. In some embodiments, the insertion site is not essential for the functionality of the inserted construct, nor for the viability of the resulting genetically modified yeast.
        When the yeast is for example Saccharomyces cerevisiae, genes introduced in the yeast genome and originating from other organisms than Saccharomyces cerevisiae are generally “transcoded” (generally codon-optimized”), meaning that these genes are synthesized with an optimal codon usage for expression in S. cerevisiae. The nucleotide sequence (and not the protein sequence) of some genes from S. cerevisiae has also been modified (“transcoded”) to minimize recombination with an endogenous copy of the said gene.
        Genes may be deleted through standard procedures used in yeast genetic engineering. In some embodiments, the genes targeted for deletion may be interrupted by insertion of one of the above described gene constructs, or alternatively the genes targeted for deletion are replaced by a short stretch of nucleotide.
        Down regulating gene expression may be obtained by disrupting the endogenous copy of the gene and replacing it with a copy of the ORF under the control of a weak promoter. A list and sequences of weak promoters is described elsewhere in the present specification.
        A gene may be rendered “inducible or repressible” by deleting the endogenous copy of the gene (if necessary) and placing a new copy of the ORF under the control of an inducible or repressible promoter. An inducible or repressible promoter is a promoter which activity is modulated or controlled, i.e. either increased or decreased upon a change in the environmental conditions or external stimuli. Induction or repression may be artificially controlled, which encompasses induction or repression by abiotic factors such as chemical compounds not found naturally in the organism of interest, light, oxygen levels, heat or cold. A list and sequence of inducible or repressible promoters is described elsewhere in the present specification.
        As already specified elsewhere herein, a protein may be underexpressed by destabilization by using “the degron” technology which is described in Yu et al. 2015, (Current Opinion in Biotechnology, Vol. 36: 199-204). In brief this technology consists in introducing in the protein sequence a modification that targets it for degradation. It can consist only in the two first amino acids following the principle known as the N-end rule, or a larger sequence targeting the whole protein to the ubiquitin-preoteasome degradation pathway.

Recombinant Yeast According to the Invention

    
    


The inventors have conceived recombinant microorganisms, and especially recombinant yeasts, having an increased ability of producing ectoine.
The present invention relates to recombinant yeasts having an increased ectoine production, and wherein the increased ectoine production is obtained through a plurality of alterations that have been introduced in the genome thereof, by genetic engineering methods.
This invention pertains to an ectoine-producing recombinant yeast, in the genome of which:

    
    
        (A) (i) at least one nucleic acid encoding an aspartokinase HOM3 is overexpressed and/or is under the control of an inducible or repressible promoter; and/or
        
            (ii) at least one nucleic acid encoding an aspartate kinase AK is overexpressed and/or is under the control of an inducible or repressible promoter;
        
        
        (B) at least one nucleic acid encoding an aspartate semi-aldehyde dehydrogenase HOM2 and/or at least one nucleic acid encoding an aspartate semi-aldehyde dehydrogenase HOM2 that can use as coenzyme both NAD and NADP is overexpressed and/or is under the control of an inducible or repressible promoter;
        (C) at least one nucleic acid encoding a diaminobutyrate aminotransferase EctB is overexpressed and/or is under the control of an inducible or repressible promoter;
        (D) (i) at least one nucleic acid encoding an homoserine-O-acetyltransferase MET2 is overexpressed and/or is under the control of an inducible or repressible promoter;
        
            (ii) at least one nucleic acid encoding an homoserine-O-acetyltransferase METX is overexpressed and/or is under the control of an inducible or repressible promoter, and/or
            (iii) at least one nucleic acid encoding a diaminobutyric acid acetyltransferase EctA is overexpressed and/or is under the control of an inducible or repressible promoter;
        
        
        (E) at least one nucleic acid encoding an ectoine synthase EctC is overexpressed and/or is under the control of an inducible or repressible promoter;
        (F) (i) at least one, preferably all, endogenous nucleic acid encoding an homoserine dehydrogenase HOM6 has been deleted, and/or
        
            (ii) at least one, preferably all, nucleic acid encoding an homoserine dehydrogenase HOM6 is independently:
        
        
        under the control of an inducible or repressible promoter;
        under the control of a weak promoter; and/or
        in a destabilized form.
    
    


The inventors have found that an increased production of ectoine by yeast cells may be reached by introducing in the genome of these yeast cells a plurality of genetic alterations. As it is fully described herein, the said plurality of genetic alterations encompass an overexpression of certain genes, a controlled expression of certain other genes, as well as repression or deletion of further other genes.
The increased ectoine production by yeast cells has been reached by the inventors by optimizing the metabolism of oxaloacetate and acetyl-CoA, so as to direct the subsequent artificially modified metabolic pathway mainly towards ectoine production whereas in the same time maintaining an optimal viability of the resulting genetically modified yeast cells.
After a lengthy research time period, the present inventors have determined that a high ectoine production by yeast cells is obtained by increasing the conversion of oxaloacetate into the successive intermediate metabolites phospho-aspartyl and aspartyl-semialdehyde, and additionally enhancing the conversion of aspartyl-semialdehyde into ectoine, while, notably, maintaining a redox status and more specifically an adapted NADH/NADPH balance allowing a good viability of the resulting recombinant yeast cells. This last point is essential and represented a significant challenge for the inventors throughout their research work.
The proposed solution according to the invention unexpectedly allows maintaining a viable NADH/NADPH equilibrium in the yeast cells throughout the ectoine-production pathway through the consumption of less reducing power, the comsumption of reducing power in the form of NADH rather than NADPH, and/or the production of NADH instead of NADPH.
As disclosed in detail in the present specification, the resulting recombinant yeast cells are genetically modified so as to effect an over expression and/or a controlled expression of (i) an aspartokinase-encoding gene (HOM3) and/or of (ii) an aspartate kinase-encoding gene (AK), in particular of an aspartate kinase-encoding gene (AK), preferably an over expression of an aspartate kinase gene (AK).
Further, a recombinant yeast according to the invention comprises further genetic modifications for an optimal use of the intermediate metabolite phospho-aspartyl for aspartyl-semialdehyde production, the said further genetic modifications comprising an over expression and/or the controlled expression of an aspartate semi-aldehyde dehydrogenase-encoding gene (HOM2) and/or of a gene encoding an aspartate semi-aldehyde dehydrogenase that can use as coenzyme both NADH and NADPH.
Moreover, a recombinant yeast according to the invention comprises further genetic modifications for an optimal use of the intermediate metabolite aspartyl-semialdehyde for ectoine production, the said further genetic modifications comprising (i) an over expression and/or the controlled expression of a diaminobutyrate aminotransferase gene (EctB), (ii) an over expression and/or the controlled expression of a homoserine O-acetyltransferase-encoding gene (MET2; METX) and/or of a diaminobutyric acid acetyltransferase gene (EctA), (iii) an over expression and/or the controlled expression of an ectoine synthase gene (EctC) and (iv) the under expression and/or the controlled expression of an homoserine dehydrogenase gene (HOME).
In some embodiments of a recombinant yeast according to the invention, the said yeast comprises further genetic modifications for an optimal use of the intermediate metabolite oxaloacetate for aspartate production, the said further genetic modifications comprising (i) an over expression and/or the controlled expression of an aspartate transaminase gene (AAT2) and/or (ii) an over expression and/or the controlled expression of a glutamate dehydrogenase that converts oxo-glutarate to glutamate gene (GDH).
In some embodiments of a recombinant yeast according to the invention, the said yeast comprises further genetic modifications for an optimal secretion of the produced ectoine, the said further genetic modifications comprising (i) the under expression and/or the controlled expression of a general amino acid permease gene (AGP3), (ii) the under expression and/or the controlled expression of a branched-chain amino-acid permease 3 gene (BAP3), (iii) the under expression and/or the controlled expression of a branched-chain amino-acid permease 2 gene (BAP2), (iv) the under expression and/or the controlled expression of a general amino acid permease gene (GAP1), (v) the under expression and/or the controlled expression of a high-affinity glutamine permease gene (GNP1), (vi) the under expression and/or the controlled expression of a general amino acid permease gene (AGP1), (vii) the under expression and/or the controlled expression of a low-affinity methionine permease gene (MUP3; MUP1), (viii) the over expression and/or the controlled expression of a probable transporter gene (AQR1) and/or (ix) the over expression and/or the controlled expression of a polyamine transporter 1 gene (TPO1).
In a particular embodiment, the at least one nucleic acid encoding a general amino acid permease, a branched-chain amino-acid permease 3, a branched-chain amino-acid permease 2, a general amino acid permease GAP1, a high-affinity glutamine permease GNP1, a general amino acid permease AGP1, a low-affinity methionine permease MUP3 and a high-affinity methionine permease MUP1 are, independently, nucleic acid from a yeast, preferably from Saccharomyces cerevisiae. 
A recombinant yeast according to the invention produces ectoine with a higher yield than the parent yeast which does not contain the genetic modifications described above.
A recombinant yeast according to the invention has been genetically engineered so as to promote the expression of enzymes utilizing NADH rather than NADPH, such as an appropriate glutamate dehydrogenase or an appropriate aspartate semialdehyde dehydrogenase.
In some embodiments of a recombinant yeast according to the invention, the aspartate-semialdehyde dehydrogenase that are over expressed consist of the S. cerevisiae endogenous gene that is placed under the control of strong promoters and/or of inducible or repressible promoters.
In some embodiments, the aspartate-semialdehyde dehydrogenase is preferably encoded by the S. cerevisiae HOM2 gene.
In some embodiments, the aspartate-semialdehyde dehydrogenase is most preferably encoded by a variant of the S. cerevisiae HOM2 gene, which genes codes for a mutated HOM2 protein that uses both NAD and NADP, as it is shown in the examples herein. Such gene variant is for example illustrated in the examples and is called HOM2-2. It corresponds to the S. cerevisiae HOM2 gene mutated as discussed here-under.
The nature of the mutations aiming several amino acid residues in the aspartate-semialdehyde dehydrogenase variant in order to relaxe the high selectivity of HOM2 for NADP as coenzyme and enhance the affinity of the enzyme for NAD are known to the man skilled in the art and can for example be found in Faehnle, C. R. et al., Journal of Molecular Biology 1055-1068 (2005). In particular, the mutation S39 to E39 corresponding to the replacement of the nucleotides TCT in position 115 to 117 of the nucleotide sequence by the nucleotides GAG can be mentioned.
According to the nomenclature of the amino acids well known to the man skilled in the art, S represents a Serine and E represents a Glutamic acid.
In some embodiments, the aspartokinase is most preferably encoded by the S. cerevisiae HOM3 gene, as it is shown in the examples herein.
Aspartokinase-Encoding Gene Over Expression and/or Controlled Expression
In some embodiments of a recombinant yeast according to the invention, over expression of an aspartokinase-encoding gene is obtained by inserting, at selected location(s) of the yeast genome, one or more copies of an expression cassette comprising an aspartokinase coding sequence. Aspartokinase and an aspartokinase-encoding gene that are encompassed by the invention are detailed elsewhere in the present specification.
In some of these embodiments, the said one or more copies of an expression cassette comprising an aspartokinase coding sequence comprise(s) regulatory sequences allowing a strong expression of the aspartokinase, such as a strong promoter that is functional in yeast cells.
In addition to or as an alternative to these embodiments of a recombinant yeast according to the invention, at least one aspartokinase-encoding gene can be under the control of an inducible or repressible promoter that is functional in yeast cells.
In these embodiments, a controlled expression of an aspartokinase-encoding gene can be obtained by inserting, at the location of the natural yeast aspartokinase open reading frame, an inducible regulatory sequence, such as an inducible or repressible promoter, that replaces the endogenous promoter initially present in the yeast genome at this genome location.
Without wishing to be bound by any particular theory, the inventors believe that with over expression of an aspartokinase-encoding gene, a controlled level of conversion of aspartate into aspartyl phosphate (Aspartyl-P), also termed phospho-aspartyl, is obtained that shall contribute to the high level of viability of a recombinant yeast according to the invention. The same applies when at least one aspartokinase coding sequence is under the control of an inducible or repressible promoter.
In some preferred embodiments, the said aspartokinase-encoding gene is the HOM3 gene from Saccharomyces cerevisiae, as shown in the examples herein.
In preferred embodiments, the said aspartokinase-encoding gene is placed under the control of the strong promoter pCCW12 or of the inducible or repressible promoter pCUP-1-1.
Illustratively, the aspartokinase gene may be inserted within the HOME gene and/or within the SAM3 gene, as it is shown in the examples herein.
Aspartate Kinase-Encoding Gene Over Expression and/or Controlled Expression
Alternatively or in complement to the over expression and/or controlled expression of an aspartokinase as discussed here-above, a recombinant yeast according to the invention can also be such that it comprises the over expression and/or controlled expression of an aspartate kinase-encoding gene.
Accordingly, in preferred embodiments of a recombinant yeast according to the invention, over expression of an aspartate kinase-encoding gene is obtained by inserting, at selected location(s) of the yeast genome, one or more copies of an expression cassette comprising a aspartate kinase coding sequence. Aspartate kinase and an aspartate kinase-encoding gene that are encompassed by the invention are detailed elsewhere in the present specification.
In some of these embodiments, the said one or more copies of an expression cassette comprising an aspartate kinase-coding sequence comprise regulatory sequences allowing a strong expression of the aspartate kinase, such as a strong promoter that is functional in yeast cells.
In addition to or as an alternative to these embodiments of a recombinant yeast according to the invention, at least one aspartate kinase-encoding gene can be under the control of an inducible or repressible promoter that is functional in yeast cells.
Without wishing to be bound by any particular theory, the inventors believe that with a controlled expression of an aspartate kinase-encoding gene, a controlled level of conversion of aspartate into aspartyl phosphate (Aspartyl-P) is obtained that shall contribute to the high level of viability of a recombinant yeast according to the invention.
In preferred embodiments, the said aspartate kinase-encoding gene is the AK gene from Bacillus subtilis, as shown in the examples herein.
In preferred embodiments, the said aspartate kinase-encoding gene is placed under the control of the inducible or repressible promoter pACU7.
Illustratively, the aspartate kinase gene may be inserted within the TRP1 gene, as it is shown in the examples herein.
Aspartate-Semialdehyde Dehydrogenase-Encoding Gene Over Expression and/or Controlled Expression
In preferred embodiments of a recombinant yeast according to the invention, over expression of an aspartate-semialdehyde dehydrogenase-encoding gene is obtained by inserting, at selected location(s) of the yeast genome, one or more copies of an expression cassette comprising an aspartate-semialdehyde dehydrogenase coding sequence. Aspartate-semialdehyde dehydrogenase and an aspartate-semialdehyde dehydrogenase-encoding gene that are encompassed by the invention are detailed elsewhere in the present specification.
In some of these embodiments, the said one or more copies of an expression cassette comprising an aspartate-semialdehyde dehydrogenase coding sequence comprise(s) regulatory sequences allowing a strong expression of the aspartate-semialdehyde dehydrogenase, such as a strong promoter that is functional in yeast cells.
In addition to or as an alternative to these embodiments of a recombinant yeast according to the invention, at least one aspartate-semialdehyde dehydrogenase-encoding gene can be under the control of an inducible or repressible promoter that is functional in yeast cells.
Without wishing to be bound by any particular theory, the inventors believe that over expression of an aspartate-semialdehyde dehydrogenase may enhance the conversion of the intermediate metabolite aspartyl phosphate (Aspartyl-P) into aspartyl-semialdehyde. The same applies when at least one aspartate-semialdehyde dehydrogenase coding sequence is under the control of an inducible or repressible promoter.
In some embodiments, the aspartate-semialdehyde dehydrogenase may be an enzyme variant that uses both NADH or NADPH for catalyzing the conversion of aspartyl phosphate (Aspartyl-P) into aspartyl-semialdehyde.
In some preferred embodiments, the said aspartate-semialdehyde dehydrogenase-encoding gene is the HOM2 gene from Saccharomyces cerevisiae, or alternatively a variant of HOM2 utilizing both NADH and NADPH as shown in the examples herein and discussed previously.
In preferred embodiments, the said aspartate semi-aldehyde dehydrogenase-encoding gene is placed under the control of the inducible or repressible promoter pACU5 or the strong promoter pCCW12.
Illustratively, the aspartate-semialdehyde dehydrogenase gene may be inserted within the HIS3 gene, and/or within the MUP3 gene, as it is shown in the examples herein.
Diaminobutyrate Aminotransferase-Encoding Gene Over Expression and/or Controlled Expression
In preferred embodiments of a recombinant yeast according to the invention, over expression of a diaminobutyrate aminotransferase-encoding gene is obtained by inserting, at selected location(s) of the yeast genome, one or more copies of an expression cassette comprising a diaminobutyrate aminotransferase coding sequence. Diaminobutyrate aminotransferase and a diaminobutyrate aminotransferase-encoding gene that are encompassed by the invention are detailed elsewhere in the present specification.
In some of these embodiments, the said one or more copies of an expression cassette comprising a diaminobutyrate aminotransferase coding sequence comprise(s) regulatory sequences allowing a strong expression of the diaminobutyrate aminotransferase, such as a strong promoter that is functional in yeast cells.
In addition to or as an alternative to these embodiments of a recombinant yeast according to the invention, at least one diaminobutyrate aminotransferase-encoding gene can be under the control of an inducible or repressible promoter that is functional in yeast cells.
Without wishing to be bound by any particular theory, the inventors believe that over expression of an diaminobutyrate aminotransferase may enhance the conversion of the intermediate metabolite aspartyl-semialdehyde into 2,4-diaminobutyrate. The same applies when at least one diaminobutyrate aminotransferase coding sequence is under the control of an inducible or repressible promoter.
In preferred embodiments, the said diaminobutyrate aminotransferase-encoding gene is the EctB gene from Pseudomonas aeruginosa, or the EctB gene from Halomonas elongata (sometimes also named Chromohalobacter salexigens), as shown in the examples herein.
In preferred embodiments, the said diaminobutyrate aminotransferase-encoding gene is placed under the control of the strong promoter pCCW12 and/or the strong promoter pTDH3.
Illustratively, the diaminobutyrate aminotransferase gene may be inserted within the HOME gene and/or within the SAM3 gene and/or within the MUP3 gene and/or within the URA3 gene, as it is shown in the examples herein.
Homoserine-O-Acetyltransferase-Encoding Gene Over Expression and/or Controlled Expression
In preferred embodiments of a recombinant yeast according to the invention, over expression of a homoserine-O-acetyltransferase-encoding gene is obtained by inserting, at selected location(s) of the yeast genome, one or more copies of an expression cassette comprising a homoserine-O-acetyltransferase coding sequence. Homoserine-O-acetyltransferase and a homoserine-O-acetyltransferase-encoding gene that are encompassed by the invention are detailed elsewhere in the present specification.
In some of these embodiments, the said one or more copies of an expression cassette comprising a homoserine-O-acetyltransferase coding sequence comprise regulatory sequences allowing a strong expression of the homoserine-O-acetyltransferase, such as a strong promoter that is functional in yeast cells.
In addition to or as an alternative to these embodiments of a recombinant yeast according to the invention, at least one homoserine-O-acetyltransferase-encoding gene can be under the control of an inducible or repressible promoter that is functional in yeast cells.
Without wishing to be bound by any particular theory, the inventors believe that over expression of a homoserine-O-acetyltransferase allows, and consequently increases, the level of conversion of the intermediate metabolite 2,4-diaminobutyrate into acetyl-2,4-diaminobutyrate, in the presence of acetyl-CoA. The same applies when at least one homoserine-O-acetyltransferase coding sequence is under the control of an inducible or repressible promoter.
In preferred embodiments, the said homoserine-O-acetyltransferase-encoding gene is the MET2 gene from Saccharomyces cerevisiae, as shown in the examples herein.
In preferred embodiments, the said homoserine-O-acetyltransferase-encoding gene is the METX gene from Corynebacterium glutamicum, as shown in the examples herein.
In a particularly preferred embodiment, a recombinant yeast according to the invention comprises at least one homoserine-O-acetyltransferase-encoding gene which is the MET2 gene from Saccharomyces cerevisiae and at least one homoserine-O-acetyltransferase-encoding gene which is the METX gene from Corynebacterium glutamicum. 
In preferred embodiments, the said homoserine-O-acetyltransferase-encoding gene is, independently for each copy of said gene if multiple copies are present, placed under the control of a strong promoter pPDC1 or the inducible or repressible promoter pACU6.
Illustratively, the homoserine-O-acetyltransferase gene may be inserted within the SAM3 gene and/or within the HIS3 gene, as it is shown in the examples herein.
Diaminobutyric Acid Acetyltransferase-Encoding Gene Over Expression and/or Controlled Expression
In preferred embodiments of a recombinant yeast according to the invention, over expression of a diaminobutyric acid acetyltransferase-encoding gene is obtained by inserting, at selected location(s) of the yeast genome, one or more copies of an expression cassette comprising a diaminobutyric acid acetyltransferase coding sequence. A diaminobutyric acid acetyltransferase and a diaminobutyric acid acetyltransferase-encoding gene that are encompassed by the invention are detailed elsewhere in the present specification.
In some of these embodiments, the said one or more copies of an expression cassette comprising a diaminobutyric acid acetyltransferase coding sequence comprise(s) regulatory sequences allowing a strong expression of the diaminobutyric acid acetyltransferase, such as a strong promoter that is functional in yeast cells.
In addition to or as an alternative to these embodiments of a recombinant yeast according to the invention, at least one diaminobutyric acid acetyltransferase-encoding gene can be under the control of an inducible or repressible promoter that is functional in yeast cells.
Without wishing to be bound by any particular theory, the inventors believe that over expression of a diaminobutyric acid acetyltransferase allows, and consequently increases, the level of conversion of the intermediate metabolite 2,4-diaminobutyrate into acetyl-2,4-diaminobutyrate, in the presence of acetyl-CoA. The same applies when at least one diaminobutyric acid acetyltransferase coding sequence is under the control of an inducible or repressible promoter.
In preferred embodiments, the said diaminobutyric acid acetyltransferase-encoding gene is the EctA gene from Halomonas elongata (sometimes also named Chromohalobacter salexigens), as shown in the examples herein.
In preferred embodiments, the said diaminobutyric acid acetyltransferase-encoding gene is placed under the control of the strong promoter pPDC1.
Illustratively, the diaminobutyric acid acetyltransferase gene may be inserted within the LYP1 gene and/or within the MUP3 gene, as it is shown in the examples herein.
In a particular embodiment, a recombinant yeast according to the invention is such that its genome comprises:

    
    
        at least one nucleic acid encoding an homoserine-O-acetyltransferase METX over expressed and/or under the control of an inducible or repressible promoter, and preferably under the control of an inducible or repressible promoter; and
        at least one nucleic acid encoding a diaminobutyric acid acetyltransferase EctA over expressed and/or under the control of an inducible or repressible promoter, and preferably under the control of an inducible or repressible promoter.

Ectoine Synthase-Encoding Gene Over Expression and/or Controlled Expression

    
    


In preferred embodiments of a recombinant yeast according to the invention, over expression of an ectoine synthase-encoding gene is obtained by inserting, at selected location(s) of the yeast genome, one or more copies of an expression cassette comprising an ectoine synthase coding sequence. An ectoine synthase and an ectoine synthase-encoding gene that are encompassed by the invention are detailed elsewhere in the present specification.
In some of these embodiments, the said one or more copies of an expression cassette comprising an ectoine synthase coding sequence comprise(s) regulatory sequences allowing a strong expression of the ectoine synthase, such as a strong promoter that is functional in yeast cells.
In addition to or as an alternative to these embodiments of a recombinant yeast according to the invention, at least one ectoine synthase-encoding gene can be under the control of an inducible or repressible promoter that is functional in yeast cells.
Without wishing to be bound by any particular theory, the inventors believe that over expression of an ectoine synthase allows, and consequently increases, the level of conversion of the intermediate metabolite acetyl-2,4-diaminobutyrate into ectoine. The same applies when at least one ectoine synthase coding sequence is under the control of an inducible or repressible promoter.
In preferred embodiments, the said ectoine synthase-encoding gene is the EctC gene from Halomonas elongata, as shown in the examples herein.
In preferred embodiments, the said ectoine synthase-encoding gene is placed under the control of the strong promoter pTDH3 and/or the strong promoter pTEF1.
Illustratively, the ectoine synthase gene may be inserted within the LYP1 gene and/or within the MUP3 gene and/or within the URA3 gene, as it is shown in the examples herein.
Deletion or Under Expression of Homoserine Dehydrogenase
A recombinant yeast according to the invention is further defined as having a genome in which:

    
    
        (i) at least one, preferably all, endogenous nucleic acid encoding an homoserine dehydrogenase HOM6 has been deleted and/or interrupted, and/or
        (ii) at least one, preferably all, nucleic acid encoding an homoserine dehydrogenase HOM6 is independently:
        
            under the control of an inducible or repressible promoter;
            under the control of a weak promoter; and/or
            in a destabilized form.
        
        
    
    


Without wishing to be bound by any particular theory, the inventors believe that an under expression of an homoserine dehydrogenase gene shall increase 2,4-diaminobutyrate production by the recombinant yeast by reducing the consumption of the produced aspartyl-semialdehyde by its conversion into homoserine.
In some embodiments, under expression of an homoserine dehydrogenase may be rendered conditional, for example by placing the expression of this gene under the control of repressible regulatory sequences, such as inducible or repressible promoters.
Methods for repressing gene expression, for interrupting target genes or for deleting target genes, are well known from the one skilled in the art.
Homoserine dehydrogenase under expression also encompasses the insertion of a nucleic acid encoding a destabilized homoserine dehydrogenase. A destabilized homoserine dehydrogenase is a variant of homoserine dehydrogenase that is more rapidly degraded within the yeast cell than the parent homoserine dehydrogenase.
In preferred embodiments, a destabilized homoserine dehydrogenase consists of a degron-tagged homoserine dehydrogenase protein.
For example, the homoserine dehydrogenase gene can be interrupted by loxP, or for example by URA3.K1-loxP, and is thus deleted (which can also be termed inactivated).
It can alternatively be interrupted by a cassette comprising genes of interest, as illustrated in the examples as filed.
Aspartokinase (Hom3)
The aspartokinase enzyme is a protein which is described in the art for catalyzing the conversion of L-aspartate in the presence of ATP into 4-phospho-L-aspartate. The aspartokinase encoded by the genome of Saccharomyces cerevisiae may be termed HOM3.
A method implemented to measure the activity level of aspartokinase belongs to the general knowledge of the one skilled in the art.
In this regard, the one skilled in the art may advantageously refer to the method described by Stadtman et al. (1961, J Biol Chem, Vol. 236 (7): 2033-2038).
Preferred aspartokinase in the present specification is an enzyme having an EC number of n° EC 2.7.2.4.
According to a preferred embodiment, the nucleic acid(s) encoding an aspartokinase may be nucleic acid(s) originating from organisms preferably selected in a group comprising prokaryotic organisms and eukaryotic organisms. In some embodiments, the nucleic acid(s) encoding an aspartokinase may be nucleic acid(s) originating from archaebacteria. In some embodiments, the nucleic acid(s) encoding an aspartokinase may be nucleic acid(s) originating from organisms preferably selected from Bacillus subtilis, and yeasts. In some other preferred embodiments, the nucleic acid(s) encoding an aspartokinase may be nucleic acid(s) originating from a yeast, and especially from Saccharomyces cerevisiae. 
According to a yet preferred embodiment, the nucleic acid(s) encoding an aspartokinase may be nucleic acid(s) selected from the group consisting of sequences having at least 25%, advantageously at least 65%, preferably at least 80%, nucleic acid identity with a nucleic acid of SEQ ID NO: 1, and also a biological activity of the same nature. The nucleic acid of SEQ ID NO: 1 encodes an aspartokinase originating from Saccharomyces cerevisiae, that may also be termed HOM3.
A biological activity of the same nature regarding this sequence is the capacity to code for an enzyme that catalyzes the conversion of L-aspartate in the presence of ATP into 4-phospho-L-aspartate.
As described herein, a nucleic acid sequence having at least 25% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.
As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.
As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.
For the amino acid sequence of the aspartokinase from Saccharomyces cerevisiae, the one skilled in the art may refer to the accession number NP010972 in the UniProt database, or to SEQ ID NO. 2 described herein.
According to another particular embodiment, the nucleic acid(s) encoding aspartokinase may be nucleic acid(s) encoding an amino acid sequence selected from the group consisting of sequences having at least 25%, advantageously at least 65%, preferably at least 80%, amino acid identity with the amino acid sequence of SEQ ID NO: 2, and also a biological activity of the same nature. Illustratively, the aspartokinase originating from Aquamarina atlantica has 25% amino acid identity with the aspartokinase of SEQ ID NO. 2.
A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the conversion of L-aspartate in the presence of ATP into 4-phospho-L-aspartate.
As described herein, an amino acid sequence having at least 25% amino acid identity with a reference nucleic acid sequence encompasses amino acid sequences having at least 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference nucleic acid sequence.
As described herein, an amino acid sequence having at least 65% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence.
As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence.
As above-mentioned, the expression level of the aspartokinase in the present invention is regulated by at least one promoter and at least one terminator, such as herein after defined more in details, which are present in 5′ and 3′ position respectively of the nucleic acid sequence encoding the said aspartokinase.
As it is specified elsewhere in the present description, the aspartokinase is overexpressed and/or under the control of an inducible or repressible promoter in a recombinant yeast according to the invention.
In some embodiments, overexpression of the aspartokinase may result from the control of the corresponding gene by a strong promoter within the said recombinant yeast.
In some other embodiments, overexpression of the aspartokinase may result from the presence of a plurality of copies of an aspartokinase-encoding sequence within the genome of the said recombinant yeast.
In still further embodiments, overexpression of aspartokinase may result from both (i) the control of the corresponding gene by a strong promoter within the said recombinant yeast and (ii) the presence of a plurality of copies of an aspartokinase-encoding sequence within the genome the said recombinant yeast.
Aspartate Kinase (AK)
The aspartate kinase enzyme is a protein which is described in the art for catalyzing the conversion of L-aspartate in the presence of ATP into 4-phospho-L-aspartate. The aspartate kinase encoded by the genome of Bacillus subtilis may be termed AK.
A method implemented to measure the activity level of aspartate kinase belongs to the general knowledge of the one skilled in the art and is the same as the one indicated previously for aspartokinase.
According to a preferred embodiment, the nucleic acid(s) encoding an aspartate kinase may be nucleic acid(s) originating from organisms preferably selected in a group comprising prokaryotic organisms and eukaryotic organisms. In some embodiments, the nucleic acid(s) encoding an aspartate kinase may be nucleic acid(s) originating from archaebacteria. In some embodiments, the nucleic acid(s) encoding an aspartate kinase may be nucleic acid(s) originating from organisms preferably selected from Bacillus subtilis, and yeasts. In some other preferred embodiments, the nucleic acid(s) encoding an aspartate kinase may be nucleic acid(s) originating from yeast, and especially from Saccharomyces cerevisiae. 
For the nucleic acid sequence, it may be referred to the one disclosed in the access number NC_000964.3 in the NCBI database.
According to a yet preferred embodiment, the nucleic acid(s) encoding an aspartate kinase may be nucleic acid(s) selected from the group consisting of sequences having at least 25%, advantageously at least 65%, preferably at least 80%, nucleic acid identity with a nucleic acid of SEQ ID NO. 3, and also a biological activity of the same nature. The nucleic acid of SEQ ID NO. 3 encodes an aspartate kinase originating from Bacillus subtilis, that may also be termed AK.
A biological activity of the same nature regarding this sequence is the capacity to code for an enzyme that catalyzes the conversion of L-aspartate in the presence of ATP into 4-phospho-L-aspartate.
As described herein, a nucleic acid sequence having at least 25% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.
As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.
As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.
For the amino acid sequence of the aspartate kinase from Bacillus substilis, the one skilled in the art may refer to the accession number NP_389558.2 in the UniProt database, or to SEQ ID NO. 4 described herein.
According to another particular embodiment, the nucleic acid(s) encoding aspartate kinase may be nucleic acid(s) encoding an amino acid sequence selected from the group consisting of sequences having at least 25%, advantageously at least 65%, preferably at least 80%, amino acid identity with the amino acid sequence of SEQ ID NO. 4, and also a biological activity of the same nature. Illustratively, the aspartate kinase originating from Aquamarina atlantica has 25% amino acid identity with the aspartokinase of SEQ ID NO. 4.
A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the conversion of L-aspartate in the presence of ATP into 4-phospho-L-aspartate.
As described herein, an amino acid sequence having at least 25% amino acid identity with a reference nucleic acid sequence encompasses amino acid sequences having at least 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.
As described herein, an amino acid sequence having at least 65% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature.
As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature.
As above-mentioned, the expression level of the aspartate kinase in the present invention is regulated by at least one promoter and at least one terminator, such as herein after defined more in details, which are present in 5′ and 3′ position respectively of the nucleic acid sequence encoding the said aspartate kinase.
As it is specified elsewhere in the present description, the aspartate kinase is overexpressed and/or under the control of an inducible or repressible promoter in a recombinant yeast according to the invention.
In some embodiments, overexpression of the aspartate kinase may result from the control of the corresponding gene by a strong promoter within the said recombinant yeast.
In some other embodiments, overexpression of the aspartate kinase may result from the presence of a plurality of copies of an aspartate kinase-encoding sequence within the genome of the said recombinant yeast.
In still further embodiments, overexpression of aspartate kinase may result from both (i) the control of the corresponding gene by a strong promoter within the said recombinant yeast and (ii) the presence of a plurality of copies of an aspartate kinase-encoding sequence within the genome the said recombinant yeast.
Aspartate-Semialdehyde Dehydrogenase (HOM2)
The aspartate-semialdehyde dehydrogenase is a protein which is known in the art to catalyze the NADPH-dependent formation of L-aspartate-semialdehyde by the reductive dephosphorylation of L-aspartyl-4-phosphate. The aspartate-semialdehyde dehydrogenase encoded by the genome of Saccharomyces cerevisiae may be termed HOM2.
A method implemented to measure the activity level of aspartate-semialdehyde dehydrogenase belongs to the general knowledge of the one skilled in the art.
Preferred aspartate-semialdehyde dehydrogenase in the present specification is an enzyme having an EC number 1.2.1.11.
According to a preferred embodiment, the nucleic acid(s) encoding an aspartate-semialdehyde dehydrogenase may be nucleic acid(s) originating from organisms preferably selected in a group comprising prokaryotic organisms and eukaryotic organisms. In some embodiments, the nucleic acid(s) encoding an aspartate-semialdehyde dehydrogenase may be nucleic acid(s) originating from archaebacteria. In some preferred embodiments, the nucleic acid(s) encoding an aspartate-semialdehyde dehydrogenase may be nucleic acid(s) originating from yeast, and especially from Saccharomyces cerevisiae. 
According to other preferred embodiment, the nucleic acid encoding an aspartate-semialdehyde dehydrogenase may be a variant or a mutant of the aspartate-semialdehyde dehydrogenase from Saccharomyces cerevisiae, wherein the said variant enzyme or the said mutant enzyme uses both NADH or NADPH for catalyzing reactions. Such variant or mutant enzymes are known in the art and are previously discussed in the present text.
According to a yet preferred embodiment, the nucleic acid(s) encoding an aspartate-semialdehyde dehydrogenase may be nucleic acid(s) selected from the group consisting of sequences having at least 27%, advantageously at least 65%, preferably at least 80%, nucleic acid identity with a nucleic acid selected in a group consisting of the reference nucleic acid sequences of SEQ ID NO: 5 and SEQ ID NO. 6, and also a biological activity of the same nature. The nucleic acids of SEQ ID NO: 5 and SEQ ID NO. 6 encode an aspartate-semialdehyde dehydrogenase originating from Saccharomyces cerevisiae, that may also be collectively termed HOM2 herein.
A biological activity of the same nature regarding this sequence is the capacity to code for an enzyme that catalyzes the NADPH-dependent formation of L-aspartate-semialdehyde by the reductive dephosphorylation of L-aspartyl-4-phosphate.
As described herein, a nucleic acid sequence having at least 27% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequences, and also a biological activity of the same nature.
As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequences, and also a biological activity of the same nature.
As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.
For the amino acid sequence of the aspartate-semialdehyde dehydrogenase from Saccharomyces cerevisiae, the one skilled in the art may refer to the accession number NP010442 in the UniProt database, or to SEQ ID NO. 7 described herein.
According to another particular embodiment, the nucleic acid(s) encoding an aspartate-semialdehyde dehydrogenase may be nucleic acid(s) encoding an amino acid sequence selected from the group consisting of sequences having at least 27%, advantageously at least 65%, preferably at least 80%, amino acid identity with the amino acid sequence of SEQ ID NO: 7, and also a biological activity of the same nature. Illustratively, the aspartate-semialdehyde dehydrogenase originating from Lactobacillus wasatchensis has 27% amino acid identity with the aspartate-semialdehyde dehydrogenase of SEQ ID NO. 7.
A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the NADPH-dependent formation of L-aspartate-semialdehyde by the reductive dephosphorylation of L-aspartyl-4-phosphate.
As described herein, an amino acid sequence having at least 27% amino acid identity with a reference nucleic acid sequence encompasses amino acid sequences having at least 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.
As described herein, an amino acid sequence having at least 65% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature.
As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature.
As above-mentioned, the expression level of the aspartate-semialdehyde dehydrogenase in the present invention is regulated by at least one promoter and at least one terminator, such as herein after defined more in details, which are present in 5′ and 3′ position respectively of the nucleic acid sequence encoding the said aspartate-semialdehyde dehydrogenase.
As it is specified elsewhere in the present description, the aspartate-semialdehyde dehydrogenase is overexpressed and/or under the control of an inducible or repressible promoter in a recombinant yeast according to the invention.
In some embodiments, overexpression of the aspartate-semialdehyde dehydrogenase may result from the control of the corresponding gene by a strong promoter within the said recombinant yeast.
In some other embodiments, overexpression of the aspartate-semialdehyde dehydrogenase may result from the presence of a plurality of copies of a aspartate-semialdehyde dehydrogenase-encoding sequence within the genome of the said recombinant yeast.
In still further embodiments, overexpression of the aspartate-semialdehyde dehydrogenase may result from both (i) the control of the corresponding gene by a strong promoter within the said recombinant yeast and (ii) the presence of a plurality of copies of an aspartate-semialdehyde dehydrogenase-encoding sequence within the genome the said recombinant yeast.
Diaminobutyrate Aminotransferase (EctB)
The diaminobutyrate aminotransferase enzyme is a protein which is described in the art for catalyzing the conversion of aspartyl semialdehyde in the presence of glutamate into 2,4-diaminobutyrate. The diaminobutyrate aminotransferase encoded by the genome of Halomonas elongata may be termed EctB or EctB.He.
A method implemented to measure the activity level of diaminobutyrate aminotransferase belongs to the general knowledge of the one skilled in the art.
In this regard, the one skilled in the art may advantageously refer to the method described by Ono H et al., 1999, Journal of Bacteriology, p 91-99.
Preferred diaminobutyrate aminotransferase in the present specification is an enzyme having an EC number of n° 2.6.1.76.
According to a preferred embodiment, the nucleic acid(s) encoding an diaminobutyrate aminotransferase may be nucleic acid(s) originating from organisms preferably selected in a group comprising prokaryotic organisms and eukaryotic organisms. In some embodiments, the nucleic acid(s) encoding a diaminobutyrate aminotransferase may be nucleic acid(s) originating from archaebacteria. In some embodiments, the nucleic acid(s) encoding a diaminobutyrate aminotransferase may be nucleic acid(s) originating from organisms preferably selected from bacteria, and especially from Pseudomonas aeruginosa, Halomonas elongata or Sporocarcina newyorkensisn, and preferably from Pseudomonas aeruginosa or Halomonas elongata. 
According to a yet preferred embodiment, the nucleic acid(s) encoding a diaminobutyrate aminotransferase may be nucleic acid(s) selected from the group consisting of sequences having at least 35%, advantageously at least 65%, preferably at least 80%, nucleic acid identity with a nucleic acid of SEQ ID NO. 8 or SEQ ID NO. 9, and also a biological activity of the same nature.
A biological activity of the same nature regarding this sequence is the capacity to code for an enzyme that catalyzes the conversion of aspartyl semialdehyde in the presence of glutamate into 2,4-diaminobutyrate.
As described herein, a nucleic acid sequence having at least 35% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.
As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.
As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.
For the amino acid sequence of the diaminobutyrate aminotransferase from Halomonas elongata, the one skilled in the art may refer to the accession number WP_013332345.1 in the UniProt database, or to SEQ ID NO. 10 or SEQ ID NO. 11 described herein.
According to another particular embodiment, the nucleic acid(s) encoding diaminobutyrate aminotransferase may be nucleic acid(s) encoding an amino acid sequence selected from the group consisting of sequences having at least 35%, advantageously at least 65%, preferably at least 80%, amino acid identity with the amino acid sequence of SEQ ID NO. 10 or SEQ ID NO. 11, and also a biological activity of the same nature.
A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the conversion of aspartyl semialdehyde in the presence of glutamate into 2,4-diaminobutyrate.
As described herein, an amino acid sequence having at least 35% amino acid identity with a reference nucleic acid sequence encompasses amino acid sequences having at least 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.
As described herein, an amino acid sequence having at least 65% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature.
As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature.
As above-mentioned, the expression level of the diaminobutyrate aminotransferase in the present invention is regulated by at least one promoter and at least one terminator, such as herein after defined more in details, which are present in 5′ and 3′ position respectively of the nucleic acid sequence encoding the said diaminobutyrate aminotransferase.
As it is specified elsewhere in the present description, the diaminobutyrate aminotransferase is overexpressed and/or under the control of an inducible or repressible promoter in a recombinant yeast according to the invention.
In some embodiments, overexpression of the diaminobutyrate aminotransferase may result from the control of the corresponding gene by a strong promoter within the said recombinant yeast.
In some other embodiments, overexpression of the diaminobutyrate aminotransferase may result from the presence of a plurality of copies of a diaminobutyrate aminotransferase-encoding sequence within the genome of the said recombinant yeast.
In still further embodiments, overexpression of diaminobutyrate aminotransferase may result from both (i) the control of the corresponding gene by a strong promoter within the said recombinant yeast and (ii) the presence of a plurality of copies of a diaminobutyrate aminotransferase-encoding sequence within the genome of the said recombinant yeast.
Homoserine O-Acetyltransferase (MET2; METX)
The homoserine O-acetyl transferase enzyme is a protein which is described in the art for catalyzing the reaction between Acetyl-CoA and 2,4-diaminobutyrate into CoA and Acetyl-2,4-diaminobutyrate. The homoserine O-acetyl transferase encoded by the genome of Saccharomyces cerevisiae may be termed MET2.
A method implemented to measure the activity level of homoserine O-acetyltransferase belongs to the general knowledge of the one skilled in the art.
In this regard, the one skilled in the art may advantageously refer to the method described by Shuzo Yamagata (1987, The Journal of Bacteriology, Vol. 169(8): 3458-3463.
Preferred homoserine O-acetyltransferase in the present specification is an enzyme having an EC number of n° EC 2.3.1.31.
According to a preferred embodiment, the nucleic acid(s) encoding a homoserine O-acetyltransferase may be nucleic acid(s) originating from organisms preferably selected in a group comprising prokaryotic organisms and eukaryotic organisms. In some embodiments, the nucleic acid(s) encoding a homoserine O-acetyltransferase may be nucleic acid(s) originating from archaebacteria. In some embodiments, the nucleic acid(s) encoding a homoserine O-acetyltransferase may be nucleic acid(s) originating from organisms preferably selected from Corynebacterium glutamicum, and yeasts. In some other preferred embodiments, the nucleic acid(s) encoding a homoserine O-acetyltransferase may be nucleic acid(s) originating from yeast, and especially from Saccharomyces cerevisiae. 
According to a particular embodiment, the nucleic acid encoding an homoserine-O-acetyltransferase METX are nucleic acid from a bacterium, in particular from a bacterium selected, independently, from the group consisting of Corynebacterium glutamicum, Escherichia coli, Haemophilius influenza, Streptomyces lavendulae, Leptospira interrogans, Streptococcus pneumonia and Mycobacterium tuberculosis. 
According to a yet preferred embodiment, the nucleic acid(s) encoding a homoserine O-acetyltransferase may be nucleic acid(s) selected from the group consisting of sequences having at least 27%, advantageously at least 65%, preferably at least 80%, nucleic acid identity with a nucleic acid of SEQ ID NO: 12, and also a biological activity of the same nature. The nucleic acid of SEQ ID NO: 12 encodes a homoserine O-acetyltransferase originating from Saccharomyces cerevisiae, that may also be termed MET2. The homoserine O-acetyltransferase originating from Corynebacterium glutamicum is usually termed METX.
A biological activity of the same nature regarding this sequence is the capacity to code for an enzyme that catalyzes the reaction between Acetyl-CoA and 2,4-diaminobutyrate into CoA and Acetyl-2,4-diaminobutyrate.
As described herein, a nucleic acid sequence having at least 27% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.
As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.
As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.
For the amino acid sequence of the the homoserine O-acetyltransferase from Saccharomyces cerevisiae, the one skilled in the art may refer to the accession number NP014122 in the UniProt database, or to SEQ ID NO. 13 described herein.
According to another particular embodiment, the nucleic acid(s) encoding a homoserine O-acetyltransferase may be nucleic acid(s) encoding an amino acid sequence selected from the group consisting of sequences having at least 27%, advantageously at least 65%, preferably at least 80%, amino acid identity with the amino acid sequence of SEQ ID NO: 13, and also a biological activity of the same nature. Illustratively, the homoserine O-acetyltransferase originating from Aquamarina atlantica has 27% amino acid identity with the homoserine O-acetyltransferase of SEQ ID NO. 13.
A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the reaction between Acetyl-CoA and 2,4-diaminobutyrate into CoA and Acetyl-2,4-diaminobutyrate.
As described herein, an amino acid sequence having at least 27% amino acid identity with a reference nucleic acid sequence encompasses amino acid sequences having at least 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.
As described herein, an amino acid sequence having at least 65% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature.
As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature.
As above-mentioned, the expression level of a homoserine O-acetyltransferase in the present invention is regulated by at least one promoter and at least one terminator, such as herein after defined more in details, which are present in 5′ and 3′ position respectively of the nucleic acid sequence encoding the said homoserine O-acetyltransferase.
As it is specified elsewhere in the present description, in some embodiments of the invention, the homoserine O-acetyltransferase is overexpressed and/or under the control of an inducible or repressible promoter in a recombinant yeast according to the invention.
In some embodiments, overexpression of the homoserine O-acetyltransferase may result from the control of the corresponding gene by a strong promoter within the said recombinant yeast.
In some other embodiments, overexpression of the homoserine O-acetyltransferase may result from the presence of a plurality of copies of a homoserine O-acetyltransferase-encoding sequence within the genome the said recombinant yeast.
In still further embodiments, overexpression of the homoserine O-acetyltransferase may result from both (i) the control of the corresponding gene by a strong promoter within the said recombinant yeast and (ii) the presence of a plurality of copies of a homoserine O-acetyltransferase-encoding sequence within the genome the said recombinant yeast.
Diaminobutyric Acid Acetyltransferase (EctA)
The diaminobutyric acid acetyltransferase enzyme is a protein which is described in the art for catalyzing the conversion of 2,4-diaminobutyrate in the presence of Acetyl-CoA into acetyl-2,4-diaminobutyrate. The diaminobutyric acid acetyltransferase encoded by the genome of Halomonas elongata may be termed EctA or EctA.He.
A method implemented to measure the activity level of diaminobutyric acid acetyltransferase belongs to the general knowledge of the one skilled in the art.
In this regard, the one skilled in the art may advantageously refer to the method described by Ono H et al., 1999, Journal of Bacteriology, p 91-99.
Preferred diaminobutyric acid acetyltransferase in the present specification is an enzyme having an EC number of n° 2.3.1.178.
According to a preferred embodiment, the nucleic acid(s) encoding a diaminobutyric acid acetyltransferase may be nucleic acid(s) originating from organisms preferably selected in a group comprising prokaryotic organisms and eukaryotic organisms. In some embodiments, the nucleic acid(s) encoding a diaminobutyric acid acetyltransferase may be nucleic acid(s) originating from archaebacteria. In some embodiments, the nucleic acid(s) encoding a diaminobutyric acid acetyltransferase may be nucleic acid(s) originating from organisms preferably selected from bacteria, and especially from Chromohalobacter salexigens, Pseudomonas aeruginosa, Thaurea sp.28, or Halomonas elongata. 
According to a yet preferred embodiment, the nucleic acid(s) encoding a diaminobutyric acid acetyltransferase may be nucleic acid(s) selected from the group consisting of sequences having at least 30%, advantageously at least 65%, preferably at least 80%, nucleic acid identity with a nucleic acid of SEQ ID NO. 14, and also a biological activity of the same nature.
A biological activity of the same nature regarding this sequence is the capacity to code for an enzyme that catalyzes the conversion of 2,4-diaminobutyrate in the presence of Acetyl-CoA into acetyl-2,4-diaminobutyrate.
As described herein, a nucleic acid sequence having at least 30% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.
As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.
As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.
For the amino acid sequence of the diaminobutyric acid acetyltransferase from Halomonas elongata, the one skilled in the art may refer to the accession number WP_035409657.1 in the UniProt database, or to SEQ ID NO. 15 described herein.
According to another particular embodiment, the nucleic acid(s) encoding diaminobutyric acid acetyltransferase may be nucleic acid(s) encoding an amino acid sequence selected from the group consisting of sequences having at least 30%, advantageously at least 65%, preferably at least 80%, amino acid identity with the amino acid sequence of SEQ ID NO. 15, and also a biological activity of the same nature.
A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the conversion of 2,4-diaminobutyrate in the presence of Acetyl-CoA into acetyl-2,4-diaminobutyrate.
As described herein, an amino acid sequence having at least 30% amino acid identity with a reference nucleic acid sequence encompasses amino acid sequences having at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.
As described herein, an amino acid sequence having at least 65% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature.
As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature.
As above-mentioned, the expression level of the diaminobutyric acid acetyltransferase in the present invention is regulated by at least one promoter and at least one terminator, such as herein after defined more in details, which are present in 5′ and 3′ position respectively of the nucleic acid sequence encoding the said diaminobutyric acid acetyltransferase.
As it is specified elsewhere in the present description, the diaminobutyric acid acetyltransferase is overexpressed and/or under the control of an inducible or repressible promoter in a recombinant yeast according to the invention.
In some embodiments, overexpression of the diaminobutyric acid acetyltransferase may result from the control of the corresponding gene by a strong promoter within the said recombinant yeast.
In some other embodiments, overexpression of the diaminobutyric acid acetyltransferase may result from the presence of a plurality of copies of a diaminobutyric acid acetyltransferase-encoding sequence within the genome of the said recombinant yeast.
In still further embodiments, overexpression of diaminobutyric acid acetyltransferase may result from both (i) the control of the corresponding gene by a strong promoter within the said recombinant yeast and (ii) the presence of a plurality of copies of a diaminobutyric acid acetyltransferase-encoding sequence within the genome the said recombinant yeast.
Ectoine Synthase (EctC)
The ectoine synthase enzyme is a protein which is described in the art for catalyzing the conversion of acetyl-2,4-diaminobutyrate into ectoine. The ectoine synthase encoded by the genome of Halomonas elongata may be termed EctC or EctC.He.
A method implemented to measure the activity level of ectoine synthase belongs to the general knowledge of the one skilled in the art.
In this regard, the one skilled in the art may advantageously refer to the method described by Ono H et al., 1999, Journal of Bacteriology, p 91-99.
Preferred ectoine synthase in the present specification is an enzyme having an EC number of n° 4.2.1.108.
According to a preferred embodiment, the nucleic acid(s) encoding an ectoine synthase may be nucleic acid(s) originating from organisms preferably selected in a group comprising prokaryotic organisms and eukaryotic organisms. In some embodiments, the nucleic acid(s) encoding an ectoine synthase may be nucleic acid(s) originating from archaebacteria. In some embodiments, the nucleic acid(s) encoding an ectoine synthase may be nucleic acid(s) originating from organisms preferably selected from bacteria, and especially from Pseudomonas aeruginosa, Halomonas elongata or Micrococcus luteus. 
According to a yet preferred embodiment, the nucleic acid(s) encoding an ectoine synthase may be nucleic acid(s) selected from the group consisting of sequences having at least 35%, advantageously at least 65%, preferably at least 80%, nucleic acid identity with a nucleic acid of SEQ ID NO. 16, and also a biological activity of the same nature.
A biological activity of the same nature regarding this sequence is the capacity to code for an enzyme that catalyzes the conversion of acetyl-2,4-diaminobutyrate into ectoine.
As described herein, a nucleic acid sequence having at least 35% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.
As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.
As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.
For the amino acid sequence of the ectoine synthase from Halomonas elongata, the one skilled in the art may refer to the accession number WP_013332346 in the UniProt database, or to SEQ ID NO. 17 described herein.
According to another particular embodiment, the nucleic acid(s) encoding ectoine synthase may be nucleic acid(s) encoding an amino acid sequence selected from the group consisting of sequences having at least 35%, advantageously at least 65%, preferably at least 80%, amino acid identity with the amino acid sequence of SEQ ID NO. 17, and also a biological activity of the same nature.
A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the conversion of acetyl-2,4-diaminobutyrate into ectoine.
As described herein, an amino acid sequence having at least 35% amino acid identity with a reference nucleic acid sequence encompasses amino acid sequences having at least 36%, 37%, 38%, 39%, 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference nucleic acid sequence, and also a biological activity of the same nature.
As described herein, an amino acid sequence having at least 65% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature.
As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence, and also a biological activity of the same nature.
As above-mentioned, the expression level of the ectoine synthase in the present invention is regulated by at least one promoter and at least one terminator, such as herein after defined more in details, which are present in 5′ and 3′ position respectively of the nucleic acid sequence encoding the said ectoine synthase.
As it is specified elsewhere in the present description, the ectoine synthase is overexpressed and/or under the control of an inducible or repressible promoter in a recombinant yeast according to the invention.
In some embodiments, overexpression of the ectoine synthase may result from the control of the corresponding gene by a strong promoter within the said recombinant yeast.
In some other embodiments, overexpression of the ectoine synthase may result from the presence of a plurality of copies of an ectoine synthase-encoding sequence within the genome of the said recombinant yeast.
In still further embodiments, overexpression of ectoine synthase may result from both (i) the control of the corresponding gene by a strong promoter within the said recombinant yeast and (ii) the presence of a plurality of copies of an ectoine synthase-encoding sequence within the genome the said recombinant yeast.
Homoserine Dehydrogenase (HOM6)
The homoserine dehydrogenase enzyme is a protein which is described in the art for catalyzing the conversion of L-homoserine into L-aspartate 4-semialdehyde, in the presence of NAD or NADP. The homoserine dehydrogenase encoded by the genome of Saccharomyces cerevisiae may be termed HOM6.
A method implemented to measure the activity level of homoserine dehydrogenase belongs to the general knowledge of the one skilled in the art.
In this regard, the one skilled in the art may advantageously refer to the method described by Calnyanto et al. (2006, Microbiology, Vol. 152: 105-112).
Preferred homoserine dehydrogenase in the present specification is an enzyme having an EC number of n° 1.1.1.3.
According to a preferred embodiment, the nucleic acid(s) encoding a homoserine dehydrogenase may be nucleic acid(s) originating from organisms preferably selected in a group comprising prokaryotic organisms and eukaryotic organisms. In some preferred embodiments, the nucleic acid(s) encoding a homoserine dehydrogenase may be nucleic acid(s) originating from a yeast, and especially from Saccharomyces cerevisiae. 
According to a particular embodiment, the nucleic acid(s) encoding a homoserine dehydrogenase may be nucleic acid of SEQ ID NO: 18. The nucleic acid of SEQ ID NO: 18 encodes a homoserine dehydrogenase originating from Saccharomyces, that may also be termed HOM6.
For the amino acid sequence of the homoserine dehydrogenase from Saccharomyces cerevisiae, the one skilled in the art may refer to the accession number AJR75529 or NP012673 in the UniProt database, or to SEQ ID NO. 19 described herein.
As above-mentioned, the expression level of the homoserine dehydrogenase in the present invention is regulated by at least one promoter and at least one terminator, such as herein after defined more in details, which are present in 5′ and 3′ position respectively of the nucleic acid sequence encoding the said homoserine dehydrogenase.
As it is specified elsewhere in the present description, in some embodiments of the invention, the homoserine dehydrogenase is (a) fully or partially deleted or interrupted, or (b) under the control of an inducible or repressible promoter; under the control of a weak promoter; and/or in a destabilized form, in a recombinant yeast according to the invention.
Specific Embodiments of an Ectoine-Producing Recombinant Yeast
Aspartate Transaminase Over Expression and/or Controlled Expression
In preferred embodiments of a recombinant yeast according to the invention, at least one nucleic acid encoding an aspartate transaminase is overexpressed and/or is under the control of an inducible or repressible promoter.
The aspartate transaminase enzyme (also known as aspartate aminotransferase) is a protein which is described in the art for catalyzing the reaction of L-aspartate and 2-oxoglutarate for producing oxaloacetate and L-glutamate. The aspartate transaminase enzyme encoded by the genome Saccharomyces cerevisiae may be termed AAT2.
According to these embodiments, over expression of an aspartate transaminase-encoding gene is obtained by inserting, at selected location(s) of the yeast genome, one or more copies of an expression cassette comprising an aspartate transaminase coding sequence. Aspartate transaminase and aspartate-transaminase-encoding gene that are encompassed by the invention are detailed elsewhere in the present specification.
In some of these embodiments, the said one or more copies of an expression cassette comprising an aspartate transaminase coding sequence comprise regulatory sequences allowing a strong expression of the aspartate transaminase, such as a strong promoter that is functional in yeast cells.
In addition to or as an alternative to these embodiments of a recombinant yeast according to the invention, at least one aspartate transaminase-encoding gene can be under the control of an inducible or repressible promoter that is functional in yeast cells.
Without wishing to be bound by any particular theory, the inventors believe that an over expression of an aspartate transaminase may induce a high level of conversion of oxaloacetate into aspartate. The same applies when at least one aspartate transaminase coding sequence is under the control of an inducible or repressible promoter.
A method implemented to measure the activity level of an aspartate transaminase belongs to the general knowledge of the one skilled in the art.
In this regard, the one skilled in the art may advantageously refer to the method described in Yagi et al. (1982, Biochem, VOl. 92: 35-43).
In some embodiments, the said aspartate transaminase-encoding gene is the AAT2 gene from Saccharomyces cerevisiae, as shown in the examples herein.
In preferred embodiments, the aspartate aminotransferase is encoded by the A. Thaliana AAT2-gene.
In preferred embodiments, the said aspartate transaminase-encoding gene is placed under the control of the inducible or repressible promoter pACU1 or of the strong promoter pADH1 or of the strong promoter pPGK1.
Illustratively, the AAT2 gene may be inserted within the CAN1 gene and/or within the GNP1 gene and/or within the MUP3 gene, as it is shown in the examples herein.
Preferred aspartate transaminase in the present specification is known by the EC number 2.6.1.1.
The nucleic acid(s) encoding an aspartate transaminase may be nucleic acid(s) originating from organisms preferably selected in a group comprising prokaryotic organisms and eukaryotic organisms. In some embodiments, the nucleic acid(s) encoding an aspartate transaminase may be nucleic acid(s) originating from archaebacteria. In some preferred embodiments, the nucleic acid(s) encoding an aspartate transaminase may be nucleic acid(s) originate(s) from a yeast organism, and most preferably Saccharomyces cerevisiae. 
According to a yet preferred embodiment, the nucleic acid(s) encoding an aspartate transaminase or AAT2 may be nucleic acid(s) selected from the group consisting of sequences having at least 39%, advantageously at least 65%, and preferably at least 80%, nucleic acid identity with the nucleic acid sequences of SEQ ID NO: 20, and also a biological activity of the same nature.
A biological activity of the same nature regarding this sequence is the capacity to code for an enzyme that catalyzes the reaction of L-aspartate and 2-oxoglutarate for producing oxaloacetate and L-glutamate.
As described herein, a nucleic acid sequence having at least 39% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the nucleic acid sequence of SEQ ID NO: 20, and also a biological activity of the same nature.
As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the nucleic acid sequence of SEQ ID NO: 20, and also a biological activity of the same nature.
As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the nucleic acid sequence of SEQ ID NO: 20, and also a biological activity of the same nature.
For the amino acid sequence of the aspartate transaminase AAT2 from Saccharomyces cerevisiae, the one skilled in the art may refer to the accession number NP013127 in the UniProt database, or to SEQ ID NO. 21 described herein. Illustratively, the aspartate transaminase originating from E. coli has 39% amino acid identity with the aspartate transaminase AAT2 of SEQ ID NO. 21.
According to another particular embodiment, the nucleic acid(s) encoding an aspartate transaminase may be nucleic acid(s) encoding an amino acid sequence selected from the group consisting of sequences having at least 39%, advantageously at least 65%, preferably at least 80%, identity with the amino acid sequence of SEQ ID NO: 21, and also a biological activity of the same nature.
A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the reaction of L-aspartate and 2-oxoglutarate for producing oxaloacetate and L-glutamate.
As described herein, an amino acid sequence having at least 39% amino acid identity with a reference nucleic acid sequence encompasses amino acid sequences having at least 40% 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the amino acid sequence of SEQ ID NO: 21, and also a biological activity of the same nature.
As described herein, an amino acid sequence having at least 65% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the amino acid sequence of SEQ ID NO: 21, and also a biological activity of the same nature.
As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the amino acid sequence of SEQ ID NO: 21, and also a biological activity of the same nature.
As above-mentioned, the expression level of the aspartate transaminase in the present invention is regulated by at least one promoter and at least one terminator, such as herein after defined more in details, which are present in 5′ and 3′ position respectively of the nucleic acid sequence encoding the aspartate transaminase.
As it is specified elsewhere in the present description, aspartate transaminase is overexpressed in a recombinant yeast according to the invention.
In some embodiments, overexpression of aspartate transaminase may result from the control of the corresponding gene by a strong promoter within the said recombinant yeast.
In some other embodiments, overexpression of aspartate transaminase may result from the presence of a plurality of copies of an aspartate transaminase-encoding sequence within the genome the said recombinant yeast.
In still further embodiments, overexpression of aspartate transaminase may result from both (i) the control of the corresponding gene by a strong promoter within the said recombinant yeast and (ii) the presence of a plurality of copies of an aspartate transaminase-encoding sequence within the genome the said recombinant yeast.
Glutamate Dehydrogenase Over Expression and/or Controlled Expression
In preferred embodiments of a recombinant yeast according to the invention, at least one nucleic acid encoding a glutamate dehydrogenase that converts oxo-glutarate to glutamate-encoding gene is overexpressed and/or under the control of an inducible or repressible promoter.
Accordingly, in a particular embodiment, the genome of a recombinant yeast of the invention is such that at least one nucleic acid encoding a glutamate dehydrogenase that converts oxo-glutarate to glutamate is overexpressed and/or is under the control of an inducible or repressible promoter.
The glutamate dehydrogenase enzyme (also known as NAD-specific glutamate dehydrogenase) is a protein which is described in the art for catalyzing the transformation of 2-oxoglutarate for producing L-glutamate. Thus, glutamate dehydrogenase is an enzyme specifically involved in the chemical reaction involving the conversion of 2-oxoglutarate to L-glutamate, in the presence of NADH.
According to these embodiments, over expression of a glutamate dehydrogenase enzyme-encoding gene is obtained by inserting, at selected location(s) of the yeast genome, one or more copies of an expression cassette comprising a glutamate dehydrogenase coding sequence. Glutamate dehydrogenase and a glutamate dehydrogenase-encoding gene that are encompassed by the invention are detailed elsewhere in the present specification.
In some of these embodiments, the said one or more copies of an expression cassette comprising a glutamate dehydrogenase coding sequence comprise regulatory sequences allowing a strong expression of the glutamate dehydrogenase, such as a strong promoter that is functional in yeast cells.
In addition to or as an alternative to these embodiments of a recombinant yeast according to the invention, at least one glutamate dehydrogenase-encoding gene can be under the control of an inducible or repressible promoter that is functional in yeast cells.
Without wishing to be bound by any particular theory the inventors believe that the over expression of the glutamate dehydrogenase, by converting oxoglutarate into glutamate, simultaneously generates NAD. The same applies when at least one glutamate dehydrogenase coding sequence is under the control of an inducible or repressible promoter.
A method implemented to measure the activity level of glutamate dehydrogenase belongs to the general knowledge of the one skilled in the art.
In this regard, the one skilled in the art may advantageously refer to the method described in Noor and Punekar (2005, Microbiology, Vol. 151: 1409-1419).
In preferred embodiments, the said glutamate dehydrogenase-encoding gene encodes for a glutamate dehydrogenase which uses NADH instead of NADPH, is more particularly the GDH gene from Entodinium caudatum (represented as GDH.Eca or GDH2.Eca) or from Saccharomyces cerevisiae (represented as GDH2), as shown in the examples herein. In a particular embodiment, the said glutamate dehydrogenase-encoding gene encodes for a glutamate dehydrogenase from Entodinium caudatum. 
Preferred glutamate dehydrogenase in the present specification can in particular be the enzyme having the EC number n° EC 1.4.1.2.
In preferred embodiments, the said glutamate dehydrogenase-encoding gene is placed under the control of the strong promoter pTDH3 or the inducible or repressible promoter pCUP1-1.
Illustratively, the glutamate dehydrogenase gene may be inserted within the HIS3 gene and/or within the MUP3 gene, as it is shown in the examples herein.
According to a preferred embodiment, the nucleic acid(s) encoding a glutamate dehydrogenase may be nucleic acid(s) originating from organisms preferably selected in a group comprising prokaryotic organisms and eukaryotic organisms. In some embodiments, the nucleic acid(s) encoding a glutamate dehydrogenase may be nucleic acid(s) originating from archaebacteria. In some embodiments, the nucleic acid(s) encoding a glutamate dehydrogenase may be nucleic acid(s) originating from organisms preferably selected from Entodinium caudatum, Bacillus subtilis, Clostridium symbiosium. 
According to a yet preferred embodiment, the nucleic acid(s) encoding a glutamate dehydrogenase may be nucleic acid(s) selected from the group consisting of sequences having at least 49%, advantageously at least 65%, preferably at least 80%, nucleic acid identity with the nucleic acid sequences of SEQ ID NO: 22, and also a biological activity of the same nature. The nucleic acid of SEQ ID NO. 22 encodes a glutamate dehydrogenase originating from Entodinium caudatum, the said nucleic acid sequence being codon-optimized for its expression in yeast, and especially in Saccharomyces cerevisiae. 
A biological activity of the same nature regarding this sequence is the capacity to code for an enzyme that catalyzes the transformation of 2-oxoglutarate for producing

    L-glutamate.


As described herein, a nucleic acid sequence having at least 49% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the nucleic acid sequence of SEQ ID NO: 22, and also a biological activity of the same nature.
As described herein, a nucleic acid sequence having at least 65% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the nucleic acid sequence of SEQ ID NO: 22, and also a biological activity of the same nature.
As described herein, a nucleic acid sequence having at least 80% nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the nucleic acid sequence of SEQ ID NO: 22, and also a biological activity of the same nature.
For the amino acid sequence of the glutamate dehydrogenase from Entodinium caudatum, the one skilled in the art may refer to the accession number AAF15393 in the UniProt database, or to SEQ ID NO. 23 described herein. Illustratively, the glutamate dehydrogenase originating from Giardia intestinalis has 49% amino acid identity with the glutamate dehydrogenase of SEQ ID NO. 23.
According to another particular embodiment, the nucleic acid(s) encoding a glutamate dehydrogenase may be nucleic acid(s) encoding an amino acid sequence selected from the group consisting of sequences having at least 49%, advantageously at least 65%, preferably at least 80%, amino acid identity with the amino acid sequence of SEQ ID NO: 23, and also a biological activity of the same nature.
A biological activity of the same nature regarding this sequence is as described previously, i.e. the capacity to catalyze the transformation of 2-oxoglutarate for producing

    L-glutamate.


As described herein, an amino acid sequence having at least 49% amino acid identity with a reference nucleic acid sequence encompasses amino acid sequences having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the amino acid sequence of SEQ ID NO: 23, and also a biological activity of the same nature.
As described herein, an amino acid sequence having at least 65% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the amino acid sequence of SEQ ID NO: 23, and also a biological activity of the same nature.
As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the amino acid sequence of SEQ ID NO: 23, and also a biological activity of the same nature.
As above-mentioned, the expression level of the glutamate dehydrogenase in the present invention is regulated by at least one promoter and at least one terminator, such as herein after defined more in details, which are present in 5′ and 3′ position respectively of the nucleic acid sequence encoding the said glutamate dehydrogenase.
As it is specified elsewhere in the present description, the glutamate dehydrogenase is overexpressed in a recombinant yeast according to the invention.
In some embodiments, overexpression of the glutamate dehydrogenase may result from the control of the corresponding gene by a strong promoter within the said recombinant yeast.
In some other embodiments, overexpression of the glutamate dehydrogenase may result from the presence of a plurality of copies of a glutamate dehydrogenase-encoding sequence within the genome the said recombinant yeast.
In still further embodiments, overexpression of the glutamate dehydrogenase may result from both (i) the control of the corresponding gene by a strong promoter within the said recombinant yeast and (ii) the presence of a plurality of copies of a glutamate dehydrogenase-encoding sequence within the genome the said recombinant yeast.
Export of the Compounds of Interest
In further embodiments of a recombinant yeast according to the invention, the export of the produced ectoine outside of the yeast cell may be enhanced by (i) under expression of genes encoding yeast permeases, by (ii) under expression of genes encoding amino acid exporter proteins, or by (iii) both under expression of genes encoding yeast permeases and under expression of genes encoding amino acid exporter proteins.
Under Expression of Permease-Encoding Gene(s)
As it is described below, permease-encoding genes that may be under expressed in a recombinant yeast according to the invention encompass AGP1, AGP3, BAP3, BAP2, GAP1, GNP1, MUP3 and MUP1.
AGP1 is the general amino acid permease 1 from Saccharomyces cerevisiae. For the amino acid sequence of AGP1 it may be referred to the access number NP_009905 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001178671 in the NCBI database.
AGP3 is the general amino acid permease 3 from Saccharomyces cerevisiae. For the amino acid sequence of AGP3 it may be referred to the access number NP_116600 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001179912 in the NCBI database.
BAP3 is the valine amino acid permease from Saccharomyces cerevisiae. For the amino acid sequence of BAP3 it may be referred to the access number NP_010331 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001180354 in the NCBI database.
BAP2 is the Leu/Val/Ile amino acid permease from Saccharomyces cerevisiae. For the amino acid sequence of BAP2 it may be referred to the access number NP_009624 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001178416 in the NCBI database.
GAP1 is the general amino-acid permease from Saccharomyces cerevisiae. For the amino acid sequence of GAP1 it may be referred to the access number NP_012965.3 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001179829 in the NCBI database.
GNP1 is the high-affinity glutamine permease from Saccharomyces cerevisiae. For the amino acid sequence of GNP1 it may be referred to the access number NP_010796 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001180816 in the NCBI database.
MUP3 is the low-affinity methionine permease from Saccharomyces cerevisiae. For the amino acid sequence of MUP3 it may be referred to the access number NP_011827 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001179116 in the NCBI database.
MUP1 is the low-affinity methionine permease from Saccharomyces cerevisiae. For the amino acid sequence of MUP it may be referred to the access number NP_011569 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001181184 in the NCBI database.
In some embodiments of a recombinant yeast according to the invention, the said recombinant yeast is further defined as having an under expression one or more genes encoding a permease, that encompasses AGP1, AGP3, BAP3, BAP2, GAP1, GNP1, MUP3 and MUP1 permeases.
Accordingly, in a particular embodiment, the genome of a recombinant yeast of the invention is such that at least one of the following modifications has been performed:

    
    
        (A) at least one, preferably all, endogenous nucleic acid encoding a general amino acid permease AGP3 have been deleted from the genome of the yeast, and optionally:
        
            (i) at least one nucleic acid encoding a general amino acid permease AGP3 has been inserted and is under the control of an inducible or repressible promoter, and/or
            (ii) at least one nucleic acid encoding a destabilized general amino acid permease AGP3 has been inserted;
        
        
        (B) at least one, preferably all, endogenous nucleic acid encoding a branched-chain amino-acid permease 3 has been deleted from the genome of the yeast, and, optionally:
        
            (i) at least one nucleic acid encoding a branched-chain amino-acid permease 3 has been inserted and is under the control of an inducible or repressible promoter, and/or
            (ii) at least one nucleic acid encoding a destabilized branched-chain amino-acid permease 3 has been inserted;
        
        
        (C) at least one, preferably all, endogenous nucleic acid encoding a branched-chain amino-acid permease 2 has been deleted from the genome of the yeast, and, optionally:
        
            (i) at least one nucleic acid encoding a branched-chain amino-acid permease 2 has been inserted and is under the control of an inducible or repressible promoter, and/or
            (ii) at least one nucleic acid encoding a destabilized branched-chain amino-acid permease 2 has been inserted;
        
        
        (D) at least one, preferably all, endogenous nucleic acid encoding a general amino acid permease GAP1 has been deleted from the genome of the yeast, and, optionally:
        
            (i) at least one nucleic acid encoding a general amino acid permease GAP1 has been inserted and is under the control of an inducible or repressible promoter, and/or
            (ii) at least one nucleic acid encoding a destabilized general amino acid permease GAP1 has been inserted;
        
        
        (E) at least one, preferably all, endogenous nucleic acid encoding a high-affinity glutamine permease GNP1 has been deleted from the genome of the yeast, and, optionally:
        
            (i) at least one nucleic acid encoding a high-affinity glutamine permease GNP1 has been inserted and is under the control of an inducible or repressible promoter, and/or
            (ii) at least one nucleic acid encoding a destabilized high-affinity glutamine permease GNP1 has been inserted;
        
        
        (F) at least one, preferably all, endogenous nucleic acid encoding a general amino acid permease AGP1 has been deleted from the genome of the yeast, and, optionally:
        
            (i) at least one nucleic acid encoding a general amino acid permease AGP1 has been inserted and is under the control of an inducible or repressible promoter, and/or
            (ii) at least one nucleic acid encoding a destabilized general amino acid permease AGP1 has been inserted;
        
        
        (G) at least one, preferably all, endogenous nucleic acid encoding a low-affinity methionine permease MUP3 has been deleted from the genome of the yeast, and, optionally:
        
            (i) at least one nucleic acid encoding a low-affinity methionine permease MUP3 has been inserted and is under the control of an inducible or repressible promoter, and/or
            (ii) at least one nucleic acid encoding a destabilized low-affinity methionine permease MUP3 has been inserted;
        
        
        (H) at least one, preferably all, endogenous nucleic acid encoding a high-affinity methionine permease MUP1 has been deleted from the genome of the yeast, and, optionally:
        
            (i) at least one nucleic acid encoding a high-affinity methionine permease MUP1 has been inserted and is under the control of an inducible or repressible promoter, and/or
            (ii) at least one nucleic acid encoding a destabilized high-affinity methionine permease MUP1 has been inserted;
        
        
        (I) at least one nucleic acid encoding a probable transporter AQR1 is overexpressed; and/or
        (J) at least one nucleic acid encoding a polyamine transporter 1 is overexpressed.
    
    


In a particular embodiment, at least two, in particular at least three of these modifications have been performed.
Without wishing to be bound by any particular theory, the inventors believe that an under expression of any of the permease genes shall increase the excretion of the produced ectoine outside the yeast cell, e.g. in the culture medium.
As regards permeases under expression of one or more of these genes encompasses a complete repression of their expression, e.g. by interruption or deletion of the said one or more permease genes.
In some embodiments, under expression of a permease-encoding gene may be rendered conditional, for example by placing the expression of this gene under the control of repressible regulatory sequences, such as inducible or repressible promoters.
Methods for repressing gene expression, for interrupting target genes or for deleting target genes, are well known from the one skilled in the art.
As regards a permease gene, under expression also encompasses the insertion of a nucleic acid encoding a destabilized permease protein or the insertion of a nucleic acid encoding a destabilized permease protein, or both.
A destabilized permease is a variant of a permease that is more rapidly degraded within the yeast cell than the parent permease.
In preferred embodiments, a destabilized permease consists of a degron-tagged permease protein.
As illustrated in the examples, the AGP3 gene, the BAP3 gene, the GAP1 gene, the GNP1 gene and the MUP3 gene can be interrupted by loxP and are thus deleted.
Over Expression of Amino Acid Exporter Protein-Encoding Gene(s)
As it is described below, exporter protein-encoding genes that may be over expressed in a recombinant yeast according to the invention encompass AQR1 and TPO1.
AQR1 is a transporter from Saccharomyces cerevisiae. For the amino acid sequence of AQR1 it may be referred to the access number NP_014334 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001182903 in the NCBI database.
TPO1 is a polyamine transporter from Saccharomyces cerevisiae. For the amino acid sequence of TPO1 it may be referred to the access number NP_013072 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001181848 in the NCBI database.
In preferred embodiments of a recombinant yeast according to the invention, over expression of a transporter-encoding gene is obtained by inserting, at selected location(s) of the yeast genome, one or more additional copies of an expression cassette comprising the said transporter coding sequence.
Without wishing to be bound by any particular theory, the inventors believe that an over expression of a transporter-encoding gene shall increase the excretion of the produced ectoine outside the yeast cell, e.g. in the culture medium.
In some embodiments, over expression of a transporter-encoding gene is obtained by inserting, at selected location(s) of the yeast genome, one or more additional copies of an expression cassette comprising a transporter gene coding sequence. In some of these embodiments, the said one or more copies of an expression cassette comprising a transporter coding sequence comprise regulatory sequences allowing a strong expression of the said transporter, such as a strong promoter that is functional in yeast cells.
In some other embodiments, one copy of a transporter-encoding gene is inserted at a selected location of the yeast genome. In these other embodiments, the said one or more copies of an expression cassette comprising a transporter coding sequence comprise regulatory sequences allowing a strong expression of the said transporter, such as a strong promoter that is functional in yeast cells.
In preferred embodiments, the said amino acid exporter protein-encoding gene AQR1 is placed under the control of the strong promoter pTEF3.
Illustratively, the AQR1 gene may be inserted within the PYK1 gene, as it is shown in the examples herein.
In preferred embodiments, the said amino acid exporter protein-encoding gene_TPO1 is placed under the control of the strong inducible or repressible promoter pSAM4 or the strong constitutive promoter pTEF1.
TPO1-1 can be used instead of TPO1. TPO1-1 is an artificial allele in which the lysines 10, 49, 86, 143, 144 and 145 are replaced by arginines.
It is believed by the inventors that these modifications protect TP01 from degradation through the ubiquitin-proteasome pathway, thus stabilizing it.
In preferred embodiments, the said amino acid exporter protein-encoding gene_TPO1 is placed under the control of the strong promoter pTEF3.
Illustratively, the TPO1 gene may be inserted within the PYK1 gene, as it is shown in the examples herein.
In view of further increasing ectoine production, a recombinant yeast according to the invention may comprise additional genetic changes, such that they produce large quantities of the intermediate product oxaloacetate. These optional genetic changes are described here below.
Further Embodiments of an Ectoine-Producing Recombinant Yeast
According to some embodiments of a recombinant yeast according to the invention, production of ectoine may be further increased by placing the said recombinant yeast in conditions leading to an increase production of the intermediate metabolite oxaloacetate.
Placing the said recombinant yeast in conditions leading to an increased production of oxaloacetate may be performed by introducing further genetic modifications in the yeast genome.
The present inventors have found that an optimally increased ectoine production may be reached by introducing further genetic changes to the ectoine-producing recombinant yeast, that are described below.
First Further Embodiments of a Ectoine-Producing Recombinant Yeast
According to these first further embodiments of a ectoine-producing recombinant yeast according to the invention, further genetic engineering of the recombinant yeast is performed with the aim of increasing the production of the intermediate product phosphoenol-pyruvate (PEP).
Without wishing to be bound by any particular theory, the inventors believe that the further genetic changes introduced in the ectoine-producing recombinant yeast (i) cause an over-production of NADPH, (ii) cause a controlled and balanced conversion of phosphoenol pyruvate into oxaloacetate and pyruvate, respectively, and (iii) cause a reduced conversion of pyruvate into ethanol and a redirection towards conversion of phosphoenol pyruvate into oxaloacetate.
These further genetic changes introduced by genetic engineering in a ectoine-producing recombinant yeast according to the invention are specified in more detail below.
According to these embodiments, genetic changes are introduced so as to over-express a glucose-6-phosphate-1-dehydrogenase (also termed MET19 or ZWF1) and a
6-phosphogluconate dehydrogenase, decarboxylating 1 (also termed GND1). Without wishing to be bound by any particular theory, the inventors believe that an over expression of MET19 and GND1 causes an increase in NADPH production.
According to these embodiments, genetic changes are introduced so as to over-express a phosphoenolpyruvate carboxylase (also termed PEPC ou PPC) and/or a phosphoenolpyruvate carboxykinase [ATP] (also termed PCK1 or PEPCK).
According to these embodiments, genetic changes are introduced so as to under-express a pyruvate kinase 1 (also termed PYK1 or CDC19) and a pyruvate kinase 2 (also termed (PYK2). In some of these embodiments, PYK2 gene may be deleted rather than being under-expressed.
In some of these embodiments, one or more of the genes encoding a pyruvate decarboxylase is (are) inactivated, preferably by deletion. Pyruvate decarboxylase-encoding genes encompass those termed PDC1, PDC5 and PDC6, respectively. According to some of these embodiments, PDC1 and/or PDC6 genes are inactivated, preferably by interruption or deletion, whereas the other pyruvate decarboxylase-encoding gene PDC5 is left unaltered; Or its expression is reduced by controlling it with a weak promoter.
In some of these embodiments, alcohol dehydrogenase activity of the recombinant yeast is reduced by altering the expression of one or more of the alcohol dehydrogenase-encoding genes. In some of these embodiments, the expression of ADH1 is reduced by placing the gene under the control of a weak promoter or by producing a destabilized ADH1 enzyme. In some of these embodiments, one or more of ADH3, ADH4 and ADH5 may be inactivated, preferably by interruption or deletion.
In some of these embodiments, an exogenous acetyl dehydrogenase-encoding gene (also termed MHPF) may be introduced in the yeast genome and over-expressed.
In some of these embodiments, an exogenous acetate kinase-encoding gene (also termed ACKA) may be introduced in the yeast genome and over-expressed.
In some of these embodiments, an exogenous phosphate acetyl transferase-encoding gene (also termed PTA) may be introduced in the yeast genome and over-expressed.
Glucose-6-phosphate-1-dehydrogenase
The glucose-6-phosphate-1-dehydrogenase enzyme is a protein which is described in the art for catalyzing D-glucose 6-phosphate to 6-phospho-D-glucono-1,5-lactone, with concomitant reduction of NADP to NADPH.
A method implemented to measure the activity level of glucose-6-phosphate-1-dehydrogenase belongs to the general knowledge of the one skilled in the art.
In this regard, the one skilled in the art may advantageously refer to the method described by Kuby, S. et al. (1966) Dehydrogenases and Oxidases Methods in Enzymology 9, 116-117.
Preferred glucose-6-phosphate-1-dehydrogenase in the present specification is an enzyme having an EC number of n° 1.1.1.49.
For the amino acid sequence of glucose-6-phosphate-1-dehydrogenase (also termed MET19), it may be referred to the access number NP_014158.1 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001183079.1 in the UniProt database.
6-phosphogluconate dehydrogenase, decarboxylating/The 6-phosphogluconate dehydrogenase, decarboxylating 1 enzyme is a protein which is described in the art for catalyzing the oxidative decarboxylation of 6-phosphogluconate to ribulose 5-phosphate and CO2, with concomitant reduction of NADP to NADPH.
A method implemented to measure the activity level of 6-phosphogluconate dehydrogenase, decarboxylating 1 belongs to the general knowledge of the one skilled in the art.
In this regard, the one skilled in the art may advantageously refer to the method described by He W. et al. (2007) BMC Structural Biology, 7:38.
Preferred 6-phosphogluconate dehydrogenase, decarboxylating 1 in the present specification is an enzyme having an EC number of n° 1.1.1.44.
For the amino acid sequence of 6-phosphogluconate dehydrogenase, decarboxylating 1 (also termed GND1), it may be referred to the access number NP_012053 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001179314 in the NCBI database.
Pyruvate Kinase 1
The pyruvate kinase 1 enzyme is a protein which is described in the art for catalyzing the conversion of pyruvate into phosphoenolpyruvate, in the presence of ATP.
A method implemented to measure the activity level of pyruvate kinase 1 belongs to the general knowledge of the one skilled in the art.
In this regard, the one skilled in the art may advantageously refer to the method described by Susan-resiga and Nowak (biochemistry, 2004, 43, 15230-15245).
Preferred pyruvate kinase 1 in the present specification is an enzyme having an EC number of n° 2.7.1.40.
For the amino acid sequence of pyruvate kinase 1 (also termed PYK1) it may be referred to the access number NP_009362 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001178183 in the NCBI database.
Pyruvate Kinase 2
The pyruvate kinase 2 enzyme is a protein which is described in the art for catalyzing the conversion of pyruvate into phosphoenolpyruvate, in the presence of ATP. Pyruvate kinase 2 may be used by the yeast cell under conditions in which the level of glycolytic flux is very low.
A method implemented to measure the activity level of pyruvate kinase 2 belongs to the general knowledge of the one skilled in the art.
In this regard, the one skilled in the art may advantageously refer to the method described by Susan-resiga and Nowak (biochemistry, 2004, 43, 15230-15245).
Preferred pyruvate kinase 2 in the present specification is an enzyme having an EC number of n° 2.7.1.40.
For the amino acid sequence of pyruvate kinase 2 (also termed PYK2) it may be referred to the access number NP_014992 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001183767 in the NCBI database.
Pyruvate Decarboxylase Isozyme 1
The pyruvate decarboxylase isozyme 1 is a protein which is described in the art for being involved in the non-oxidative conversion of pyruvate to acetaldehyde and carbon dioxide during alcoholic fermentation.
A method implemented to measure the activity level of the pyruvate decarboxylase isozyme 1 belongs to the general knowledge of the one skilled in the art.
In this regard, the one skilled in the art may advantageously refer to the method described by Wang et al. (Biochemistry, 2001, 40:1755-1763).
Preferred pyruvate decarboxylase isozyme 1 in the present specification is an enzyme having an EC number of n° 4.1.1.1.
For the amino acid sequence of pyruvate decarboxylase isozyme 1 (also termed PDC1) it may be referred to the access number NP_013145 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001181931 in the NCBI database.
Pyruvate Decarboxylase Isozyme 5
The pyruvate decarboxylase isozyme 5 is a protein which is described in the art for being involved in the nonoxidative conversion of pyruvate to acetaldehyde and carbon dioxide during alcoholic fermentation.
A method implemented to measure the activity level of the pyruvate decarboxylase isozyme 5 belongs to the general knowledge of the one skilled in the art.
In this regard, the one skilled in the art may advantageously refer to the method described by Wang et al. (Biochemistry, 2001, 40:1755-1763).
Preferred pyruvate decarboxylase isozyme 5 in the present specification is an enzyme having an EC number of n° 4.1.1.1.
For the amino acid sequence of pyruvate decarboxylase isozyme 5 (also termed PDC5) it may be referred to the access number NP_013235 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001182021 in the NCBI database.
Pyruvate Decarboxylase Isozyme 6
The pyruvate decarboxylase isozyme 6 is a protein which is described in the art for being involved in the nonoxidative conversion of pyruvate to acetaldehyde and carbon dioxide during alcoholic fermentation.
A method implemented to measure the activity level of the pyruvate decarboxylase isozyme 5 belongs to the general knowledge of the one skilled in the art. In this regard, the one skilled in the art may advantageously refer to the method described by Wang et al. (Biochemistry, 2001, 40:1755-1763).
Preferred pyruvate decarboxylase isozyme 6 in the present specification is an enzyme having an EC number of n° 4.1.1.1.
For the amino acid sequence of pyruvate decarboxylase isozyme 6 (also termed PDC6) it may be referred to the access number NP_013235 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001182021 in the NCBI database.
Acetaldehyde Dehydrogenase
The acetaldehyde dehydrogenase is a protein which is described in the art for catalyzing the conversion of acetaldehyde to acetyl-CoA, using NAD and coenzyme A.
A method implemented to measure the activity level of acetaldehyde dehydrogenase belongs to the general knowledge of the one skilled in the art.
In this regard, the one skilled in the art may advantageously refer to the method described by Fisher et al. (2013) Chemi. Biol. Interact. 202 70-77.
Preferred acetaldehyde dehydrogenase in the present specification is an enzyme having an EC number of n° 1.1.1.10.
For the amino acid sequence of acetaldehyde dehydrogenase (also termed MHPF) it may be referred to the access number NP_414885 in the UniProt database. For the nucleic acid sequence, it may be referred to the one disclosed in the access number NC_000913.3 in the NCBI database.
Acetate Kinase
The acetate kinase is a protein which is described in the art for the formation of acetyl phosphate from acetate and ATP.
A method implemented to measure the activity level of acetate kinase belongs to the general knowledge of the one skilled in the art.
In this regard, the one skilled in the art may advantageously refer to the method described by Sagers et al. J. Bacteriology (1961) 82 233-238.
For the amino acid sequence of acetate kinase (also termed ACKA) it may be referred to the access number NP_416799 in the UniProt database. For the nucleic acid sequence, it may be referred to the one disclosed in the access number NC_000913.3 in the NCBI database.
Phosphate Acetyltransferase
The phosphate acetyltransferase is a protein which is described in the art for catalyzing the reversible interconversion of acetyl-CoA and acetyl phosphate.
A method implemented to measure the activity level of the phosphate acetyltransferase belongs to the general knowledge of the one skilled in the art.
In this regard, the one skilled in the art may advantageously refer to the method described by Castano-Cerezo and Canovas, Microbial Cell Factories 2009, 8:54.
Preferred phosphate acetyltransferase in the present specification is an enzyme having an EC number of n° 2.3.1.8.
For the amino acid sequence of phosphate acetyltransferase (also termed PTA) it may be referred to the access number NP_416800 in the UniProt database. For the nucleic acid sequence, it may be referred to the one disclosed in the access number NC_000913 in the NCBI database.
Alcohol Dehydrogenase 1
The alcohol dehydrogenase 1 is a protein which is described in the art for catalyzing the conversion of primary unbranched alcohols to their corresponding aldehydes.
A method implemented to measure the activity level of the alcohol dehydrogenase 1 belongs to the general knowledge of the one skilled in the art.
In this regard, the one skilled in the art may advantageously refer to the method described by Ganzhorn et al. (1987) The Journal of Biological Chemistry, 262, 3754-61
Preferred alcohol dehydrogenase 1 in the present specification is an enzyme having an EC number of n° 1.1.1.1.
For the amino acid sequence of alcohol dehydrogenase 1 (also termed ADH1) it may be referred to the access number NP_014555 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001183340 in the NCBI database.
Alcohol Dehydrogenase 3
The alcohol dehydrogenase 3 is a protein which is described in the art for catalyzing the conversion of primary unbranched alcohols to their corresponding aldehydes.
A method implemented to measure the activity level of the alcohol dehydrogenase 3 belongs to the general knowledge of the one skilled in the art.
In this regard, the one skilled in the art may advantageously refer to the method described by Ganzhorn et al. (1987) The Journal of Biological Chemistry, 262, 3754-61.
Preferred alcohol dehydrogenase 3 in the present specification is an enzyme having an EC number of n° 1.1.1.1.
For the amino acid sequence of alcohol dehydrogenase 3 (also termed ADH3) it may be referred to the access number NP_013800 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001182582 in the NCBI database.
Alcohol Dehydrogenase 4
The alcohol dehydrogenase 4 is a protein which is described in the art for catalyzing the conversion of primary unbranched alcohols to their corresponding aldehydes.
A method implemented to measure the activity level of the alcohol dehydrogenase 4 belongs to the general knowledge of the one skilled in the art.
In this regard, the one skilled in the art may advantageously refer to the method described by Ganzhorn et al. (1987) The Journal of Biological Chemistry, 262, 3754-61.
Preferred alcohol dehydrogenase 4 in the present specification is an enzyme having an EC number of n° 1.1.1.1.
For the amino acid sequence of alcohol dehydrogenase 4 (also termed ADH4) it may be referred to the access number NP_011258 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001181122 in the NCBI database.
Alcohol Dehydrogenase 5
The alcohol dehydrogenase 5 is a protein which is described in the art for catalyzing the conversion of primary unbranched alcohols to their corresponding aldehydes.
A method implemented to measure the activity level of the alcohol dehydrogenase 5 belongs to the general knowledge of the one skilled in the art.
In this regard, the one skilled in the art may advantageously refer to the method described by Ganzhorn et al. (1987) The Journal of Biological Chemistry, 262, 3754-61.
Preferred alcohol dehydrogenase 5 in the present specification is an enzyme having an EC number of n° 1.1.1.1.
For the amino acid sequence of alcohol dehydrogenase 5 (also termed ADH5) it may be referred to the access number NP_009703 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_001178493 in the NCBI database.
Second Further Embodiments of a Ectoine-Producing Recombinant Yeast
According to these further embodiments of a ectoine-producing recombinant yeast according to the invention, further genetic engineering of the recombinant yeast is performed with the aim of increasing the production of the intermediate product phosphoenol-pyruvate (PEP).
Without wishing to be bound by any particular theory, the inventors believe that the further genetic changes introduced in the ectoine-producing recombinant yeast (i) cause an over-production of NADPH, (ii) cause a controlled and balanced conversion of phosphoenol pyruvate into oxaloacetate and pyruvate, respectively, and (iii) cause a reduced conversion of pyruvate into ethanol and a redirection towards conversion of phosphoenol pyruvate into oxaloacetate.
For this purpose, the inventors have conceived a completely novel metabolic pathway, starting from phosphenolpyruvate and ending with the production of oxaloacetate.
These further genetic changes introduced by genetic engineering in a ectoine-producing recombinant yeast according to the invention are specified in more detail below.
According to these embodiments, genetic changes are introduced so as to under express the pyruvate kinase 1 (also termed PYK1), and optionally also pyruvate kinase 2 (also termed PYK2). In some of these embodiments, PYK1 may be under-expressed by placing the gene under the control of a weak promoter or of an inducible or repressible promoter. In some of these embodiments, PYK2 may be inactivated, e.g. by interruption or deletion. In some of these embodiments, PYK1 gene may be deleted rather than being under-expressed. In some of these embodiments, PYK1 gene and PYK2 gene may be deleted rather than being under-expressed.
According to these embodiments, genetic changes are introduced so as to over-express a phosphoenolpyruvate carboxykinase [ATP] (also termed PCK or PCKA or PEPCK), either (i) by constitutive over-expression or (ii) by inducible over-expression.
According to these embodiments, genetic changes are introduced so as over-express in the cytoplasm a malate dehydrogenase, such as a peroxisomal malate dehydrogenase (also termed MDH3), either (i) by constitutive over-expression or (ii) by inducible over-expression.
According to these embodiments, genetic changes are introduced so as over-express a NADP-dependent malic enzyme 3 (also termed ME3 or NADP-ME3), either (i) by constitutive over-expression or (ii) by inducible over-expression.
According to these embodiments, genetic changes are introduced so as to reduce expression of one or more alcohol dehydrogenase(s), preferably one or more alcohol dehydrogenase(s) selected in a group comprising alcohol dehydrogenase 1 (also termed ADH1), alcohol dehydrogenase 3 (also termed ADH3), alcohol dehydrogenase 4 (also termed ADH4) and alcohol dehydrogenase 5 (also termed ADH5), e.g. (i) by placing the corresponding coding sequence under the control of a weak promoter or of an inducible or repressible promoter, or (ii) by production of a destabilized form of the said alcohol dehydrogenase(s).
Still according to these embodiments, genetic changes are introduced so as to over-express an exogenous acetaldehyde dehydrogenase (also termed MHPF), either (i) by constitutive over-expression or (ii) by inducible over-expression.
Phosphoenolpyruvate Carboxykinase (PPCK)
The phosphoenol carboxykinase [ATP] enzyme is a protein which is described in the art for catalyzing the conversion of oxaloacetate to phosphoenolpyruvate through direct phosphoryl transfer between the nucleoside triphosphate and oxaloacetate.
A method implemented to measure the activity level of phosphoenol carboxykinase [ATP] belongs to the general knowledge of the one skilled in the art.
In this regard, the one skilled in the art may advantageously refer to the method described by Bazaes S. et al. (2007) The Protein Journal, 26, 265-269 and Mariet J. Van der Werf et al. (1997) Arch Microbiol 167: 332-342.
Preferred phosphoenol carboxykinase [ATP] in the present specification is an enzyme having an EC number of n° 4.1.1.49.
For the amino acid sequence of phosphoenol carboxykinase [ATP] (also termed PCKA) it may be referred to the access number NP_417862 in the UniProt database. For the nucleic acid sequence, it may be referred to the one disclosed in the access number NC 000913 in the NCBI database.
Preferred phosphoenol carboxykinase according to the invention can be selected from phosphoenolpyruvate carboxykinase PPCK such as PEPCK having an EC number of n° 4.1.1.32.
Malate Dehydrogenase
The malate dehydrogenase enzyme is a protein which is described in the art for catalyzing the conversion of malate to oaxaloacetate, in the presence of NADH.
A method implemented to measure the activity level of malate dehydrogenase belongs to the general knowledge of the one skilled in the art. Mention can for example be made of the commercial kit sold by Sigma entitled “Malate dehydrogenase assay kit” under the reference MAK196-1KT.
For the amino acid sequence of malate dehydrogenase (also termed MDH3) it may be referred to the access number NP_010205 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_00118037 in the NCBI database.
NADP-Dependent Malic Enzyme 3
The NADP-dependent malic enzyme 3 enzyme is a protein which is described in the art for catalyzing the conversion of malate to pyruvate, in the presence of NADP.
A method implemented to measure the activity level of NADP-dependent malic enzyme 3 belongs to the general knowledge of the one skilled in the art.
In this regard, the one skilled in the art may advantageously refer to the method described by Gerrard-Wheeler et al. FEBS Journal 276 (2009) 5665-5677.
Preferred NADP-dependent malic enzyme 3 in the present specification is an enzyme having an EC number of n° 1.1.1.40.
For the amino acid sequence of NADP-dependent malic enzyme 3 (also termed NADP-ME3 or ME3) it may be referred to the access number NP_197960 in the UniProt database. For the nucleic acid sequence, it may be referred to the access number NM_122489 in the NCBI database.
Alcohol dehydrogenase 1, alcohol dehydrogenase 3, alcohol dehydrogenase 4, alcohol dehydrogenase 5 and acetaldehyde dehydrogenase are as indicated here-above.
Promoters
As it is disclosed herein, the expression of the genes of interest that have been genetically engineered for obtaining a recombinant yeast according to the invention comprise appropriate regulatory sequences that are functional in yeast cells, including in Saccharomyces cerevisiae. 
As disclosed in the present specification, various promoters may be used for the desired expression of the coding sequences of interest, which include (i) constitutive strong promoters (also called strong promoters in the present text), (ii) constitutive weak promoters (also called weak promoters in the present text) and (iii) inducible or repressible promoters. A list of yeast promoter with their relative activities in different media can be found in Keren et al. (2013) Molecular Systems Biology 9:701.
Promoters allowing the constitutive over-expression of a given gene, may be found in literature (Velculescu et al. (1997) Cell 88, 243-251).
Strong promoters more particularly interesting in the present invention may be selected from the group comprising:

    
    
        pTDH3 (SEQ ID N° 24),
        pENO2 (SEQ ID N° 25),
        pTEF K1 (SEQ ID N° 26),
        pTEF3 (SEQ ID N° 27),
        pTEF1 (SEQ ID N° 28),
        pADH1 (SEQ ID N° 29),
        pGMP1 (SEQ ID N° 30),
        pFBA1 (SEQ ID N° 31),
        pPDC1 (SEQ ID N° 32),
        pCCW12 (SEQ ID N° 33), and
        pGK1 (SEQ ID N° 34).
    
    


According to a particular embodiment, the strong promoter according to the invention is, independently, selected from the group consisting of pTDH3, pENO2, pTEF-K1, pTEF3, pTEF1, pADH1, pGMP1, pFBA1, pPDC1, pCCW12 and pGK1.
Weak promoters more particularly interesting in the present invention may be selected from the group comprising:

    
    
        pURA3 (SEQ ID N° 36),
        pRPLA1 (SEQ ID N° 37),
        pNUP57 (SEQ ID N° 119), and
        pGAP1 (SEQ ID N° 120).
    
    


According to a particular embodiment, the weak promoter according to the invention is, independently, selected from the group consisting of pURA3, pRPLA1, pNUP57 and pGAP1.
As previously mentioned, inducible or repressible promoters are promoters whose activity is controlled by the presence or absence of biotic or abiotic factors and also by the quantity of said factor. Accordingly, for some promoters, their activity will in particular be induced and thus increased when the quantity of a given factor increases or is increased, and, accordingly, the activity of these same promoters can be repressed and thus reduced when the quantity of said factor diminishes or is reduced. The quantity of said factor(s) in the culture medium of a recombinant yeast of the invention comprising inducible or repressible promoters can be decided and thus controlled by the man skilled in the art.
For example, increasing the quantity of methionine in a culture medium of a recombinant yeast according to the invention comprising a pSAM4 promoter will induce and thus increase transcription of the gene under the control of this promoter. On the contrary, reducing the quantity of methionine in said culture medium will lead to a repression, and thus a reduced, transcription of the gene under the control of this promoter.
In another example, increasing the quantity of copper in a culture medium of a recombinant yeast according to the invention comprising a pCTR1 promoter will represse and thus decrease transcription of the gene under the control of this promoter. On the contrary, reducing the quantity of copper in said culture medium will lead to an induced, and thus an increased, transcription of the gene under the control of this promoter.
For this reason, the following promoters are referred to in the present text as being “inducible or repressible promoters”.
According to a first embodiment, inducible or repressible promoters according to the invention may be selected from the group comprising promoters inducible or repressible with copper, promoters inducible or repressible with methionine and promoters inducible or repressible with threonine, and are in particular selected from the group consisting of:

    
    
        pSAM4—methionine inducible or repressible (SEQ ID N° 38),
        pCUP1-1—copper inducible or repressible (SEQ ID N° 39),
        pCUP1.cgla—copper inducible or repressible (SEQ ID N° 40),
        pCUP1.sba—copper inducible or repressible (SEQ ID N° 41),
        pACU1—copper inducible or repressible (SEQ ID N° 42),
        pACU2—copper inducible or repressible (SEQ ID N° 43),
        pACU3p—copper inducible or repressible (SEQ ID N° 44),
        pACU4p—copper inducible or repressible (SEQ ID N° 45),
        pACU5—copper inducible or repressible (SEQ ID N° 46),
        pACU6—copper inducible or repressible (SEQ ID N° 47),
        pACU7—copper inducible or repressible (SEQ ID N° 48),
        pACU8—copper inducible or repressible (SEQ ID N° 49),
        pACU9—copper inducible or repressible (SEQ ID N° 50),
        pACU10p—copper inducible or repressible (SEQ ID N° 51),
        pACU11—copper inducible or repressible (SEQ ID N° 52),
        pACU12—copper inducible or repressible (SEQ ID N° 53),
        pACU13—copper inducible or repressible (SEQ ID N° 54),
        pACU14—copper inducible or repressible (SEQ ID N° 55),
        pACU15—copper inducible or repressible (SEQ ID N° 56),
        pGAL/CUPlp—copper inducible or repressible (SEQ ID N° 57),
        pCRS5—copper inducible or repressible (SEQ ID N° 58), and
        pCHA1—threonine inducible or repressible (SEQ ID N° 59).
    
    


According to this embodiment, the inducible or repressible promoter according to the invention can in particular, independently, be selected from the group consisting of pSAM4, pCUP1-1, pCUP1.Cgla, pCUP1.Sba, pACU1, pACU2, pACU3p, pACU4p, pACU5, pACU6, pACU7, pACU8, pACU9, pACU10p, pACU11, pACU12, pACU13, pACU14, pACU15, pGAL/CUP1p, pCRS5, and pCHA1.
The activity of these promoters is thus induced by the increasing presence of methionine, copper or threonine as indicated above, and their activity diminishes, i.e. is repressed, when the quantity of methionine, copper or threonine is reduced.
According to a second embodiment, inducible or repressible promoters according to the invention may be selected from the group comprising promoters inducible or repressible with copper, promoters inducible or repressible with lysine and promoters inducible or repressible with methionine, and in particular selected from the group consisting of:

    
    
        pCTR1—copper inducible or repressible (SEQ ID N° 60),
        pCTR3—copper inducible or repressible (SEQ ID N° 61),
        pCUR1—copper inducible or repressible (SEQ ID N° 62),
        pCUR2—copper inducible or repressible (SEQ ID N° 63),
        pCUR3—copper inducible or repressible (SEQ ID N° 64),
        pCUR4—copper inducible or repressible (SEQ ID N° 65),
        pCUR5p—copper inducible or repressible (SEQ ID N° 66),
        pCUR6—copper inducible or repressible (SEQ ID N° 67),
        pCUR7—copper inducible or repressible (SEQ ID N° 68),
        pCUR8—copper inducible or repressible (SEQ ID N° 69),
        pCUR9—copper inducible or repressible (SEQ ID N° 70),
        pCUR10—copper inducible or repressible (SEQ ID N° 71),
        pCUR11—copper inducible or repressible (SEQ ID N° 72),
        pCUR12—copper inducible or repressible (SEQ ID N° 73),
        pCUR13—copper inducible or repressible (SEQ ID N° 74),
        pCUR14—copper inducible or repressible (SEQ ID N° 75),
        pCUR15—copper inducible or repressible (SEQ ID N° 76),
        pCUR16—copper inducible or repressible (SEQ ID N° 77),
        pCUR17—copper inducible or repressible (SEQ ID N° 78),
        pLYS1—lysine inducible or repressible (SEQ ID N° 79),
        pLYS4—lysine inducible or repressible (SEQ ID N° 80),
        pLYS9—lysine inducible or repressible (SEQ ID N° 81),
        pLYR1p—lysine inducible or repressible (SEQ ID N° 82),
        pLYR2p—lysine inducible or repressible (SEQ ID N° 83),
        pLYR3p—lysine inducible or repressible (SEQ ID N° 84),
        pLYR4p—lysine inducible or repressible (SEQ ID N° 85),
        pLYR5p—lysine inducible or repressible (SEQ ID N° 86),
        pLYR6p—lysine inducible or repressible (SEQ ID N° 87),
        pLYR7p—lysine inducible or repressible (SEQ ID N° 88),
        pLYR8—lysine inducible or repressible (SEQ ID N° 89),
        pLYR9—lysine inducible or repressible (SEQ ID N° 90),
        pLYR10—lysine inducible or repressible (SEQ ID N° 91),
        pLYR11—lysine inducible or repressible (SEQ ID N° 92),
        pMET17—methionine inducible or repressible (SEQ ID N° 93),
        pMET6—methionine inducible or repressible (SEQ ID N° 94),
        pMET14—methionine inducible or repressible (SEQ ID N° 95),
        pMET3—methionine inducible or repressible (SEQ ID N° 96),
        pSAM1—methionine inducible or repressible (SEQ ID N° 97),
        pSAM2—methionine inducible or repressible (SEQ ID N° 98),
        pMDH2—glucose inducible or repressible (SEQ ID N° 35),
        pJEN1—glucose inducible or repressible (SEQ ID N° 118),
        pICL1—glucose inducible or repressible (SEQ ID N° 119),
        pADH2—glucose inducible or repressible (SEQ ID N° 120), and
        pMLS1—glucose inducible or repressible (SEQ ID N° 121).
    
    


According to this particular embodiment, the inducible or repressible promoter according of the invention can, independently, be selected from the group consisting of pCTR1, pCTR3, pCUR1, pCUR2, pCUR3, pCUR4, pCUR5p, pCUR6, pCUR7, pCUR8, pCUR9, pCUR10, pCUR11, pCUR12, pCUR13, pCUR14, pCUR15, pCUR16, pCUR17, pLYS1, pLYS4, pLYS9, pLYR1p, pLYR2p, pLYR3p, pLYR4p, pLYR5p, pLYR6p, pLYR7p, pLYR8, pLYR9, pLYR10, pLYR11, pMET17, pMET6, pMET14, pMET3, pSAM1, pSAM2, pMDH2, pJEN1, pICL1, pADH2 and pMLS1.
The activity of these promoters is thus repressed by the increasing presence of methionine, copper, lysine or glucose as indicated above, and their activity increases, i.e. is induced, when the quantity of methionine, copper, lysine or glucose is reduced.
In a particular embodiment, inducible or repressible promoters according to the invention may be selected from the group comprising promoters inducible or repressible with copper, promoters inducible or repressible with glucose, promoters inducible or repressible with lysine, promoters inducible or repressible with methionine and promoters inducible or repressible with threonine.
In a more particular embodiment, the inducible or repressible promoter according to the invention can, independently, be selected from the group consisting of pSAM4, pCUP1-1, pCUP1.Cgla, pCUP1.Sba, pACU1, pACU2, pACU3p, pACU4p, pACU5, pACU6, pACU7, pACU8, pACU9, pACU10p, pACU11, pACU12, pACU13, pACU14, pACU15, pGAL/CUP1p, pCRS5, pCHA1, pCTR1, pCTR3, pCUR1, pCUR2, pCUR3, pCUR4, pCUR5p, pCUR6, pCUR7, pCUR8, pCUR9, pCUR10, pCUR11, pCUR12, pCUR13, pCUR14, pCUR15, pCUR16, pCUR17, pLYS1, pLYS4, pLYS9, pLYR1p, pLYR2p, pLYR3p, pLYR4p, pLYR5p, pLYR6p, pLYR7p, pLYR8, pLYR9, pLYR10, pLYR11, pMET17, pMET6, pMET14, pMET3, pSAM1, pSAM2, pMDH2, pJEN1, pICL1, pADH2 and pMLS1.
More particularly, said promoters, identical or different, may be preferably characterized by a sequence of nucleic acid selected from the group consisting of sequences having at least 80% identity with sequences SEQ ID NO: 24 to 98 and 116-121.
Synthetic promoters as described in Blazeck & Alper (2013) Biotechnol. J. 8 46-58 can also be used.
The strong, weak and inductible or repressible promoters of the invention can originate from any organism from the Saccharomycetes class and can in particular originate, independently, from an organism selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces castelii, Saccharomyces bayanus, Saccharomyces arboricola, Saccharomyces kudriavzevii, Ashbya gossypii, Kluveromyces lactis, Pichia pastoris, Candida glabrata, Candida tropicalis, Debaryomyces castelii, Yarrowia lipolitica and Cyberlindnera jadinii. 
The strong, weak and inductible or repressible promoters of the invention can preferably originate from an organism selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces castelii, Saccharomyces bayanus, Saccharomyces arboricola, Saccharomyces kudriavzevii and Kluveromyces lactis. 
Terminators
As it is disclosed herein, the expression of the genes of interest that have been genetically engineered for obtaining a recombinant yeast according to the invention comprise appropriate transcription terminator sequences that are functional in yeast cells, including in Saccharomyces cerevisiae. 
Said transcription terminators, identical or different, may be found in literature Yamanishi et al., (2013) ACS synthetic biology 2, 337-347.
Terminators more particularly interesting in the present invention may be selected from the group comprising:

    
    
        tTDH2 from the gene coding for Glyceraldehyde-3-phosphate dehydrogenase, isozyme 2 (TDH2 gene=Sequence SEQ ID N° 99),
        tCYC1 (=Sequence SEQ ID N° 100),
        tTDH3 (=Sequence SEQ ID N° 101), and
        tADH1 from gene coding for the alcohol dehydrogenase (ADH1 gene=Sequence SEQ ID N° 102),
        tADH2 from gene coding for the alcohol dehydrogenase (ADH2 gene=Sequence SEQ ID N° 103),
        tTPI1 from the gene encoding for the Triose Phosphate Isomerase (TPI1 gene=Sequence SEQ ID N° 104),
        tMET17 from the gene encoding for the O-acetyl homoserine-O-acetyl serine sulfhydrylase (Met17 gene=Sequence SEQ ID N° 105),
        tENO2 from the gene coding for Enolase II (ENO2 gene=Sequence SEQ ID N° 106),
        tMET3 (=Sequence SEQ ID N° 107), and
        tPGK1 from the gene encoding for the 3-phosphoglycerate kinase (PGK1 gene=Sequence SEQ ID N° 108),
        tDIT1 (=Sequence SEQ ID N° 109)
        tRPL3 (=Sequence SEQ ID N° 110)
        tRPL41B (=Sequence SEQ ID N° 111)
        tRPL15A (=Sequence SEQ ID N° 112)
        tIDP1 (=Sequence SEQ ID N° 113).
        More particularly, said terminator, identical or different, may be preferably characterized by a sequence of nucleic acid selected from the group consisting of sequences having at least 80% identity with sequences SEQ ID NO: 99 to 113.

Recombinant Yeast

    
    


Generally, yeast can grow rapidly and can be cultivated at higher density as compared with bacteria, and does not require an aseptic environment in the industrial setting. Furthermore, yeast cells can be more easily separated from the culture medium compared to bacterial cells, greatly simplifying the process for product extraction and purification.
Preferentially, the yeast of the invention may be selected among the genus Saccharomyces, CandidaAshbya, Dekkera, Pichia (Hansenula), Debaryomyces, Clavispora, Lodderomyces, Yarrowia, Zigosaccharomyces, Schizosaccharomyces, Torulaspora, Kluyveromyces, Brettanomycces, Cryptococcus or Malassezia. 
More preferentially, the yeast may be Crabtree positive yeast of genus of Saccharomyces, Dekkera, Schizosaccharomyces, Kluyveromyces, Torulaspora Zigosaccharomyces, or. Brettanomycces. 
More preferentially, the yeast may be from the species Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces douglasii, Saccharomyces bayanus, Zigosaccharomyces bailii, Schizosaccharomyces pombe, Dekkera brucelensis, Dekkera intermedia, Brettanomycces custersii, Brettanomycces intermedius, Kluyveromyces themotolerens, Torulaspora globosa or Torulaspora glabrata. 
More preferentially, the recombinant yeast may belong to the Saccharomyces genus, and preferably to the Saccharomyces cerevisiae species.
As above-mentioned, a recombinant yeast according to the invention has a pyruvate decarboxylase activity which is reduced by insertion of at least one DNA construct(s) selected from those disclosed in the present specification.
Methods implemented to insert a specific DNA construct within a gene belong to the general knowledge of a man skilled in the art. A related method is described in more details in the herein after examples.
Culture Conditions
The present invention also relates to the use of a recombinant yeast such as above-defined, for the production of ectoine.
The present invention further relates to a method of production of ectoine comprising the following steps:

    
    
        providing a recombinant microorganism as previously described, cultivating the recombinant microorganism in a culture medium containing a source of carbon, and
        recovering the ectoine.
    
    


Typically, microorganisms of the invention are grown at a temperature in the range of about 20° C. to about 37° C., preferably at a temperature ranging from 27 to 34° C., in an appropriate culture medium.
When the recombinant yeast according to the invention belongs to the S. cerevisiae species, the temperature may advantageously range from 27 to 34° C., in an appropriate culture medium.
Suitable growth media for yeast are common commercially prepared media such as broth that includes yeast nitrogen base, ammonium sulfate, and dextrose as the carbon/energy source) or YPD Medium, a blend of peptone, yeast extract, and dextrose in optimal proportions for growing most. Other defined or synthetic growth media may also be used and the appropriate medium for growth of the particular microorganism will be known by one skilled in the art of microbiology or fermentation science.
The term “appropriate culture medium” is above-defined.
Examples of known culture media for a recombinant yeast according to the present invention are known to the person skilled in the art, and are presented in the following publication D. Burke et al., Methods in yeast Genetics—A cold spring harbor laboratory course Manual (2000).
Suitable pH ranges for the fermentation may be between pH 3.0 to pH 7.5, where pH 4.5 to pH 6.5 is preferred as the initial condition.
Fermentations may be performed under aerobic conditions or micro-aerobic conditions.
The amount of product in the fermentation medium can be determined using a number of methods known in the art, for example, high performance liquid chromatography (HPLC) or gas chromatography (GC).
The present process may employ a batch method of fermentation. A classical batch fermentation is a closed system where the composition of the medium is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation. Thus, at the beginning of the fermentation, the medium is inoculated with the desired organism or organisms, and fermentation is permitted to occur without adding anything to the system. Typically, however, a “batch” fermentation is batch with respect to the addition of carbon source and attempts are often made at controlling factors such as temperature, pH and oxygen concentration. In batch systems, the metabolite and biomass compositions of the system change constantly up to the time when the fermentation is stopped. Within batch cultures cells progress through a static lag phase to a high growth log phase and finally to a stationary phase where growth rate is diminished or halted. If untreated, cells in the stationary phase will eventually die. Cells in log phase generally are responsible for the bulk of production of end product or intermediate.
A Fed-Batch system may also be used in the present invention. A Fed-Batch system is similar to a typical batch system with the exception that the carbon source substrate is added in increments as the fermentation progresses. Fed-Batch systems are useful when catabolite repression (e.g. glucose repression) is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media.
Measurement of the actual substrate concentration in Fed-Batch systems is difficult and is therefore estimated on the basis of the changes of measurable factors such as pH, dissolved oxygen and the partial pressure of waste gases such as CO2.
Fermentations are common and well known in the art and examples may be found in Sunderland et al., (1992), herein incorporated by reference. Although the present invention is performed in batch mode it is contemplated that the method would be adaptable to continuous fermentation.
Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth.
Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. For example, one method will maintain a limiting nutrient such as the carbon source or nitrogen level at a fixed rate and allow all other parameters to vary. In other systems a number of factors affecting growth can be altered continuously while the cell concentration, measured by media turbidity, is kept constant. Continuous systems strive to maintain steady state growth conditions and thus the cell loss due to the medium being drawn off must be balanced against the cell growth rate in the fermentation. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology.
It is contemplated that the present invention may be practiced using either batch, fed-batch or continuous processes and that any known mode of fermentation would be suitable. Additionally, it is contemplated that cells may be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for production.
In order to still improve the ectoine production, a particular embodiment may consist of culturing the recombinant yeast cells in an appropriate culture medium, such as above-mentioned, wherein the said culture medium comprises an optimal amount of carbon source, especially glucose.
Preferably, the cells are cultured in such an optimal culture medium during only a part of the whole culture duration. In some embodiments, the yeast cells are incubated in the said optimal culture medium 10 hours or more after initiation of the culture, which encompasses 11, 12, 13, 14, 15 or 16 hours or more after initiation of the culture.
Preferably, the cells are cultured in such an optimal culture medium during a time period ranging from 5 hours to 15 hours, which includes from 6 hours to 10 hours, e.g. 8 hours after initiation of the culture.
In preferred embodiments, the carbon source comprised in said optimal culture medium consists of glucose. In preferred embodiments, the said optimal culture medium comprises 12% w/w or more glucose, including 15% w/w or more glucose. In preferred embodiments, the said optimal culture medium comprises at most 40% w/w glucose, which includes at most 35% w/w glucose.
Thus, in the preferred embodiments described above, a method for producing ectoine according to the invention may further comprise, between steps (a) and (c), an intermediate step (b) consisting of cultivating the yeast cells in the said optimal culture medium.
Purification of Ectoine
According to a specific aspect of the invention, the fermentative production of ectoine comprises a step of isolation of the ectoine from the culture medium. Recovering the ectoine from the culture medium is a routine task for a man skilled in the art. It may be achieved by a number of techniques well known in the art including but not limiting to distillation, gas-stripping, pervaporation, selective precipitation or liquid extraction. The expert in the field knows how to adapt parameters of each technique dependent on the characteristics of the material to be separated.
The yeast as model of microorganism in the present invention has been retained in that the synthesized ectoine is/are entirely exported outside the cells, thus simplifying the purification process.
The synthesized ectoine may be collected by distillation. Distillation may involve an optional component different from the culture medium in order to facilitate the isolation of ectoine by forming azeotrope and notably with water. This optional component is an organic solvent such as cyclohexane, pentane, butanol, benzene, toluene, trichloroethylene, octane, diethylether or a mixture thereof.
Gas stripping is achieved with a stripping gas chosen among helium, argon, carbon dioxide, hydrogen, nitrogen or mixture thereof.
Liquid extraction is achieved with organic solvent as the hydrophobic phase such as pentane, hexane, heptane or dodecane.
The terms “between . . . and . . . ” and “ranging from . . . to . . . ” should be understood as being inclusive of the limits, unless otherwise specified.
The examples and figures which follow are presented by way of illustration and without implied limitation of the invention.
EXAMPLES
Example 1: Protocol for Making a Recombinant Saccharomyces cerevisiae Strain According to the Invention
All the hereinafter implemented recombinant Saccharomyces cerevisiae strains were constructed from standard strains using standard yeast molecular genetics procedure (Methods in yeast Genetics—A cold spring harbor laboratory course Manual (2000) by D. Burke, D. Dawson, T. Stearns CSHL Press).
Cluster of the following-mentioned genes were integrated in recombinant yeast at once using the ability of yeast to efficiently recombine free DNA ends which have sequence homology.
In addition, for a better comprehension of following genotypes:

    
    
        ade2, his3, leu2, trp1 and ura3 are auxotrophy marker genes.
        Lowercase letters mean that the considered gene is inactive, uppercase letters reflect an active gene.
        “::”: following a gene name means that the gene is interrupted by what follows (if more than one gene are inserted, they are noted in brackets [ ]). The interruption of the gene is concomitant with an entire deletion of the coding sequence but preserves the promoter. In consequence the gene followed by “::” is inactive and is noted in lowercase. If not specified the transcription of the gene inserted is controlled by the promoter of the disrupted gene.
        “gene.K1” means that the gene originates from Kluyveromyces lactis. 
    
    


More particularly, the coding sequences to be cloned were artificially synthetized. For heterologous sequences (non-yeast), the nucleic sequences were modified in order to obtain a synonymous coding sequence using the yeast codon usage. Using restriction enzyme and classical cloning technology, each synthetic sequence was cloned in between a transcription promoter and a transcription terminator. Each promoter sequence is preceded by a 50 to 200 nucleotide sequence homologous to the sequence of the terminator of the upstream gene. Similarly, the terminator of each gene (a gene comprising the promoter-coding sequence-terminator) is followed by sequences homologous to the gene immediately following. So that each of the unit to be integrated have a 50-200 nucleotide overlap with both the unit upstream and the unit downstream. For the first unit, the promoter is preceded by 50-200 nucleotides homologous to the yeast chromosome nucleotide for the locus in which it will be integrated. Similarly, for the last unit, the terminator is followed by 50-200 nucleotides homologous to the yeast chromosome nucleotide for the locus in which it will be integrated.
Each unit are then PCR amplified from the plasmids constructs, yielding X unit of linear DNA having overlapping sequences. At least one of this gene is an auxotrophic marker, in order to select for recombination event. All the linear fragments are transformed in the yeast at once, and recombinant yeast are selected for the auxotrophy related to the marker used. The integrity of the sequence is then verified by PCR and sequencing.
Example 2: Comparative Examples for the Production of Ectoine
A. Firstly, two recombinant strains are obtained: YA3370-20 and YA3371-46. These two strains have been recombined in order to only comprise a part of the modifications according to the invention.
Accordingly, these two strains are as follows:
YA3370-20: ade2, can1::[pACU1-AAT2-tRPL3-pCUP1-1-PPC-5.Ec-tTP11]x4, his3::[pACU5-HOM2-2-tRPL3-pTDH3-GDH-2.Eca-tIDP1]x4, hom6::[URA3-pCCW12-ECTB.He-tIDP1]x5, leu2, lyp1::[pPDC1-ECTA.He-tCYC1-pTDH3-ECTC.He-tTDH3]x2, pyk1::[LEU2.K1-RS,pTDH3-PEPCK-1.Ec-tIDP1,pTEF3-AQR1-tRPL41B, pCUR3-PYK1-tPYK1], sam3::[pPDC1-METX.Cg-tRPL3-pTDH3-MHPF.ec-tIDP1]x2, trp1::[pPDC1-PPC-5.Ec-tRPL3-pACU7-AK.Bs-tIDP1-TRP1]x6, ura3
YA3371-46: ade2, can1::[pACU1-AAT2-tRPL3-pCUP1-1-PPC-5.Ec-tTP11]x4, his3::[pACU5-HOM2-2-tRPL3-pTDH3-GDH-2.Eca-tIDP1]x4, hom6::[URA3-pCCW12-ECTB.He-tIDP1]x5, leu2, lyp1::[pPDC1-ECTA.He-tCYC1-pTDH3-ECTC.He-tTDH3]x2, pyk1::[LEU2.K1-RS,pTDH3-PEPCK-1.Ec-tIDP1,pTEF3-AQR1-tRPL41B, pCUR3-PYK1-tPYK1], sam3::[pCCW12-ECTB.Pa-tRPL3-pTDH3-MHPF.Ec-tRPL41B]x4, trp1::[pPDC1-PPC-5.Ec-tRPL3-pACU7-AK.Bs-tIDP1-TRP1]x6, ura3
PEPCK-1 is a form of PEPCK stabilized by modification of the Arginine amino acid in position 2 by a Glycine.
PPC-5 is a more stable form of PPC wherein an alanine has been added in N+1.
All these strains were grown for 24 hours in YE (Yeast Extract) 2% and Glucose 8%, and 500 μM of CuSO4 was added after 8 hours. The content of ectoine in the medium was assayed after 24 hours using the AccQ-Tag precolumn derivatization method for amino acid determination using a AccQ-Tag Ultra Derivatization Kit from Waters as advised by the manufacturer.
The ectoine amounts obtained with these different strains are respectively:

    
    
        YA3370-20: 1 g/L−1.
        YA3371-46: 1.55 g/L−1.
    
    


In comparison, a native strain does not produce ectoine.
It results from this comparative experiment that a recombinant strain comprising the modifications according to the invention produces a greater amount of ectoine when cultured in the same conditions as other recombinant strains not comprising all the genetic modifications according to the invention.
B. Five other recombinant strains have also been obtained: YA3380-40B, YA3595-25 and YA3595-34.
These three strains are as follows:
YA3380-40B: gnp1::[LEU2.K1-RS, pADH1-AAT2-tRPL15A, pTEF3-MDH3-1-tRPL3, pPDC1-PEPCK-1.Ec-tMET25, pTDH3-MHPF.Ec-tTP11, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tIDP1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tTPI1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tIDP1, pCCW12-ME3.At-tRPL3], his3::[pACU5-ME3.At-tRPL3-pACU6-METX-1.Cg-tIDP1]x11, hom6::[TRP1.K1, pCCW12.Sba-HOM3-tDIT1], leu2, mup3::[HIS5. Sp, pACU7-PEPCK-1.Ec-tRPL3, pCCW12-HOM2-1-tTDH3, pPGK1-AAT2-tTDH2, pENO2-MDH3-1-tRPL15A, pCUP1-1-GDH-2.Eca-tTPI1, pTDH3.Sba-ECTB.He-tIDP1, pPDC1-ECTA.He-tRPL41B, pTEF1.Sba-ECTC.He-tRPL15A], pyk1::[LEU2.K1, pTDH3-PEPCK-1.Ec-tIDP1, pPDC1-MDH3-1-tRPL15A, pTEF3-TP01-tEN02, pCUR3-PYK1-7-tCYC1], trp1, ura3
YA3595-25: ade2, gnp1::[LEU2.K1-RS,pADH1-AAT2-tRPL15A, pTEF3-MDH3-1-tRPL3, pPDC1-PEPCK-1.Ec-tMET25, pTDH3-MHPF.Ec-tTPI1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tIDP1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tTPI1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tIDP1, pCCW12-ME3.At-tRPL3], his3, hom6::[TRP1.K1, pCCW12.Sba-HOM3-tDIT1], leu2, mup3::[HIS5.Sp, pACU7-PEPCK-1.Ec-tRPL3, pCCW12-HOM2-1-tTDH3, pPGK1-AAT2-tTDH2, pEN02-MDH3-1-tRPL15A, pCUP1-1-GDH-2.Eca-tTPI1, pTDH3.Sba-ECTB.He-tIDP1, pPDC1-ECTA.He-tRPL41B, pTEF1.Sba-ECTC.He-tRPL15A], pyk1::[LEU2.K1, pTDH3-PEPCK-1.Ec-tIDP1, pPDC1-MDH3-1-tRPL15A, pTEF3-TP01-tEN02, pCUR3-PYK1-7-tCYC1], sam3::[pACU7-PEPCK-1.Ec-tRPL3-pCUP1-1-HOM3-tDIT1]x7, trp1, ura3::[pCCW12-ECTB.Ab-tRPL3-pTDH3-ECTC.He-tRPL41B.Sba]x14
YA3595-34: ade2, gnp1::[LEU2.K1-RS,pADH1-AAT2-tRPL15A, pTEF3-MDH3-1-tRPL3, pPDC1-PEPCK-1.Ec-tMET25, pTDH3-MHPF.Ec-tTPI1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tIDP1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tTPI1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tIDP1, pCCW12-ME3.At-tRPL3], his3, hom6::[TRP1.K1, pCCW12.Sba-HOM3-tDIT1], leu2, mup3::[HIS5.Sp, pACU7-PEPCK-1.Ec-tRPL3, pCCW12-HOM2-1-tTDH3, pPGK1-AAT2-tTDH2, pEN02-MDH3-1-tRPL15A, pCUP1-1-GDH-2.Eca-tTPI1, pTDH3.Sba-ECTB.He-tIDP1, pPDC1-ECTA.He-tRPL41B, pTEF1.Sba-ECTC.He-tRPL15A], pyk1::[LEU2.K1, pTDH3-PEPCK-1.Ec-tIDP1, pPDC1-MDH3-1-tRPL15A, pTEF3-TPO1-tENO2, pCUR3-PYK1-7-tCYC1], sam3::[pACU7-PEPCK-1.Ec-tRPL3-pCUP1-1-HOM3-tDIT1]x7, trp1, ura3::[pCCW12-ECTB.Ab-tRPL3-pTDH3-ECTC.He-tRPL41B.Sba]x9
The strains YA3380-40B and YA3595-25 were grown for 24 hours in YPA medium (1% yeast extract, 2% peptone, 0.01% adenine hemisulfate), Glucose 8%, (NH4)2SO4 50 mM, and Methionine 0.5 mM and Threonine 0.85 mM. The content of ectoine in the medium was assayed after 24 hours using the AccQ-Tag precolumn derivatization method for amino acid determination using a AccQ-Tag Ultra Derivatization Kit from Waters as advised by the manufacturer.
PEPCK-1 is a form of PEPCK stabilized by modification of the Arginine amino acid in position 2 by a Glycine.
The ectoine amounts obtained with these two strains are respectively:

    
    
        YA3380-40B: 211 mg/L−1.
        YA3595-25: 1.29 g/L−1.
    
    


In comparison, a native strain does not produce ectoine.
The strain YA3595-34, as well as the strain YA3595-25, were grown for 24 hours in YPA medium (1% yeast extract, 2% peptone, 0.01% adenine hemisulfate), Saccharose 8%, (NH4)2SO4 50 mM, and Methionine 0.5 mM and Threonine 0.85 mM. The content of ectoine in the medium was assayed after 24 hours using the AccQ-Tag precolumn derivatization method for amino acid determination using a AccQ-Tag Ultra Derivatization Kit from Waters as advised by the manufacturer.
The ectoine amounts obtained with these two strains are respectively:

    
    
        YA3595-25: 2.63 g/L−1.
        YA3595-34: 2.58 g/L−1.
    
    


In comparison, a native strain does not produce ectoine.
It results from this comparative experiment that a recombinant strain comprising the modifications according to the invention produces a greater amount of ectoine when cultured in the same conditions as other recombinant strains not comprising all the genetic modifications according to the invention.
C. Three other recombinant strains have also been obtained: YA4440, YA4442 and YA4444.
These three strains are as follows:
YA4440: MAT-α, gnp1::[LEU2.K1-RS, pADH1-AAT2-tRPL15A, pTEF3-MDH3-1-tRPL3, pPDC1-PEPCK-1.Ec-tMET17, pTDH3-MHPF.Ec-tTPI1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tIDP1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tTPI1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tIDP1, pCCW12-ME3.At-tRPL3], his3::[HIS3-pACU5-ME3.At-tRPL3, pACU6-METX-1.Cg-tIDP1]x5, hom6::[TRP1.K1-RS, pCCW12.Sba-HOM3-tDIT1], leu2, lys2Δ201, mup3::[HIS5.sp-RS, pACU7-PEPCK-1.Ec-tRPL3, pCCW12-HOM2-1-tTDH3, pPGK1-AAT2-tTDH2, pENO2-MDH3 tRPL15A, pCUP1-1-GDH-21.Eca-tTPI1, pTDH3.Sba-ECTB.He-tIDP1, pPDC1-ECTA.He-tRPL41B, pTEF1.Sba-ECTC.He-tRPL15A], pyk1::[LEU2.K1-RS, pTDH3-PEPCK-1.Ec-tIDP1, pPDC1-MDH3-1-tRPL15A, pTEF3-TPO1-tENO2, pCUR3-PYK1-7-tCYC1], sam3:: [pACU7-PEPCK-1.Ec-tRPL3, pCUP1-1-HOM3-tDIT1-sam3]x5, trp1, trp4::[LYS2-loxP, pCCW12-PEPCK-1.Ec-tTPI1, pCCW12-GDH2-tRPL3, pCCW12-METX-1.Cg-tRPL41B.Sba, pCCW12.Sba-HOM3-tRPL15A], ura3::[ECTB.Ab-ECTC.He-URA3]x7
YA4442: MAT-α, gnp1::[LEU2.K1-RS, pADH1-AAT2-tRPL15A, pTEF3-MDH3-1-tRPL3, pPDC1-PEPCK-1.Ec-tMET17, pTDH3-MHPF.Ec-tTPI1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tIDP1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tTPI1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tIDP1, pCCW12-ME3.At-tRPL3], his3::[HIS3-pACU5-ME3.At-tRPL3, pACU6-METX-1.Cg-tIDP1]x5, hom6::[TRP1.K1-RS, pCCW12.Sba-HOM3-tDIT1], leu2, lys2Δ201, mup3::[HIS5.sp-RS, pACU7-PEPCK-1.Ec-tRPL3, pCCW12-HOM2-1-tTDH3, pPGK1-AAT2-tTDH2, pENO2-MDH3-1-tRPL15A, pCUP1-1-GDH-21.Eca-tTPI1, pTDH3.Sba-ECTB.He-tIDP1, pPDC1-ECTA.He-tRPL41B, pTEF1.Sba-ECTC.He-tRPL15A], pyk1::[LEU2.K1-RS, pTDH3-PEPCK-1.Ec-tIDP1, pPDC1-MDH3-1-tRPL15A, pTEF3-TPO1-tENO2, pCUR3-PYK1-7-tCYC1], sam3::[pACU7-PEPCK-1.Ec-tRPL3, pCUP1-1-HOM3-tDIT1-sam3]x5, trp1, trp4::[LYS2-loxP, pCCW12-PEPCK-1.Ec-tTPI1, pCCW12-GDH1-tRPL3, pCCW12-METX-1.Cg-tRPL41B.Sba, pCCW12.Sba-HOM3-tRPL15A], ura3::[ECTB.Ab-ECTC.He-URA3]x7
YA4444: MAT-α, gnp1::[LEU2.K1-RS, pADH1-AAT2-tRPL15A, pTEF3-MDH3-1-tRPL3, pPDC1-PEPCK-1.Ec-tMET17, pTDH3-MHPF.Ec-tTPI1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tIDP1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tTPI1, pCCW12-ME3.At-tRPL3, pTDH3-MHPF.Ec-tIDP1, pCCW12-ME3.At-tRPL3], his3::[HIS3-pACU5-ME3.At-tRPL3, pACU6-METX-1.Cg-tIDP1]x5, hom6::[TRP 1.K1-RS, pCCW12.Sba-HOM3-tDIT1], leu2, lys2Δ201, mup3::[HIS5.sp-RS, pACU7-PEPCK-1.Ec-tRPL3, pCCW12-HOM2-1-tTDH3, pPGK1-AAT2-tTDH2, pENO2-MDH3 tRPL15A, pCUP1-1-GDH-21.Eca-tTPI1, pTDH3.Sba-ECTB.He-tIDP1, pPDC1-ECTA.He-tRPL41B, pTEF1.Sba-ECTC.He-tRPL15A], pyk1::[LEU2.K1-RS, pTDH3-PEPCK-1.Ec-tIDP1, pPDC1-MDH3-1-tRPL15A, pTEF3-TPO1-tENO2, pCUR3-PYK1 tCYC1], sam3::[pACU7-PEPCK-1.Ec-tRPL3, pCUP1-1-HOM3-tDIT1-sam3]x5, trp1, trp4::[LYS2-loxP, pCCW12-PEPCK-1.Ec-tTPI1, pCCW12-GDH2.Eca-tRPL3, pCCW12-METX-1.Cg-tRPL41B.Sba, pCCW12.Sba-HOM3-tRPL15A], ura3::[ECTB.Ab-ECTC.He-URA3]x7
GDH1 and GDH2 are endogenous Saccharomyces cerevisiae enzymes, while GDH2.Eca is a GDH enzyme from Entodimium caudatum.
These strains were grown in Erlenmeyer flasks at 28° C. for 16 h in Yeast extract 2%, Sucrose 8%, Methionine 0.5 mM, Threonine 4.2 mM, Urea 50 mM, vitamin B5 4 μM, vitamin B1 6 μM, vitamin B6 10 μM, vitamin B10 1.5 μM, vitamin B3 2.9 μM, vitamin B2 0.5 μM, vitamin B8 0.08 μM, vitamin B9 4.5 nM, CuSO4 500 μM. After 16 hours 500 μM CuSO4 and urea 100 mM were added and the cultures were grown for another 8 hours.
Ectoine production was then evaluated essentially as described in Ono H, et al. (1999) Journal of Bacteriology, p, 91-99 except that ectoine was detected by HPLC-UV.
Under these conditions, YA4440 produced 6.4 g/l of ectoine, YA4442 produced 4.7 g/l of ectoine and YA4444 produced 6.1 g/l of ectoine. It is reminded that, in these same conditions, a wild-type strain (e.g. non recombinant) does not a produce a detectable amount of ectoine.
These three strains are identical but for the GDH enzyme over-expressed. The above results show that the over-expression of NADH dependent GDH (GDH2 in YA4440 and GDH2.Eca in YA4444) allows for the production of more ectoine than the overexpression of a NADPH dependent GDH (GDH1 in YA4442).
Thus, the overexpression of a NADH dependent glutamate Dehydrogenase allows for the production of more ectoine than the overexpression of a NADPH dependent glutamate Dehydrogenase (GDH1).

## Claims

1. An ectoine-producing recombinant yeast, in the genome of which:
(A) (i) at least one nucleic acid encoding an aspartokinase is overexpressed and/or is under the control of an inducible or repressible promoter; and/or
(ii) at least one nucleic acid encoding an aspartate kinase is overexpressed and/or is under the control of an inducible or repressible promoter; and

(B) at least one nucleic acid encoding an aspartate semi-aldehyde dehydrogenase that can use as coenzyme both NAD and NADP is overexpressed and/or is under the control of an inducible or repressible promoter; and
(C) at least one nucleic acid encoding a diaminobutyrate aminotransferase is overexpressed and/or is under the control of an inducible or repressible promoter; and
(D) (i) at least one nucleic acid encoding an homoserine-O-acetyltransferase METX is overexpressed and/or is under the control of an inducible or repressible promoter that is heterologous from the nucleic acid encoding METX, and
(ii) at least one nucleic acid encoding a diaminobutyric acid acetyltransferase is overexpressed and/or is under the control of an inducible or repressible promoter; and

(E) at least one nucleic acid encoding an ectoine synthase is overexpressed and/or is under the control of an inducible or repressible promoter; and
(F) (i) at least one endogenous nucleic acid encoding an homoserine dehydrogenase has been deleted and/or interrupted, and/or
(ii) at least one nucleic acid encoding a homoserine dehydrogenase is:

under the control of an inducible or repressible promoter;
under the control of a weak promoter; and/or
the homoserine dehydrogenase is degron-tagged.

2. The recombinant yeast according to claim 1, in the genome of which at least one nucleic acid encoding an aspartate transaminase is overexpressed and/or is under the control of an inducible or repressible promoter.

3. The recombinant yeast according to claim 1, in the genome of which at least one nucleic acid encoding a glutamate dehydrogenase that converts oxo-glutarate to glutamate is overexpressed and/or is under the control of an inducible or repressible promoter.

4. The recombinant yeast according to claim 1, in the genome of which at least one of the following modifications has been performed:
(A) at least one endogenous nucleic acid encoding a general amino acid permease AGP3 have been deleted from the genome of the yeast, and:
(i) at least one nucleic acid encoding a general amino acid permease AGP3 has been inserted and is under the control of an inducible or repressible promoter, and/or
(ii) at least one nucleic acid encoding a destabilized general amino acid permease AGP3 has been inserted, wherein the general amino acid permease AGP3 is degron-tagged;

(B) at least one endogenous nucleic acid encoding a branched-chain amino-acid permease 3 has been deleted from the genome of the yeast, and:
(i) at least one nucleic acid encoding a branched-chain amino-acid permease 3 has been inserted and is under the control of an inducible or repressible promoter, and/or
(ii) at least one nucleic acid encoding a destabilized branched-chain amino-acid permease 3 has been inserted, wherein the branched-chain amino-acid permease 3 is degron-tagged;

(C) at least one endogenous nucleic acid encoding a branched-chain amino-acid permease 2 has been deleted from the genome of the yeast, and:
(i) at least one nucleic acid encoding a branched-chain amino-acid permease 2 has been inserted and is under the control of an inducible or repressible promoter, and/or
(ii) at least one nucleic acid encoding a destabilized branched-chain amino-acid permease 2 has been inserted, wherein the branched-chain amino-acid permease 2 is degron-tagged;

(D) at least one endogenous nucleic acid encoding a general amino acid permease GAP1 has been deleted from the genome of the yeast, and:
(i) at least one nucleic acid encoding a general amino acid permease GAP1 has been inserted and is under the control of an inducible or repressible promoter, and/or
(ii) at least one nucleic acid encoding a destabilized general amino acid permease GAP1 has been inserted, wherein the general amino acid permease GAP1 is degron-tagged;

(E) at least one endogenous nucleic acid encoding a high-affinity glutamine permease GNP1 has been deleted from the genome of the yeast, and:
(i) at least one nucleic acid encoding a high-affinity glutamine permease GNP1 has been inserted and is under the control of an inducible or repressible promoter, and/or
(ii) at least one nucleic acid encoding a destabilized high-affinity glutamine permease GNP1 has been inserted, wherein the high-affinity glutamine permease GNP1 is degron-tagged;

(F) at least one endogenous nucleic acid encoding a general amino acid permease AGP1 has been deleted from the genome of the yeast, and:
(i) at least one nucleic acid encoding a general amino acid permease AGP1 has been inserted and is under the control of an inducible or repressible promoter, and/or
(ii) at least one nucleic acid encoding a destabilized general amino acid permease AGP1 has been inserted, wherein the general amino acid permease AGP1 is degron-tagged;

(G) at least one endogenous nucleic acid encoding a low-affinity methionine permease MUP3 has been deleted from the genome of the yeast, and:
(i) at least one nucleic acid encoding a low-affinity methionine permease MUP3 has been inserted and is under the control of an inducible or repressible promoter, and/or
(ii) at least one nucleic acid encoding a destabilized low-affinity methionine permease MUP3 has been inserted, wherein the low-affinity methionine permease MUP3 is degron-tagged;

(H) at least one endogenous nucleic acid encoding a high-affinity methionine permease MUP1 has been deleted from the genome of the yeast, and:
(i) at least one nucleic acid encoding a high-affinity methionine permease MUP1 has been inserted and is under the control of an inducible or repressible promoter, and/or
(ii) at least one nucleic acid encoding a destabilized high-affinity methionine permease MUP1 has been inserted, wherein the high-affinity methionine permease MUP1 is degron-tagged;

(I) at least one nucleic acid encoding a transporter AQR1 is overexpressed; and/or
(J) at least one nucleic acid encoding a polyamine transporter 1 is overexpressed.

5. The recombinant yeast according to claim 4, in the genome of which at least two of the modifications indicated in claim 4 have been performed.

6. The recombinant yeast according to claim 4, wherein the at least one nucleic acid encoding a general amino acid permease, a branched-chain amino-acid permease 3, a branched-chain amino-acid permease 2, a general amino acid permease GAP1, a high-affinity glutamine permease GNP1, a general amino acid permease AGP1, a low-affinity methionine permease MUP3 and a high-affinity methionine permease MUP1 are, independently, nucleic acid from a yeast.

7. The recombinant yeast according to claim 1, wherein the nucleic acid encoding an aspartokinase is nucleic acid from a yeast.

8. The recombinant yeast according to claim 1, wherein the nucleic acid encoding an homoserine-O-acetyltransferase METX is nucleic acid from a bacterium.

9. The recombinant yeast according to claim 1, wherein at least one of the overexpressed nucleic acid is under the control of a strong promoter.

10. The recombinant yeast according to claim 1, wherein all of the inducible or repressible promoters are, independently, selected from the group consisting of promoters inducible or repressible with copper, promoters inducible or repressible with methionine and promoters inducible or repressible with threonine.

11. The recombinant yeast according to claim 1, wherein the weak promoter is, independently, selected from the group consisting of pURA3, pRPLA1, pNUP57 and pGAP1.

12. The recombinant yeast according to claim 1, wherein all of the inducible or repressible promoters are, independently, selected from the group consisting of promoters inducible or repressible with copper, promoters inducible or repressible with lysine and promoters inducible or repressible with methionine.

13. The recombinant yeast according to claim 1, in the genome of which at least one nucleic acid encoding an homoserine-O-acetyltransferase MET2 is overexpressed and/or is under the control of an inducible or repressible promoter.

14. Method for producing ectoine, said method comprising the steps of:
(a) culturing a recombinant yeast as defined in claim 1 in a culture medium; and
(b) recovering the ectoine from said culture medium.

15. Method according to claim 14, wherein the culture medium comprises at least a carbon source.