Synthetic yeast promoters

Abstract

The present invention relates to novel synthetic transcriptional promoters enabling the expression of proteins or nucleic acids in a cell, in particular in a yeast cell.

Description

[0001] SYNTHETIC YEAST PROMOTERS

[0002] The present invention relates to synthetic transcriptional promoters enabling the expression of proteins in a cell, in particular in a yeast cell.

[0003] TECHNOLOGICAL BACKGROUND OF THE INVENTION

[0004] The production of molecules of interest in a cell, particularly in yeast, most often involves the implementation of one or more heterologous metabolic pathways, which themselves originate from the assembly of one or more genes. Generally, these metabolic pathways are reconstituted within a host cell by the introduction of several transcriptional units, each comprising a promoter, a coding sequence, and a terminator.

[0005] One of the problems encountered when constructing recombinant yeast strains containing multiple heterologous transcriptional units stems from the limited number of available promoters, particularly strong ones, for gene expression in yeast. The use of identical promoters or those with high sequence homology can lead to rearrangements between the nucleotide sequences of these transcriptional units, either during vector construction or during the handling of the modified yeast, thus creating unacceptable genetic instability for industrial production strains.

[0006] To limit this instability, it is important to avoid extensive nucleotide redundancy within genetic constructs. Ideally, each transcriptional unit should have a promoter different from that of the other transcriptional units in the cell. Strong natural promoters exist that can be used in yeast cells, such as pGAL1, pCCW12, or pTDH3. However, these are few in number and are controlled by different transcription factors.

[0007] There is therefore a real need to provide new transcriptional promoters usable in a yeast cell, regulated by identical transcription factors and activated by carbon sources commonly used in industrial processes, such as glucose or ethanol. SUMMARY OF THE INVENTION

[0008] According to a first aspect, the present invention relates to an isolated nucleic acid having transcriptional promoter activity in Saccharomyces cerevisiae and comprising, or consisting of, a nucleotide sequence selected from the group consisting of the nucleotide sequences SEQ ID NO: 4 to 7 and functional variants thereof, said functional variants comprising a sequence having at least 70% sequence identity with one of the nucleotide sequences SEQ ID NO: 4 to 7.

[0009] Preferably, said functional variants comprise a sequence having at least 75% or at least 80% sequence identity with one of the nucleotide sequences SEQ ID NO: 4 to 7 and / or comprise a sequence having at most 95% sequence identity with the sequence SEQ ID NO: 17.

[0010] These functional variants may include, in particular, the following sequence

[0011] R(2-3)TGTCTGGGT-X1-RGCATCC-X2-GGCATCC-X3-GAATCCCAGCACC-X4- CAACCTGC-X5-RTATATAAAG-X6-HAADWM(i-2)YCAAGAACTTA(i-2)-X7-ATG (SEQ ID NO:1) where

[0012] Xi is a nucleotide sequence of 15 nucleotides,

[0013] X2 is a nucleotide sequence of 31 nucleotides,

[0014] X3 is a nucleotide sequence of 10 to 16 nucleotides,

[0015] X4 is a nucleotide sequence of 77 to 122 nucleotides,

[0016] X5 is a nucleotide sequence of 137 to 173 nucleotides,

[0017] XÔ is a nucleotide sequence of 72 to 106 nucleotides, and

[0018] X7 is a nucleotide sequence of 20 to 100 nucleotides.

[0019] Preferably, the nucleic acid according to the invention exhibits constitutive transcriptional promoter activity and / or transcriptional promoter activity when a host cell, preferably Saccharomyces cerevisiae, containing it is cultured in a medium comprising glucose or ethanol as a carbon source.

[0020] The present invention also relates to - a recombinant expression cassette comprising a coding sequence operationally linked to a nucleic acid according to the invention,

[0021] - an expression vector comprising a nucleic acid according to the invention or an expression cassette according to the invention, and

[0022] - a microbial host cell comprising a nucleic acid according to the invention, an expression cassette according to the invention or an expression vector according to the invention.

[0023] The microbial host cell according to the invention is preferably a yeast or a bacterium, in particular a yeast of the genera Saccharomyces, Kazachstania, Naumovozyma, Nakaseomyces, Tetrapisispora, Vanderwaltozyma, Zygosaccharomyces, Schizosaccharomyces, Yarrowia, Hansenula, Kluyveromyces, Pichia or Candida or a hybrid obtained from a strain belonging to one of these genera, and more particularly preferably a yeast of the species Saccharomyces cerevisiae

[0024] According to another aspect, the present invention also relates to a method for expressing a coding sequence of interest in a microbial cell, comprising introducing into said cell a nucleic acid according to the invention, an expression cassette according to the invention or an expression vector according to the invention.

[0025] It also relates to the use of a nucleic acid according to the invention, an expression cassette according to the invention or an expression vector according to the invention, to express a coding sequence of interest in a microbial cell.

[0026] BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1: Evaluation of the promoter activity of synthetic promoters A (SEQ ID NO: 12), B (SEQ ID NO: 13), C (SEQ ID NO: 14), D (SEQ ID NO: 15), and E (SEQ ID NO: 16) in a strain of S. cerevisiae. Promoter activity was assessed by measuring the fluorescence of the YFP protein whose gene is operationally linked to the promoters being tested. The promoter activity of the synthetic promoters was compared to that of the natural promoters pTDH3 and pRLP18B. Cells were incubated for 72 hours in MSCs containing 2% glucose, with sampling every 24 hours. The results are the average of 12 independent clones.

[0028] Figure 2: Evaluation of the promoter activity of the synthetic promoters pl (SEQ ID NO: 2), p2 (SEQ ID NO: 3), p3 (SEQ ID NO: 4), p4 (SEQ ID NO: 5), p5 (SEQ ID NO: 6), p6 (SEQ ID NO: 7), p7 (SEQ ID NO: 8), p8 (SEQ ID NO: 9), p9 (SEQ ID NO: 10), and plO (SEQ ID NO: 11) in a strain of S. cerevisiae. Promoter activity is assessed by measuring the fluorescence of YFP, whose gene is operationally linked to the promoters being tested. Ruby2 protein expression is controlled by a natural constitutive promoter, thus streamlining the results. The results are therefore presented as a ratio between the fluorescence of YFP and Ruby2 proteins. The promoter activity of synthetic promoters is compared to that of natural promoters pTDH3 and pHTB2. Cells were incubated at 30°C in a CSM medium containing 2% glucose, with sampling at 72h.

[0029] Figure 3: Evaluation of the promoter activity of the synthetic promoters pl (SEQ ID NO: 2), p2 (SEQ ID NO: 3), p3 (SEQ ID NO: 4), p4 (SEQ ID NO: 5), p5 (SEQ ID NO: 6), p6 (SEQ ID NO: 7), p7 (SEQ ID NO: 8), p8 (SEQ ID NO: 9), p9 (SEQ ID NO: 10), and plO (SEQ ID NO: 11) in a strain of S. cerevisiae. Promoter activity is assessed by measuring the fluorescence of YFP, whose gene is operationally linked to the promoters being tested. Ruby2 protein expression is controlled by a natural constitutive promoter, thus streamlining the results. The results are therefore presented as a ratio between the fluorescence of YFP and Ruby2 proteins. The promoter activity of synthetic promoters is compared to that of natural promoters pTDH3 and pHTB2. Cells were incubated at 37°C in CSM medium containing 2% glucose, with sampling at 72h.

[0030] Figure 4: Evaluation of the promoter activity of the synthetic promoters pl (SEQ ID NO: 2), p2 (SEQ ID NO: 3), p3 (SEQ ID NO: 4), p4 (SEQ ID NO: 5), p5 (SEQ ID NO: 6), p6 (SEQ ID NO: 7), p7 (SEQ ID NO: 8), p8 (SEQ ID NO: 9), p9 (SEQ ID NO: 10), and plO (SEQ ID NO: 11) in a strain of S. cerevisiae. Promoter activity is assessed by measuring the fluorescence of YFP, whose gene is operationally linked to the promoters being tested. Ruby2 protein expression is controlled by a natural constitutive promoter, thus streamlining the results. The results are therefore presented as a ratio between the fluorescence of YFP and Ruby2 proteins. The promoter activity of the synthetic promoters was compared to that of the natural promoters pTDH3 and pHTB2. Cells were incubated at 30°C in a CSM medium containing 2% ethanol, with sampling at 48 hours. DETAILED DESCRIPTION OF THE INVENTION

[0031] In order to avoid any extensive nucleotide redundancy within the genetic constructs inserted into yeast host cells, and thus minimize genetic instability, it is necessary to have a sufficiently large pool of promoters so that each transcriptional unit can potentially have a promoter sufficiently different from that of other transcriptional units so as not to favor homologous recombination events in or between the different genetic constructs.

[0032] The inventors have developed novel transcriptional promoters capable of controlling the expression of a coding sequence in yeast, thereby expanding the pool of promoters available to those skilled in the art. Furthermore, these new promoters are controlled by identical transcription factors, ensuring synchronous expression of the genes under their control. They are therefore particularly well-suited for controlling the expression of heterologous metabolic pathways involving multiple genes. They are also activated by carbon sources commonly used in industrial processes, such as glucose or ethanol.

[0033] Definitions

[0034] In this description, the terms "nucleic acid," "nucleic acid sequence," "polynucleotide," "oligonucleotide," and "nucleotide sequence" are used interchangeably and refer to a polymer of deoxyribonucleotides and / or ribonucleotides. These terms may include synthetic or semi-synthetic, recombinant molecules comprising non-natural bases or modified nucleotides, such as a modified bond, a modified purine or pyrimidine base, or a modified sugar. The nucleic acid according to the invention can be obtained by any method known to those skilled in the art, for example, by chemical synthesis and / or by recombination and / or mutagenesis techniques. In some preferred embodiments, the nucleic acid according to the invention is a DNA molecule, preferably a double-stranded DNA molecule. This molecule can be obtained by recombinant techniques well known to those skilled in the art.

[0035] As used here, the term "isolated" refers to biological material that has been removed from its natural environment. More specifically, the term "isolated nucleic acid" refers to a nucleic acid molecule that is separated from adjacent nucleic acids when in its natural environment—that is, within the genome from which it was isolated. Isolated nucleic acid may be included in a vector, genome, compound, or other component, yet remain isolated, as long as that component does not constitute its natural environment.

[0036] As used here, the term "recombinant microorganism" refers to a microorganism not found in nature that contains a modified genome resulting from the insertion, modification, or deletion of one or more genetic elements. This term also includes any offspring of said microorganism that are not identical to the parent microorganism due to mutations that may occur during replication. The microorganism may be a bacterium, a filamentous fungus, or a yeast, preferably a bacterium or a yeast, and more preferably a yeast.

[0037] The term "heterologous" refers to a nucleic acid introduced into the recombinant microorganism through genetic engineering. It may be present in episomal or chromosomal form. The origin of the nucleic acid may differ from that of the cell into which it is introduced. However, the gene may also originate from the same species as the cell into which it is introduced, but it is considered heterologous because its environment is not natural. For example, the gene or nucleic acid sequence is heterologous because it is under the control of a promoter other than its natural promoter, or it is introduced in a location different from its natural position. The recombinant microorganism may contain a copy of the endogenous gene prior to the introduction of the heterologous gene, or it may not contain an endogenous copy.Furthermore, the nucleic acid sequence can be heterologous in the sense that the coding sequence has been optimized for expression in the microorganism. Preferably, in this document, a heterologous nucleic acid sequence codes for a protein that is heterologous to the microorganism, that is, one that is not naturally present in the microorganism.

[0038] As used here, the term "native" or "endogenous", in relation to the recombinant microorganism, refers to a genetic element or protein naturally present in said microorganism.

[0039] The term "gene" refers to any nucleic acid that codes for a protein. The term encompasses DNA, such as cDNA or gDNA, as well as RNA. A gene can first be prepared using recombinant, enzymatic, and / or chemical techniques, and then replicated in a host cell or an in vitro system. A gene typically comprises an open reading frame, interrupted or not by one or more introns, that codes for a desired protein. A gene may also code for a non-coding RNA. It may also contain additional sequences, such as a transcription terminator. Due to the degeneracy of the genetic code, several nucleic acids can code for a particular polypeptide. Thus, the codons in the coding sequence for a given polypeptide can be modified to achieve optimal expression in a particular microorganism, for example, by using codon translation tables appropriate for that microorganism.Nucleic acids can also be optimized for a preferred GC content for the specific microorganism and / or to reduce the number of repetitive sequences. In some embodiments, heterologous nucleic acids have been optimized codon-wise for expression in the microorganism of interest. Codon optimization can be performed using routine methods known in the field (see, for example, Welch, M., et al. (2011), Methods in Enzymology 498: 43-66).

[0040] The terms "peptide", "oligopeptide", "polypeptide" and "protein" are used interchangeably and refer to a chain of amino acids linked by peptide bonds, regardless of the number of amino acids forming said chain.

[0041] The term "control sequences" refers to the nucleic acid sequences required for gene expression. Control sequences can be native or heterologous. Control sequences that are well-known and currently used by those skilled in the art are preferred. Such control sequences include, but are not limited to, a leader, a polyadenylation sequence, a promoter, a signal peptide sequence, a ribosome-binding site, and a transcription terminator. Preferably, control sequences include both a promoter and a transcription terminator.

[0042] As used here, the term "transcriptional promoter" or simply "promoter" refers to a regulatory element capable of initiating the transcription of a nucleic acid to which it is operationally bound. The activity of such a promoter can be assessed by any method known to those skilled in the art, and in particular by any method that quantifies the mRNA or protein produced from the gene controlled by the promoter. For example, activity can be measured by assessing the amount of mRNA, for instance by Northern blot or RT-PCR, or by assessing the amount of protein translated, for instance by Western blot, ELISA, colorimetric assays, enzyme activity assays, or by using a reporter gene system.

[0043] As used here, the expression "operationally linked" refers to nucleotide sequences that are combined / arranged in such a way that the function of one acts on the other. Thus, a transcriptional promoter and a nucleotide sequence, for example a coding sequence, are operationally linked when the expression of the nucleotide sequence is controlled by that promoter.

[0044] The term "expression cassette" refers to a nucleic acid construct comprising a coding sequence and one or more control sequences required for the expression of that coding sequence. Typically, the expression cassette includes a coding sequence and regulatory sequences flanking the coding sequence. Thus, an expression cassette typically comprises a promoter sequence, a 5' untranslated region, a coding sequence, and a 3' untranslated region that usually contains a polyadenylation site and / or a transcription terminator. The expression cassette may also include additional regulatory elements such as, for example, enhancer sequences, a polylinker sequence that facilitates the insertion of a DNA fragment into a vector, and / or splicing signals.

[0045] As used here, the term "vector" refers to a nucleic acid molecule used as a vehicle to transfer genetic material, and in particular to deliver nucleic acid into a host cell, either in vitro or in vivo. The vector may be DNA or RNA, circular or non-circular, single- or double-stranded, preferably a linear or circular double-stranded DNA molecule. Vectors include, but are not limited to, plasmids, phagemides, cosmids, transposable elements, viruses, and artificial chromosomes (e.g., YACs). Preferably, the vector is a plasmid. Vectors can be constructed by classical molecular biology techniques well known to those skilled in the art.

[0046] As used here, the term "expression vector" refers to a vector that includes one or more expression cassettes. Preferably, the expression vector is a linear or circular double-stranded DNA molecule. The vector may further include an origin of replication, a selection marker, etc.

[0047] The term "genetic construct" refers to any recombinant nucleic acid molecule. This can be single-stranded or double-stranded DNA, preferably double-stranded, linear or circular. The genetic construct can be a vector or a DNA fragment. It can be constructed using classical molecular biology techniques, including ligation, cloning, artificial synthesis, amplification, etc.

[0048] The term "sequence identity percentage" or "identity percentage," as used here, refers to the percentage of identical nucleotides in an alignment of two polynucleotides. This percentage can be easily calculated by a person skilled in the art using a sequence comparison computer program. Sequence identity is determined by comparing sequences after they have been aligned, maximizing overlaps and identities while minimizing gaps. In particular, sequence identity can be determined using one of the available global or local alignment mathematical algorithms. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g., Needleman and Wunsch, 1970), which aligns sequences optimally along their entire length. Sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g.,Smith and Waterman, 1981; Altschul et al., 1997; Altschul et al., 2005). Alignments to determine the percentage of identity can be performed by any method known to a person skilled in the art, including using computer programs available on the internet (e.g., http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / ). Preferably, the percentage of identity value here refers to a value obtained with the EMBOSS Needle program which uses the Needleman-Wunsch global alignment algorithm with the default parameters, namely for proteins: Scoring matrix = BLOSUM62, Gap open = 10, Gap extend = 0.5, End gap penalty = false, End gap open = 10 and End gap extend = 0.5, and for nucleic acids: Scoring matrix = DNAfull, Gap open = 10, Gap extend = 0.5, End gap penalty = false, End gap open = 10 and End gap extend = 0.5.

[0049] In the nucleic acid sequences described in this document, nucleotides are conventionally represented by letters according to the IUP AC (International Union of Pure and Applied Chemistry) nomenclature, R is G or A; H is A, C or T; D is G, A or T; W is A or T; M is A or C; Y is T or C).

[0050] In the various embodiments described in this document, the term "comprising" may be replaced by "consisting of" or "consisting essentially of". By "consisting essentially of" is understood that the sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 1, 2, 3, 4 or 5, additions, substitutions or deletions with respect to the sequences described in the SEQ ID Nos.

[0051] According to a first aspect, the present invention therefore relates to a nucleic acid, preferably isolated, having transcriptional promoter activity and comprising, or consisting of, a nucleotide sequence selected from the group consisting of the nucleotide sequences SEQ ID NO: 2 to 16 and functional variants thereof.

[0052] As used here, the term "variant" refers to a nucleotide sequence that differs from the parent sequence but retains its essential properties. The variant sequence may differ from the parent sequence by one or more substitutions, deletions, and / or insertions. The variant may have the same length or a different length (shorter or longer) than the parent sequence. The variant preferably has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the parent sequence.In certain specific embodiments, it can differ from the parent sequence by up to 15 substitutions, insertions, and / or deletions, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The term "functional variant" refers to a variant as defined above that retains the activity of the parent, in this case, transcriptional promoter activity in yeast, and preferably in S. cerevisiae.

[0053] According to one embodiment, the functional variants have a sequence having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least

[0054] 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the parent sequence, i.e. with one of the SEQ ID NO: 2 to 16 nucleotide sequences, preferably over the entire length of the sequence.

[0055] Preferably, the functional variants have a sequence with at most 95% sequence identity with the sequence SEQ ID NO: 17. More particularly preferred, the functional variants have a sequence with at most 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% sequence identity with the sequence SEQ ID NO: 17.

[0056] According to preferred embodiments, the functional variants include the following consensus sequence:

[0057] R(2-3)TGTCTGGGT-X1-RGCATCC-X2-GGCATCC-X3-GAATCCCAGCACC-X4- CAACCTGC-X5-RTATATAAAG-X6-HAADWM(i-2)YCAAGAACTTA(i-2)-X7-ATG (SEQ ID NO:1) where

[0058] Xi is a nucleotide sequence of 10 to 20 nucleotides, preferably a nucleotide sequence of 13 to 17 nucleotides, and more particularly preferred a nucleotide sequence of 14 to 16 nucleotides,

[0059] X2 is a nucleotide sequence of 25 to 35 nucleotides, preferably a nucleotide sequence of 29 to 33 nucleotides, and more particularly preferred a nucleotide sequence of 30 to 32 nucleotides,

[0060] X3 is a nucleotide sequence of 5 to 20 nucleotides, preferably a nucleotide sequence of 10 to 16 nucleotides,

[0061] X4 is a nucleotide sequence of 70 to 130 nucleotides, preferably a nucleotide sequence of 77 to 122 nucleotides,

[0062] X5 is a nucleotide sequence of 130 to 180 nucleotides, preferably a nucleotide sequence of 137 to 173 nucleotides,

[0063] XÔ is a nucleotide sequence of 65 to 115 nucleotides, preferably a nucleotide sequence of 72 to 106 nucleotides, and

[0064] X7 is a nucleotide sequence of 15 to 110 nucleotides, preferably a nucleotide sequence of 20 to 100 nucleotides.

[0065] According to one embodiment, Xi is a nucleotide sequence of 13 to 17 nucleotides, X2 is a nucleotide sequence of 29 to 33 nucleotides, X3 is a nucleotide sequence of 5 to 20 nucleotides, preferably a nucleotide sequence of 10 to 16 nucleotides, X4 is a nucleotide sequence of 70 to 130 nucleotides, preferably a nucleotide sequence of 77 to 122 nucleotides, X5 is a nucleotide sequence of 130 to 180 nucleotides, preferably a nucleotide sequence of 137 to 173 nucleotides, X0 is a nucleotide sequence of 65 to 115 nucleotides, preferably a nucleotide sequence of 72 to 106 nucleotides, and X7 is a sequence nucleotide of 15 to 110 nucleotides, preferably a nucleotide sequence of 20 to 100 nucleotides.

[0066] According to another embodiment, Xi is a nucleotide sequence of 14 to 16 nucleotides, X2 is a nucleotide sequence of 30 to 32 nucleotides, X3 is a nucleotide sequence of 5 to 20 nucleotides, preferably a nucleotide sequence of 10 to 16 nucleotides, X4 is a nucleotide sequence of 70 to 130 nucleotides, preferably a nucleotide sequence of 77 to 122 nucleotides, X5 is a nucleotide sequence of 130 to 180 nucleotides, preferably a nucleotide sequence of 137 to 173 nucleotides, X0 is a nucleotide sequence of 65 to 115 nucleotides, preferably a nucleotide sequence of 72 to 106 nucleotides, and X7 is a sequence nucleotide of 15 to 110 nucleotides, preferably a nucleotide sequence of 20 to 100 nucleotides.

[0067] According to a preferred embodiment, Xi is a 15-nucleotide sequence, X2 is a 31-nucleotide sequence, X3 is a 5- to 20-nucleotide sequence, preferably a 10- to 16-nucleotide sequence, X4 is a 70- to 130-nucleotide sequence, preferably a 77- to 122-nucleotide sequence, X5 is a 130- to 180-nucleotide sequence, preferably a 137- to 173-nucleotide sequence, X0 is a 65- to 115-nucleotide sequence, preferably a 72- to 106-nucleotide sequence, and X7 is a nucleotide sequence of 15 to 110 nucleotides, preferably a nucleotide sequence of 20 to 100 nucleotides,

[0068] According to a particularly preferred embodiment, Xi is a nucleotide sequence of 15 nucleotides, X2 is a nucleotide sequence of 31 nucleotides, X3 is a nucleotide sequence of 10 to 16 nucleotides, X4 is a nucleotide sequence of 77 to 122 nucleotides, X5 is a nucleotide sequence of 137 to 173 nucleotides, X0 is a nucleotide sequence of 72 to 106 nucleotides, and X7 is a nucleotide sequence of 20 to 100 nucleotides.

[0069] According to a preferred embodiment, the functional variants have a sequence

[0070] - having at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the parent sequence, i.e. with one of the nucleotide sequences SEQ ID NO: 2 to 16, preferably over the entire length of the sequence; and

[0071] - at most 95% sequence identity with sequence SEQ ID NO: 17, preferably having at most 90%, at most 89%, at most 88%, at most 87%, at most 86%, at most 85%, at most 84%, at most 83%, at most 82%, at most 81% or at most 80% sequence identity with sequence SEQ ID NO: 17.

[0072] Preferably, these functional variants also include the consensus sequence SEQ ID NO: 1 as defined above, and preferably with Xi being a 15-nucleotide sequence, X2 being a 31-nucleotide sequence, X3 being a 10- to 16-nucleotide sequence, X4 being a 77- to 122-nucleotide sequence, X5 being a 137- to 173-nucleotide sequence, X0 being a 72- to 106-nucleotide sequence, and X7 being a 20- to 100-nucleotide sequence.

[0073] According to another preferred embodiment, the functional variants have a sequence having at least 70% sequence identity with the parent sequence, i.e. with one of the nucleotide sequences SEQ ID NO: 2 to 16, preferably over the entire length of the sequence and at most 95% sequence identity with the sequence SEQ ID NO: 17, preferably having at most 90% sequence identity with the sequence SEQ ID NO: 17.Preferably, these functional variants also include the consensus sequence SEQ ID NO: 1 as defined above, and preferably with Xi being a 15-nucleotide sequence, X2 being a 31-nucleotide sequence, X3 being a 10- to 16-nucleotide sequence, X4 being a 77- to 122-nucleotide sequence, X5 being a 137- to 173-nucleotide sequence, X0 being a 72- to 106-nucleotide sequence, and X7 being a 20- to 100-nucleotide sequence.

[0074] According to another preferred embodiment, the functional variants have a sequence having at least 75% sequence identity with the parent sequence, i.e. with one of the nucleotide sequences SEQ ID NO: 2 to 16, preferably over the entire length of the sequence and at most 95% sequence identity with the sequence SEQ ID NO: 17, preferably having at most 90% sequence identity with the sequence SEQ ID NO: 17.Preferably, these functional variants also include the consensus sequence SEQ ID NO: 1 as defined above, and preferably with Xi being a 15-nucleotide sequence, X2 being a 31-nucleotide sequence, X3 being a 10- to 16-nucleotide sequence, X4 being a 77- to 122-nucleotide sequence, X5 being a 137- to 173-nucleotide sequence, X0 being a 72- to 106-nucleotide sequence, and X7 being a 20- to 100-nucleotide sequence.

[0075] According to another preferred embodiment, the functional variants have a sequence having at least 75% sequence identity with the parent sequence, i.e., with one of the nucleotide sequences SEQ ID NO: 2 to 16, preferably over the entire length of the sequence, and at most 90% sequence identity with the sequence SEQ ID NO: 17. Preferably, these functional variants also include the consensus sequence SEQ ID NO: 1 as defined above, and with Xi being a 15-nucleotide sequence, X2 being a 31-nucleotide sequence, X3 being a 10- to 16-nucleotide sequence, X4 being a 77- to 122-nucleotide sequence, X5 being a 137- to 173-nucleotide sequence, and XÔ being a sequence nucleotide of 72 to 106 nucleotides, and X7 being a nucleotide sequence of 20 to 100 nucleotides.

[0076] In particular, the nucleic acid according to the invention may comprise, or consist of,

[0077] - a nucleotide sequence selected from the group consisting of the nucleotide sequence SEQ ID NO: 2 and its functional variants;

[0078] - a nucleotide sequence selected from the group consisting of the nucleotide sequence SEQ ID NO: 3 and its functional variants;

[0079] - a nucleotide sequence selected from the group consisting of the nucleotide sequence SEQ ID NO: 4 and its functional variants;

[0080] - a nucleotide sequence selected from the group consisting of the nucleotide sequence SEQ ID NO: 5 and its functional variants;

[0081] - a nucleotide sequence selected from the group consisting of the nucleotide sequence SEQ ID NO: 6 and its functional variants;

[0082] - a nucleotide sequence selected from the group consisting of the nucleotide sequence SEQ ID NO: 7 and its functional variants;

[0083] - a nucleotide sequence selected from the group consisting of the nucleotide sequence SEQ ID NO: 8 and its functional variants; - a nucleotide sequence selected from the group consisting of the nucleotide sequence SEQ ID NO: 9 and its functional variants;

[0084] - a nucleotide sequence selected from the group consisting of the nucleotide sequence SEQ ID NO: 10 and its functional variants;

[0085] - a nucleotide sequence selected from the group consisting of the nucleotide sequence SEQ ID NO: 11 and its functional variants;

[0086] - a nucleotide sequence selected from the group consisting of the nucleotide sequence SEQ ID NO: 12 and its functional variants;

[0087] - a nucleotide sequence selected from the group consisting of the nucleotide sequence SEQ ID NO: 13 and its functional variants;

[0088] - a nucleotide sequence selected from the group consisting of the nucleotide sequence SEQ ID NO: 14 and its functional variants;

[0089] - a nucleotide sequence selected from the group consisting of the nucleotide sequence SEQ ID NO: 15 and its functional variants; or

[0090] - a nucleotide sequence selected from the group consisting of the nucleotide sequence SEQ ID NO: 16 and its functional variants.

[0091] According to a particular embodiment, the nucleic acid according to the invention comprises, or consists of, a nucleotide sequence selected from the group consisting of the nucleotide sequences SEQ ID NO: 2 to 11 and functional variants thereof.

[0092] According to another particular embodiment, the nucleic acid according to the invention comprises, or consists of, a nucleotide sequence selected from the group consisting of the nucleotide sequences SEQ ID NO: 4 to 7 and functional variants thereof.

[0093] According to another particular embodiment, the nucleic acid according to the invention comprises, or consists of, a nucleotide sequence selected from the group consisting of the nucleotide sequences SEQ ID NO: 2, 3, 8, 9 and 10 and functional variants thereof.

[0094] The nucleic acids according to the invention exhibit transcriptional promoter activity when the host microorganism, preferably a yeast and in particular S. cerevisiae, containing them is cultured in a medium comprising glucose or ethanol as a carbon source. This activity can be readily measured as described in the experimental section below. In particular, the activity of a promoter can be tested by introducing into a yeast cell, for example a Saccharomyces cerevisiae cell, an expression cassette comprising said promoter and a reporter gene operationally linked to said promoter.The modified yeast can then be cultured in a suitable medium containing glucose or ethanol, for example, a culture medium comprising a nitrogenous yeast base without amino acids supplemented with CSM (Complete Supplement Mixture) containing amino acids, vitamins, and other components essential for cell growth, to which glucose or ethanol has been added. Promoter activity is measured by assessing the expression of the reporter gene. In cases where the reporter gene codes for a fluorescent protein, promoter activity is assessed by measuring the fluorescence of the culture. Preferably, promoter activity is assessed after 72 hours of batch culture at 30°C or 37°C in a culture medium containing glucose as a carbon source, or after 48 hours of batch culture at 30°C in a culture medium containing ethanol as a carbon source.

[0095] Preferably, the nucleic acids according to the invention have constitutive transcriptional promoter activity, particularly when the host microorganism, preferably a yeast and in particular Saccharomyces cerevisiae, containing them is cultured in a medium comprising glucose or ethanol as a carbon source. This means that no inducer is required for the coding sequence operationally linked to a promoter of the combination to be expressed.

[0096] Preferably, the nucleic acids according to the invention have transcriptional promoter activity that is similar to or greater than that of the strong promoter of the TDH3 gene of S. cerevisiae (SEQ ID NO: 17) when the microorganism containing said promoters is cultured in a medium comprising glucose or ethanol as a carbon source. As used herein, the term "similar" refers to an expression level of the coding sequence operationally linked to the nucleic acid according to the invention that is at least 80%, preferably at least 90%, of the expression level of the coding sequence operationally linked to the TDH3 promoter under the same culture conditions and in the same host cell.In particular, the activities of the nucleic acids according to the invention and of the pTDH3 promoter can be evaluated after 72 hours of batch culture at 30°C or 37°C in a culture medium containing glucose as a carbon source, or after 48 hours of batch culture at 30°C in a culture medium containing ethanol as a carbon source. According to one embodiment, the nucleic acid according to the invention has a transcriptional promoter activity that is superior to that of the promoter of the S. cerevisiae TDH3 gene (SEQ ID NO: 17) when the host microorganism, preferably a yeast and in particular S. cerevisiae, is cultured in a medium containing glucose or ethanol as a carbon source.Preferably, in this embodiment, the nucleic acid according to the invention comprises, or consists of, a nucleotide sequence selected from the group consisting of the nucleotide sequences SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, and 11, and functional variants thereof. Preferably, transcriptional activity is measured after 72 hours of culture.

[0097] According to another embodiment, the nucleic acid of the invention has a transcriptional promoter activity that is superior to that of the promoter of the TDH3 gene of S. cerevisiae (SEQ ID NO: 17) when the host microorganism, preferably a yeast and in particular S. cerevisiae, is cultured at 30°C in a medium containing glucose as a carbon source. Preferably, in this embodiment, the nucleic acid of the invention comprises, or consists of, a nucleotide sequence selected from the group consisting of the nucleotide sequences SEQ ID NO: 4, 5, 7, and 9, and functional variants thereof. Preferably, the transcriptional activity is measured after 72 hours of culture.

[0098] According to another embodiment, the nucleic acid of the invention has a transcriptional promoter activity that is superior to that of the promoter of the TDH3 gene of S. cerevisiae (SEQ ID NO: 17) when the host microorganism, preferably a yeast and in particular S. cerevisiae, is cultured at 37°C in a medium containing glucose as a carbon source. Preferably, in this embodiment, the nucleic acid of the invention comprises, or consists of, a nucleotide sequence selected from the group consisting of the nucleotide sequences SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, and 11, and functional variants thereof. Preferably, the transcriptional activity is measured after 72 hours of culture.

[0099] According to another embodiment, the nucleic acid of the invention has a transcriptional promoter activity that is superior to that of the promoter of the TDH3 gene of S. cerevisiae (SEQ ID NO: 17) when the host microorganism, preferably a yeast and in particular N. cerevisiae, is cultured at 30°C in a medium containing ethanol as a carbon source. Preferably, in this embodiment, the nucleic acid of the invention comprises, or consists of, a nucleotide sequence selected from the group consisting of the nucleotide sequences SEQ ID NO: 4, 5, 7, 8, 9, and 11, and functional variants thereof. Preferably, the transcriptional activity is measured after 48 hours of culture.

[0100] In a second aspect, the present invention also relates to an expression cassette comprising a coding sequence of interest operationally linked to a nucleic acid according to the invention.

[0101] The expression cassette according to the invention comprises, at a minimum, a transcriptional promoter and a coding sequence operationally linked to said promoter. Preferably, it also comprises a transcriptional terminator. As indicated above in the "definition" section, this cassette may also comprise other additional elements.

[0102] The expression cassette according to the invention is a recombinant expression cassette, that is to say that the coding sequence is operationally linked to a promoter, in this case a nucleic acid according to the invention, which is different from the promoter to which the coding sequence is functionally linked in nature and in particular in a natural genome.

[0103] The coding sequence of interest operationally linked to a nucleic acid according to the invention can code for one or more polypeptides of interest and / or one or more nucleic acids of interest.

[0104] Examples of polypeptides of interest include, but are not limited to, reporter proteins such as fluorescent proteins, antimicrobial resistance proteins, nucleases such as, for example, a CRISPR nuclease, or enzymes involved in metabolic pathways, especially metabolic synthesis pathways.

[0105] Examples of nucleic acids of interest include, but are not limited to, siRNA, shRNA, RNAi, miRNA, antisense RNA, ribozymes, DNAzymes, or guide RNAs capable of targeting the action of a CRISPR-type endonuclease.

[0106] In one embodiment, the coding sequence of interest, operationally linked to a nucleic acid according to the invention, codes for one or more polypeptides of interest. In another embodiment, the coding sequence of interest, operationally linked to a nucleic acid according to the invention, codes for one or more nucleic acids of interest.

[0107] The expression cassette according to the invention may comprise one or more coding sequences of interest operationally linked to the nucleic acid according to the invention.

[0108] All embodiments relating to other aspects of the invention are also considered in this aspect.

[0109] In a third aspect, the present invention also relates to a vector comprising a nucleic acid according to the invention or an expression cassette according to the invention.

[0110] This vector can be used to transform a host cell and enable the expression of the coding sequence of interest, operationally linked to a nucleic acid according to the invention, within said cell. The vectors can be constructed using conventional molecular biology techniques, well known to those skilled in the art.

[0111] The vector according to the invention may comprise one or more origins of replication. In particular, it may comprise a bacterial origin of replication allowing its selection during the cloning steps, for example a functional origin of replication in E. coli, and another origin of replication used during transfer into the final host cell, for example a functional origin of replication in S. cerevisiae.

[0112] Preferably, the vector according to the invention comprises a functional origin of replication in yeast. More particularly preferred, it comprises a functional origin of replication in S. cerevisiae.

[0113] The vector may also contain elements that allow its selection in a host cell, such as, for example, a gene conferring resistance to an antibiotic or a gene that complements auxotrophy. Such elements are well known to those skilled in the art and are widely described in the literature.

[0114] The expression vector may also include one or more sequences enabling targeted insertion of the vector, expression cassette, or nucleic acid according to the invention into the genome of a host cell. Preferably, the insertion is performed at a gene whose inactivation allows the selection of host cells that have integrated the vector, cassette, or nucleic acid according to the invention. The vector may be circular or linear, single- or double-stranded. It is advantageously selected from plasmids, phages, phagemids, viruses, cosmids, and artificial chromosomes. Preferably, the vector is a plasmid.

[0115] All embodiments relating to other aspects of the invention are also considered in this aspect.

[0116] In another aspect, the present invention relates to the use of a nucleic acid, an expression cassette, or a vector according to the invention to transform or transfect a host cell. It also relates to a host cell transformed or transfected by at least one nucleic acid, an expression cassette, or a vector according to the invention. The host cell thus obtained is a recombinant microorganism.

[0117] The present invention therefore relates to a microbial host cell comprising a nucleic acid, an expression cassette or a vector according to the invention.

[0118] As used here, the term "host cell" refers to a bacterium, filamentous fungus or yeast, preferably a bacterium or yeast, and more preferably a yeast.

[0119] In one embodiment, the host cell is a bacterium, in particular a bacterium suitable for use in preparing, transferring, or storing a nucleic acid, an expression cassette, or a vector according to the invention. The bacterium may be, for example, Escherichia coli, Agrobacterium tumefaciens, or Vibrio natriegens.

[0120] According to a preferred embodiment, the host cell is a yeast cell. Examples of yeasts include, but are not limited to, yeasts of the genera Saccharomyces, Kazachstania, Naumovozyma, Nakaseomyces, Tetrapisispora, Vanderwaltozyma, Zygosaccharomyces, Schizosaccharomyces, Yarrowia, Hansenula, Kluyveromyces, Pichia or Candida, as well as hybrids obtained from a strain belonging to one of these genera.

[0121] Preferably, the host cell is a yeast belonging to the genera Saccharomyces, Kazachstania, Naumovozyma, Nakaseomyces, Tetrapisispora, Vanderwaltozyma, Zygosaccharomyces, Kluyveromyces, or Pichia, or a hybrid obtained from a strain belonging to one of these genera. More particularly preferred, the host cell is a yeast belonging to the genera Saccharomyces, Kazachstania, Naumovozyma, Nakaseomyces, Tetrapisispora, Vanderwaltozyma, or Zygosaccharomyces, or a hybrid obtained from a strain belonging to one of these genera.

[0122] In particular, yeast can be:

[0123] - a yeast of the genus Saccharomyces chosen from the group consisting of Saccharomyces cerevisiae, Saccharomyces bayanus, Saccharomyces castelli, Saccharomyces eubayanus, Saccharomyces kudriavzevii, Saccharomyces mikatae, Saccharomyces uvarum, Saccharomyces paradoxus, Saccharomyces cariocanus, Saccharomyces jurei and Saccharomyces pastorianus (also called Saccharomyces carlsbergensis), or

[0124] - a yeast of the genus Kazachstania chosen from the group consisting of Kazachstania africana, Kazachstania bromeliacearum, Kazachstania kunashirensis, Kazachstania martiniae, Kazachstania molopis, Kazachstania psychrophila, Kazachstania taianensis and Kazachstania viticola, or

[0125] - a yeast of the genus Naumovozyma chosen from the group consisting of Naumovozyma castellii, Naumovozyma baii and Naumovozyma dairenensis,

[0126] - a yeast of the genus Nakaseomyces chosen from the group consisting of Nakaseomyces bracarensis, Nakaseomyces delphensis, Nakaseomyces glabratus, Nakaseomyces kungkrabaensis, Nakaseomyces nivariensis and Nakaseomyces uthaithaninus, or

[0127] - a yeast of the genus Tetrapisispora chosen from the group consisting of Tetrapisispora arboricola, Tetrapisispora fleetii, Tetrapisispora iriomotensis, Tetrapisispora namnaoensis, Tetrapisispora nanseiensis and Tetrapisispora phaffii, or

[0128] - a yeast of the genus Vanderwaltozyma chosen from the group consisting of Vanderwaltozyma polyspora, Vanderwaltozyma tropicalis, Vanderwaltozyma verrucispora and Vanderwaltozyma yarrowii, or

[0129] - a yeast of the genus Zygosaccharomyces chosen from the group consisting of Zygosaccharomyces bailii, Zygosaccharomyces bisporus, Zygosaccharomyces favi, Zygosaccharomyces gambellarensis, Zygosaccharomyces kombuchaensis, Zygosaccharomyces lentus, Zygosaccharomyces machadoi, Zygosaccharomyces mellis, Zygosaccharomyces osmophilus, Zygosaccharomyces parabailii, Zygosaccharomyces pseudobailii, Zygosaccharomyces rouxii, Zygosaccharomyces sapae, Zygosaccharomyces seidelii and Zygosaccharomyces siamensis, or - a yeast of the genus Pichia chosen from the group consisting of Pichia occidentalis, Pichia Kudriavzevii, Pichia fermentans, Pichia manshurica and Pichia terricola, or

[0130] - a yeast of the genus Kluyveromyces chosen from the group consisting of Kluyveromyces lactis and Kluyveromyces marxianus.

[0131] In a more particularly preferred manner, the said yeast is Saccharomyces cerevisiae.

[0132] According to another particular embodiment, the host cell is a filamentous fungal cell. Examples of fungi include, but are not limited to, filamentous fungal cells. Filamentous fungi include those belonging to the subdivisions Eumycota and Oomycota. Filamentous fungal cells may be selected from the group consisting of Trichoderma, Acremonium, Aspergillus, Aureohasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trametes.

[0133] The host cell can be transformed / transfected in a transient or stable manner. The nucleic acid, cassette, or vector according to the invention can be contained within the cell in a non-integrated form or integrated into the genome.

[0134] All embodiments relating to other aspects of the invention are also considered in this aspect.

[0135] According to another aspect, the present invention relates to a method for expressing a coding sequence of interest in a microbial host cell, comprising introducing into said cell a nucleic acid, an expression cassette or an expression vector according to the invention, said coding sequence being operationally linked to a nucleic acid according to the invention.

[0136] It also relates to the use of a nucleic acid, an expression cassette or an expression vector according to the invention to express a coding sequence of interest in a microbial host cell.

[0137] The coding sequence of interest may be heterologous with respect to the promoter, meaning that the promoter is different from the one to which the coding sequence is functionally linked in a natural genome, and / or heterologous with respect to the host cell, meaning a sequence that is not naturally present in the host cell. Preferably, the coding sequence of interest is heterologous with respect to both the promoter and the host cell.

[0138] The coding sequence of interest is either contained within the expression cassette or expression vector according to the invention and is operationally linked thereto to a nucleic acid according to the invention, or is already present in the host cell's genome. In the latter case, the nucleic acid according to the invention is inserted into the cell's genome so as to be operationally linked to said sequence and thus enable its expression.

[0139] Preferably, the process includes a culture step in which the host cell is grown under conditions enabling expression of the coding sequence of interest, for example in a medium including glucose or ethanol as a carbon source.

[0140] All embodiments relating to other aspects of the invention are also considered in this aspect.

[0141] The following examples are presented for illustrative purposes only and are not exhaustive.

[0142] EXAMPLES

[0143] Example 1

[0144] Materials & Methods

[0145] Strains

[0146] The yeasts were obtained from Saccharomyces cerevisiae FY1679-28A (Tettelin et al., Methods in Molecular Genetics, Volume 6, 1995, Pages 81-107). The FY1679-28A yeast is auxotrophic for uracil, tryptophan, and leucine. The inventors modified this strain to also make it auxotrophic for histidine.

[0147] The clonings were carried out in a strain of Escherichia coli.

[0148] Genetic tools

[0149] The pTDH3 (SEQ ID NO: 17) and pRLP 18B (SEQ ID NO: 18) promoters were recovered by PCR on genomic DNA from strain FY1679-28A. The synthetic promoters A (SEQ ID NO: 12), B (SEQ ID NO: 13), C (SEQ ID NO: 14), D (SEQ ID NO: 15), E (SEQ ID NO: 16), as well as the yfp gene optimized for expression in S. cerevisiae, were synthesized by Arurumolecular, Dundee, UK.

[0150] Expression cassettes, each containing one of the above promoters operationally linked to the yfp gene, were prepared and each cloned into a yeast replicative vector, composed in particular of an ARS / CEN including the his5 or leu2 gene in order to complement the auxotrophy for histidine or leucine of the yeast strains.

[0151] These vectors were then inserted into FY1679-28A yeasts auxotrophic for uracil, tryptophan, leucine and histidine.

[0152] Growing conditions

[0153] For each strain, 12 clones were cultured in 24-well plates. The cells were cultured in 1 ml of medium containing: minimum nitrogen base (Dutscher, Brumath, France) 6.7 g / L, glucose 20 g / L, and complete supplement mixture without histidine (CSM, Formedium, UK) at 30°C for 72 h with continuous shaking at 200 rpm. Each strain was inoculated at an OD of 0.3 from a pre-culture grown under the same conditions.

[0154] Fluorescence measurement

[0155] Fluorescence measurements were performed at 24, 48, and 72 hours. For each strain, 100 µL of cell culture or 25 µL diluted in 100 µL of medium were placed in a transparent 96-well Greiner plate. YFP fluorescence was measured using the TECAN microplate reader with excitation and emission wavelengths of 485 nm and 535 nm, respectively.

[0156] Results

[0157] The transcriptional promoter activity of natural promoters pRLP18B and pTDH3 and synthetic promoters A (SEQ ID NO: 12), B (SEQ ID NO: 13), C (SEQ ID NO: 14), D (SEQ ID NO: 15) and E (SEQ ID NO: 16) was evaluated in a strain of S. cerevisiae by measuring the fluorescence of YFP placed under the control of the promoters.

[0158] The results obtained with synthetic promoters A, B, C, D, and E are similar to those obtained for the strong pTDH3 promoter (see Figure 1). These promoters, which conform to the consensus sequence SEQ ID NO: 1 as defined in this document, can therefore be used in the promoter combinations according to the invention and are capable of inducing strong expression of the genes under their control.

[0159] Example 2

[0160] Materials & Methods

[0161] Strains

[0162] The yeasts used are the same as in example 1, namely the yeast FY1679-28A auxotrophic for uracil, tryptophan, leucine and histidine.

[0163] The clonings were carried out in the MH1 strain of Escherichia coli.

[0164] Genetic tools

[0165] The pTDH3 (SEQ ID NO: 17) and pHTB2 (SEQ ID NO: 19) promoters were recovered by PCR on genomic DNA from S. cerevisiae strains.

[0166] Synthetic promoters pl (SEQ ID NO: 2), p2 (SEQ ID NO: 3), p3 (SEQ ID NO: 4), p4 (SEQ ID NO: 5), p5 (SEQ ID NO: 6), p6 (SEQ ID NO: 7), p7 (SEQ ID NO: 8), p8 (SEQ ID NO: 9), p9 (SEQ ID NO: 10) and plO (SEQ ID NO: 11) and genes yjp and ruby2 optimized for expression in S. cerevisiae, were synthesized by Arurumolecular, Dundee, UK.

[0167] Expression cassettes, each containing one of the above promoters operationally linked to the yjp gene, were prepared and cloned into a yeast replicative vector. This vector included an ARS / CEN containing the leu2 gene to complement leucine auxotrophy in the yeast strains. To normalize and compare the results, a second transcriptional unit was introduced into each vector independently of the YFP expression cassette. This second transcriptional unit consists of a constitutive internal reference promoter (pRLP18B, SEQ ID NO: 18) operationally linked to the ruby2 gene.

[0168] These vectors were then inserted into FY1679-28A yeast auxotrophs for uracil, tryptophan, leucine, and histidine. Culture conditions

[0169] For each strain, 12 clones were cultured in 24-well plates. Cells were cultured in 1 ml of medium containing either minimum nitrogen base (Dutscher, Brumath, France) or complete supplement mixture without leucine (CSM, Formedium, UK) with continuous shaking at 200 rpm. The culture medium contained either 20 g / L of glucose or 20 g / L of ethanol. Cultures were carried out at 30°C or 37°C for 48 or 72 hours. Each strain was inoculated from a 1 / 20 diluted preculture and cultured in a similar medium containing 20 g / L of glucose.

[0170] Fluorescence measurement

[0171] Fluorescence measurements were performed at 48 or 72 hours. For each strain, 25 µL of cell culture were diluted in 100 µL of medium in a transparent 96-well Greiner plate. The fluorescence of YFP and RUBY2 was measured using the TECAN microplate reader with excitation and emission wavelengths of 485 nm and 535 nm, respectively, for YFP, and 530 nm and 610 nm, respectively, for RUBY2.

[0172] Results

[0173] The transcriptional promoter activity of natural promoters pTDH3 and pHTB2 and synthetic promoters pl (SEQ ID NO: 2), p2 (SEQ ID NO: 3), p3 (SEQ ID NO: 4), p4 (SEQ ID NO: 5), p5 (SEQ ID NO: 6), p6 (SEQ ID NO: 7), p7 (SEQ ID NO: 8), p8 (SEQ ID NO: 9), p9 (SEQ ID NO: 10) and plO (SEQ ID NO: 11) was evaluated in a strain of S. cerevisiae by measuring the fluorescence ratio of YFP placed under the control of the promoters and of the RUBY2 protein placed under the control of the control promoter.

[0174] The results obtained with synthetic promoters 1 to 10 are similar to or better than those obtained for the strong pTDH3 promoter, regardless of whether the yeast is cultured at 30°C or 37°C and in a culture medium with glucose or ethanol as the initial carbon source (see Figures 2 to 4). These promoters, which conform to the consensus sequence SEQ ID NO: 1 as defined in this document, can therefore be used in the promoter combinations according to the invention and are capable of inducing strong expression of the genes under their control.

Claims

DEMANDS 1. Isolated nucleic acid having transcriptional promoter activity in Saccharomyces cerevisiae and comprising, or consisting of, a nucleotide sequence selected from the group consisting of the nucleotide sequences SEQ ID NO: 4 to 7 and functional variants thereof, said functional variants comprising a sequence having at least 70% sequence identity with one of the nucleotide sequences SEQ ID NO: 4 to 7.

2. Nucleic acid according to claim 1, wherein said functional variants comprise a sequence having at least 75% sequence identity with one of the nucleotide sequences SEQ ID NO: 4 to 7.

3. Nucleic acid according to claim 1, wherein said functional variants comprise a sequence having at least 80% sequence identity with one of the nucleotide sequences SEQ ID NO: 4 to 7.

4. Nucleic acid according to any one of claims 1 to 3, wherein said functional variants comprise a sequence having at most 95% sequence identity with the sequence SEQ ID NO:

17.

5. Nucleic acid according to any one of claims 1 to 4, wherein said functional variants comprise the following sequence R(2-3)TGTCTGGGT-X1-RGCATCC-X2-GGCATCC-X3-GAATCCCAGCACC-X4- CAACCTGC-X5-RTATATAAAG-X6-HAADWM(i-2)YCAAGAACTTA(i-2)-X7-ATG (SEQ ID NO:1) where Xi is a nucleotide sequence of 15 nucleotides, X2 is a nucleotide sequence of 31 nucleotides, X3 is a nucleotide sequence of 10 to 16 nucleotides, X4 is a nucleotide sequence of 77 to 122 nucleotides, X5 is a nucleotide sequence of 137 to 173 nucleotides, XÔ is a nucleotide sequence of 72 to 106 nucleotides, and X7 is a nucleotide sequence of 20 to 100 nucleotides.

6. Nucleic acid according to any one of claims 1 to 5, characterized in that it exhibits constitutive transcriptional promoter activity.

7. Nucleic acid according to any one of claims 1 to 6, characterized in that it exhibits transcriptional promoter activity when a host cell, preferably Saccharomyces cerevisiae, containing it is cultured in a medium comprising glucose or ethanol as a carbon source.

8. Recombinant expression cassette comprising a coding sequence operationally linked to a nucleic acid according to any one of claims 1 to 7.

9. Expression vector comprising a nucleic acid according to any one of claims 1 to 7 or an expression cassette according to claim 8.

10. Expression vector according to claim 9, said vector comprising a functional origin of replication in yeast, preferably a functional origin of replication in S. cerevisiae.

11. Microbial host cell comprising a nucleic acid according to any one of claims 1 to 7, an expression cassette according to claim 8 or an expression vector according to claim 9 or 10.

12. Microbial host cell according to claim 11, said cell being a yeast or a bacterium, preferably a yeast.

13. Microbial host cell according to claim 11 or 12, said cell being a yeast of the genus Saccharomyces, Kazachstania, Naumovozyma, Nakaseomyces, Tetrapisispora, Vanderwaltozyma, Zygosaccharomyces, Schizosaccharomyces, Yarrowia, Hansenula, Kluyveromyces, Pichia or Candida or a hybrid obtained from a strain belonging to one of these genera, preferably being a yeast of the species Saccharomyces cerevisiae 14. Method for expressing a coding sequence of interest in a microbial cell, comprising introducing into said cell a nucleic acid according to any one of claims 1 to 7, an expression cassette according to claim 8 or an expression vector according to claim 9 or 10.

15. A method according to claim 14, wherein the microbial cell is a bacterium or a yeast, preferably a yeast.

16. A method according to claim 14, wherein the microbial cell is a yeast of the species Saccharomyces cerevisiae.

17. Use of a nucleic acid according to any one of claims 1 to 7, an expression cassette according to claim 8 or an expression vector according to claim 9 or 10, to express a coding sequence of interest in a microbial cell.

18. Use according to claim 17, wherein the microbial cell is a bacterium or a yeast, preferably a yeast.

19. Use according to claim 17, wherein the microbial cell is a yeast of the species Saccharomyces cerevisiae.

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