Combinations of synthetic yeast promoters

Abstract

The present invention relates to novel combinations of transcriptional promoters that exhibit similar characteristics in terms of regulation and expression and that can be used in one and the same recombinant cell or vector without risk of recombination.

Description

[0001] COMBINATIONS OF SYNTHETIC YEAST PROMOTERS

[0002] The present invention relates to tools, methods and compositions for 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 pGΦ7, CCW12, or TDH3. 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, which can be combined within the same cell or vector without risk of recombination, while being 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 a recombinant microorganism comprising a combination of at least two heterologous transcriptional promoters, wherein said at least two heterologous transcriptional promoters each comprise a nucleotide sequence comprising the following sequence

[0009] 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

[0010] Xi is a nucleotide sequence of 15 nucleotides,

[0011] X2 is a nucleotide sequence of 31 nucleotides,

[0012] X3 is a nucleotide sequence of 10 to 16 nucleotides,

[0013] X4 is a nucleotide sequence of 77 to 122 nucleotides,

[0014] X5 is a nucleotide sequence of 137 to 173 nucleotides,

[0015] XÔ is a nucleotide sequence of 72 to 106 nucleotides, and

[0016] X7 is a nucleotide sequence of 20 to 100 nucleotides, and the nucleotide sequences of said at least two heterologous transcriptional promoters do not include nucleotide sequences of 25 or more consecutive nucleotides identical to each other.

[0017] Preferably, each promoter in the combination has a sequence identity of 90% or less with the other promoters in the combination.

[0018] The combination can include between 3 and 80, preferably between 3 and 50, transcriptional promoters.

[0019] Preferably, said at least two transcriptional promoters have constitutive transcriptional promoter activity when the microorganism is grown in a culture medium comprising glucose or ethanol as a carbon source.

[0020] In particular, said at least two transcriptional promoters may comprise a nucleotide sequence selected from the group consisting of the nucleotide sequences SEQ ID NO: 2 to 16, and functional variants thereof, said functional variants comprising a sequence having at least 70% identity with one of the nucleotide sequences SEQ ID NO: 2 to 16.

[0021] These at least two transcriptional promoters are preferably each operationally linked to a coding sequence, in particular a heterologous coding sequence.

[0022] Preferably, the microorganism is a bacterium or a yeast, in particular 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 a yeast of the species Saccharomyces cerevisiae.

[0023] According to a second aspect, the present invention relates to a genetic construct comprising a combination of at least two transcriptional promoters as defined in the recombinant microorganism according to the invention, and wherein said at least two transcriptional promoters are each operationally linked to a coding sequence. The genetic construct according to the invention can be contained within an expression vector. Alternatively, it can be integrated into the genome of a recombinant microorganism. The present invention also relates to a recombinant microorganism comprising said genetic construct.

[0024] According to a third aspect, the present invention relates to a combination of at least two expression cassettes, each comprising a coding sequence of interest operationally linked to a transcriptional promoter of distinct sequence, said transcriptional promoters being a combination of at least two transcriptional promoters as defined in the recombinant microorganism according to the invention. It also relates to a recombinant microorganism comprising said combination of expression cassettes.

[0025] According to another aspect, the present invention relates to a method for expressing at least two coding sequences of interest in a microorganism, comprising introducing into said microorganism a genetic construct according to the invention or a combination of at least two expression cassettes according to the invention.

[0026] According to yet another aspect, the present invention relates to the use of a combination of at least two transcriptional promoters as defined in the recombinant microorganism according to the invention to express at least two coding sequences of interest in a microorganism, each of said promoters being operationally linked to one or more of said coding sequences of interest.

[0027] BRIEF DESCRIPTION OF THE DRAWINGS

[0028] 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 TDH3 and RLP18B. 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.

[0029] 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 )TDH3 and )HTB2. Cells were incubated at 30°C in a CSM medium containing 2% glucose with sampling at 72h.

[0030] 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 )TDH3 and )HTB2. Cells were incubated at 37°C in a CSM medium containing 2% glucose with sampling at 72h.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 synthetic promoters is compared to that of natural promoters )TDH3 and )HTB2. Cells were incubated at 30°C in a CSM medium containing 2% ethanol with sampling at 48h.

[0031] Figure 5: Expression of YFP and Ruby2 proteins induced by promoter combination activity after three subcultures. The YFP and Ruby2 genes are expressed by p10 (SEQ ID NO: 11) and p6 (SEQ ID NO: 7) in plasmid 1, and by p6 (SEQ ID NO: 7) and p7 (SEQ ID NO: 8) in plasmid 2. Three culture cycles were performed by incubating the cells at 30°C in CSM. At the end of the last cycle, protein fluorescence was measured by flow cytometry.

[0032] DETAILED DESCRIPTION OF THE INVENTION

[0033] 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.

[0034] The inventors developed a computer code, translated here into a consensus sequence, that generates novel transcriptional promoters capable of controlling the expression of a coding sequence in yeast. The multitude of transcriptional promoters that can be generated using this consensus sequence allows for the selection, for a given cell or genetic construct, of promoters with low genetic redundancy and genetically stable concatenation. Furthermore, these novel 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.

[0035] Definitions

[0036] 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.

[0037] 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.

[0038] 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. "Heterologous" means that the nucleic acid has been introduced into the recombinant microorganism by genetic engineering. It may be present in episomal or chromosomal form. The origin of the nucleic acid may be different from 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 at 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.

[0039] 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.

[0040] 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 use of codons in the sequence coding 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).

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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).

[0052] 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.

[0053] Promoter combinations

[0054] The inventors developed a computer code which, translated into a consensus sequence, generates new transcriptional promoters capable of controlling the expression of a coding sequence in yeast. The multitude of transcriptional promoters that can be generated using this consensus sequence makes it possible to select, for a given cell or genetic construct, promoters with low genetic redundancy, thus preventing or minimizing recombination events.

[0055] The inventors determined, in particular, that the occurrence of these events is minimized when the promoters do not contain identical nucleotide sequences of 25 or more consecutive nucleotides. Furthermore, thanks to the conserved motifs of the consensus sequence, the resulting promoters are similar in terms of regulation and expression and are therefore particularly well-suited for controlling the expression of heterologous metabolic pathways, especially when these pathways include several genes encoding enzymes involved in successive steps of synthesis.

[0056] According to a first aspect, the present invention therefore relates to a combination of at least two transcriptional promoters, in which said transcriptional promoters each comprise a nucleotide sequence comprising the following 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 preferably a nucleotide sequence of 14 to 16 nucleotides; X2 is a nucleotide sequence of 25 to 35 nucleotides, preferably a nucleotide sequence of 29 to 33 nucleotides, and more particularly preferably a nucleotide sequence of 30 to 32 nucleotides.

[0059] X3 is a nucleotide sequence of 5 to 20 nucleotides, preferably a nucleotide sequence of 10 to 16 nucleotides,

[0060] X4 is a nucleotide sequence of 70 to 130 nucleotides, preferably a nucleotide sequence of 77 to 122 nucleotides,

[0061] X5 is a nucleotide sequence of 130 to 180 nucleotides, preferably a nucleotide sequence of 137 to 173 nucleotides,

[0062] XÔ is a nucleotide sequence of 65 to 115 nucleotides, preferably a nucleotide sequence of 72 to 106 nucleotides, and

[0063] X7 is a nucleotide sequence of 15 to 110 nucleotides, preferably a nucleotide sequence of 20 to 100 nucleotides, and wherein the nucleotide sequences of said at least two transcriptional promoters do not include nucleotide sequences of 25 or more consecutive nucleotides identical to each other.

[0064] 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.

[0065] 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.

[0066] 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,

[0067] 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.

[0068] The promoter combination is particularly suitable for use in the same host cell, genetic construct, or vector. It may include at least 2, 3, 4, 5, 10, 20, 30, 40, or 50 promoters as defined above. In particular, the combination may include between 2 and 80, preferably between 3 and 80, between 3 and 50, or between 3 and 10, promoters as defined above. Specifically, it may include 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, preferably 2, 3, 4, 5, or 6, promoters as defined above. Most particularly preferred, it includes at least 3 promoters as defined above.

[0069] To verify that the promoters of the combination do not contain identical nucleotide sequences of 25 or more consecutive nucleotides, the complete sequence of each promoter is compared to the complete sequences of the other promoters in the combination (pairwise comparison). If a promoter contains a nucleotide sequence of 25 or more consecutive nucleotides that is identical to that of another promoter in the combination, one of these two promoters is excluded from the combination. Alternatively, the sequence of one of the promoters can be modified by introducing one or more mutations to eliminate this sequence identity.

[0070] Preferably, each promoter in the combination has 90% or less sequence identity with the other promoters in the combination. More particularly preferred, each promoter in the combination has 85% or less sequence identity with the other promoters in the combination.

[0071] Preferably, the promoters of the combination exhibit transcriptional promoter activity when the recombinant microorganism is cultured in a medium containing 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 cerevi iae cell, an expression cassette containing 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.

[0072] Preferably, the promoters of the combination exhibit constitutive transcriptional promoter activity when the microorganism containing said promoters is cultured in a medium containing 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.

[0073] Preferably, the promoters of the combination have transcriptional promoter activity that is similar to or greater than that of the strong promoter of the S. cerevisiae TDH3 gene (SEQ ID NO: 17) when the microorganism containing said promoters is cultured in a medium containing glucose or ethanol as a carbon source. As used here, the term "similar" refers to an expression level of the coding sequence operationally linked to the promoter of the combination 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 promoter activities of the combination and the >TDH3 promoter can be assessed after 72h of batch culture at 30°C or 37°C in a culture medium including glucose as a carbon source, or after 48h of batch culture at 30°C in a culture medium including ethanol as a carbon source.

[0074] According to one embodiment, said at least two transcriptional promoters of the combination are chosen from promoters comprising, or consisting of, the nucleotide sequences SEQ ID NO: 2 to 17 and functional variants thereof, said functional variants comprising 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 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with Tune of the SEQ ID NO nucleotide sequences: 2 to 17.

[0075] According to a preferred embodiment, said at least two transcriptional promoters of the combination are selected from promoters comprising, or consisting of, the nucleotide sequences SEQ ID NO: 2 to 16 and functional variants thereof, said functional variants comprising 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

[0076] 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least

[0077] 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least

[0078] 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least

[0079] 96%, at least 97%, at least 98% or at least 99% identity with one of the SEQ ID NO nucleotide sequences: 2 to 16.

[0080] According to a particularly preferred embodiment, said at least two transcriptional promoters of the combination are selected from promoters comprising, or consisting of, the nucleotide sequences SEQ ID NO: 2 to 11 and functional variants thereof, said functional variants comprising 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

[0081] 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least

[0082] 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least

[0083] 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least

[0084] 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with one of the SEQ ID NO nucleotide sequences: 2 to 11.

[0085] It is understood that the variants include the sequence

[0086] R(2-3)TGTCTGGGT-X1-RGCATCC-X2-GGCATCC-X3-GAATCCCAGCACC-X4-

[0087] CAACCTGC-X5-RTATATAAAG-X6-HAADWM(i-2)YCAAGAACTTA(i-2)-X7-ATG (SEQ

[0088] ID NO: 1) where Xi, X2, X3, X4, X5, X0 and X7 are as defined above.

[0089] According to a particular embodiment, said at least two transcriptional promoters of the combination are chosen from among the promoters comprising, or consisting of, the nucleotide sequences SEQ ID NO: 2 to 17, preferably from among the promoters comprising, or consisting of, the nucleotide sequences SEQ ID NO: 2 to 16, and more particularly preferred from among the promoters comprising, or consisting of, the nucleotide sequences SEQ ID NO: 2 to 11.

[0090] According to another particular embodiment, said at least two transcriptional promoters of the combination are chosen from promoters comprising, or consisting of, the nucleotide sequences SEQ ID NO: 2 and 3 and functional variants thereof, said functional variants comprising 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

[0091] 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least

[0092] 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least

[0093] 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least

[0094] 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with one of the SEQ ID NO: 2 and 3 nucleotide sequences. Preferably, the at least two transcriptional promoters of the combination are promoters comprising, or consisting of, the SEQ ID NO: 2 and 3 nucleotide sequences.

[0095] According to another particular embodiment, said at least two transcriptional promoters of the combination are chosen from promoters comprising, or consisting of, the nucleotide sequences SEQ ID NO: 5 to 11 and functional variants thereof, said functional variants comprising a sequence having at least 70%, at

[0096] 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least

[0097] 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least

[0098] 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least

[0099] 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least

[0100] 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with one of the SEQ ID NO: 5 to 11 nucleotide sequences. Preferably, the at least two transcriptional promoters of the combination are chosen from promoters comprising, or consisting of, the SEQ ID NO: 5 to 11 nucleotide sequences.

[0101] According to another particular embodiment, said at least two transcriptional promoters of the combination are chosen from promoters comprising, or consisting of, the nucleotide sequences SEQ ID NO: 7 and 8 and functional variants thereof, said functional variants comprising 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

[0102] 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least

[0103] 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least

[0104] 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least

[0105] 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with one of the SEQ ID NO: 7 and 8 nucleotide sequences. Preferably, the at least two transcriptional promoters of the combination are promoters comprising, or consisting of, the SEQ ID NO: 7 and 8 nucleotide sequences.

[0106] According to another particular embodiment, said at least two transcriptional promoters of the combination are chosen from promoters comprising, or consisting of, the nucleotide sequences SEQ ID NO: 7 and 11 and functional variants thereof, said functional variants comprising 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

[0107] 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least

[0108] 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least

[0109] 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least

[0110] 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with one of the nucleotide sequences SEQ ID NO: 7 and 11. Preferably, the at least two transcriptional promoters of the combination are the promoters comprising, or consisting of, the nucleotide sequences SEQ ID NO: 7 and 11. The present invention also relates to a method for selecting a combination of transcriptional promoters, preferably intended to be introduced into the same host cell or the same genetic construct.

[0111] The method includes

[0112] - the provision of a plurality of transcriptional promoters, each comprising a nucleotide sequence including the following sequence:

[0113] 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, X2, X3, X4, X5, XÔ and X7 are as defined above,

[0114] - the comparison of the nucleotide sequences of said transcriptional promoters in order to search for nucleotide sequences of 25 consecutive or more nucleotides that are identical to each other, and

[0115] - the selection of transcriptional promoters whose nucleotide sequences do not include a nucleotide sequence of 25 consecutive or more identical nucleotides.

[0116] Optionally, the method may further include calculating sequence identities between promoters and selecting transcriptional promoters whose nucleotide sequences have a sequence identity of 90% or less, preferably 85% or less, with each other, i.e., a combination in which each promoter has a sequence identity of 90% or less, preferably 85% or less, with the other promoters in the combination.

[0117] Alternatively, the method may include the generation, by bioinformatic means, of the nucleotide sequences of a plurality of transcriptional promoters, each comprising the following sequence:

[0118] 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, X2, X3, X4, X5, XÔ and X7 are as defined above, and do not include a nucleotide sequence of 25 consecutive or more identical nucleotides.

[0119] Optionally, the method also takes into account sequence identities between promoters and generates transcriptional promoters whose nucleotide sequences have a sequence identity of 90% or less, preferably 85% or less, between them.

[0120] Expression cassettes and vectors

[0121] The present invention also relates to a combination of at least two expression cassettes, each comprising a coding sequence of interest operationally linked to a transcriptional promoter of distinct sequence, said transcriptional promoters forming a combination of promoters according to the invention as defined above.

[0122] An expression cassette comprises, at a minimum, a transcriptional promoter and a coding sequence operationally linked to that promoter. Preferably, it also includes a transcriptional terminator. As indicated above in the "definition" section, a cassette may also include other additional elements.

[0123] Preferably, at least one of the expression cassettes in the combination is a recombinant expression cassette, i.e., the coding sequence is operationally linked to a promoter that is different from the promoter to which the coding sequence is functionally linked in nature and especially in a natural genome.

[0124] For each expression cassette, the coding sequence may code for one or more polypeptides of interest or one or more nucleic acids of interest.

[0125] One or more expression cassettes of the combination may include a coding sequence that codes for one or more polypeptides of interest. 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, particularly metabolic synthesis pathways.

[0126] One or more expression cassettes of the combination may include a coding sequence that codes for one or more nucleic acids of interest. Examples of nucleic acids of interest include, but are not limited to, siRNAs, shRNAs, RNAi, miRNAs, antisense RNAs, ribozymes, DNAzymes, or guide RNAs capable of targeting the action of a CRISPR-like endonuclease.

[0127] The combination of expression cassettes according to the invention is particularly suitable for use in the same host cell, in the same genetic construct, or in the same vector. It may comprise at least 2, 3, 4, 5, 10, 20, 30, 40, or 50 cassettes as defined above. In particular, the combination may comprise between 2 and 80, preferably between 3 and 80, between 3 and 50, or between 3 and 10, cassettes as defined above. It may, in particular, comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, preferably 2, 3, 4, 5, or 6, cassettes as defined above. In a more particularly preferred manner, it comprises at least 3 cassettes as defined above, preferably 3, 4, 5 or 6 cassettes.

[0128] According to one embodiment, the expression cassettes forming the combination of expression cassettes according to the invention are included in the same vector.

[0129] According to another embodiment, the expression cassettes forming the combination of expression cassettes according to the invention are distributed over at least two distinct vectors. In this embodiment, the vectors are preferably contained within the same host cell or intended to be inserted into the same host cell.

[0130] The present invention therefore relates to a vector comprising a combination of expression cassettes according to the invention.

[0131] It also relates to a combination of at least two vectors on which expression cassettes are distributed, forming the expression cassette combination according to the invention, each vector comprising at least one of said expression cassettes. Preferably, the vector combination is contained within the same host cell or intended to be inserted into the same host cell.

[0132] All embodiments relating to other aspects of the invention are also considered in these aspects. Genetic constructs

[0133] In a third aspect, the present invention also relates to a genetic construct comprising a combination according to the invention of at least two transcriptional promoters as described above.

[0134] Preferably, each of said promoters is operationally linked to one or more coding sequences. The genetic construct according to the invention therefore preferably comprises a combination according to the invention of at least two expression cassettes as defined above, namely a combination of at least two expression cassettes, each comprising one or more coding sequences of interest operationally linked to a transcriptional promoter of a combination of promoters according to the invention. In the genetic construct according to the invention, the promoters of the combination or the expression cassettes of the combination may be close to each other without risk of genetic rearrangement. For example, the promoters of the combination or the expression cassettes of the combination may be less than 1000 nucleotides apart, in particular less than 500 nucleotides apart.In the genetic construct according to the invention, the expression cassettes of the combination are preferably juxtaposed, that is to say, separated by a maximum of 50 nucleotides, preferably by a maximum of 20 nucleotides, and more particularly preferred by a maximum of 10 nucleotides.

[0135] In one particular embodiment, the genetic construct is contained within a vector or is itself a vector, in particular an expression vector. This expression vector can be used to transform a host cell and enable the expression of the coding sequences of said expression cassettes in said cell. Vectors can be constructed using conventional molecular biology techniques, well known to those skilled in the art. The present invention therefore also relates to a vector comprising a genetic construct according to the invention.

[0136] The vector according to the invention may further comprise one or more bacterial and / or eukaryotic origins of replication. In particular, it may include 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 yeast. The vector may further comprise one or more elements allowing its selection in a bacterial or eukaryotic host cell, such as, for example, an antibiotic resistance gene or a gene enabling auxotrophy complementation. Such elements are well known to those skilled in the art and are widely described in the literature.

[0137] The expression vector may also include one or more sequences allowing targeted insertion of the vector in whole or in part, including targeted insertion of one or more expression cassettes carried by the vector or of one or more promoters of the combination of promoters according to the invention into the genome of a host cell, and in particular into the genome of a yeast.

[0138] In a preferred embodiment, the vector comprises a genetic construct according to the invention, said construct comprising a combination according to the invention of at least two expression cassettes as defined above. Preferably, the vector also comprises one or more sequences enabling the targeted insertion of the genetic construct into the genome of a host cell, and in particular into the genome of a yeast cell.

[0139] All embodiments relating to other aspects of the invention are also considered in this aspect.

[0140] Recombinant microorganism

[0141] In another aspect, the present invention also relates to the use of a combination of promoters according to the invention, a combination of expression cassettes according to the invention, a combination of vectors according to the invention, a genetic construct according to the invention, or a vector according to the invention, in particular an expression vector, to transform or transfect a host cell. It also relates to a host cell transformed or transfected by a combination of promoters according to the invention, a combination of expression cassettes according to the invention, a combination of vectors according to the invention, a genetic construct according to the invention, or a vector according to the invention, in particular an expression vector.The transformed or transfected host cell may contain said combination of promoters, said combination of expression cassettes, said combination of vectors, said genetic construct or said vector, in non-integrated, integrated or partially integrated form in the genome.

[0142] The present invention therefore relates to a recombinant microorganism comprising a combination of at least two transcriptional promoters as described above. The recombinant microorganism according to the invention may comprise a genetic construct according to the invention or a combination of at least two expression cassettes according to the invention. In particular, a genetic construct according to the invention, preferably contained within an expression vector, may be introduced into the microorganism and maintained therein in episomal form. Alternatively, after introduction into the microorganism, the genetic construct may be inserted wholly or partially into the microorganism's genome in a targeted or random manner, preferably in a targeted manner, for example, by homologous recombination.

[0143] The combination of promoters included in the recombinant microorganism may include both endogenous promoters (e.g., the α-TDH3 promoter of S. cerevisiae) and heterologous promoters, provided that at least one of said promoters is heterologous. Preferably, at least two of said transcriptional promoters are heterologous.

[0144] Preferably, the recombinant microorganism comprises a combination according to the invention of transcriptional promoters including at least 2, 3, 4, 5, 10, 20, 30, 40, or 50 transcriptional promoters, said promoters preferably being heterologous. In particular, the recombinant microorganism may comprise a combination according to the invention of transcriptional promoters including between 2 and 80, preferably between 3 and 80, between 3 and 50, or between 3 and 10 promoters, said promoters preferably being heterologous. The recombinant microorganism may, in particular, include a combination according to the invention of transcriptional promoters of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, said promoters preferably being heterologous.

[0145] According to preferred embodiments, in the recombinant microorganism, said transcriptional promoters of the combination are each operationally linked to a coding sequence, preferably a heterologous coding sequence. These promoters are preferably also heterologous to the recombinant microorganism.

[0146] The recombinant microorganism can be a bacterium, a filamentous fungus or a yeast, preferably a bacterium or a yeast, and more preferably a yeast.

[0147] According to one embodiment, the recombinant microorganism is a bacterium, in particular a bacterium suitable for use in preparing, transferring, or preserving a genetic construct according to the invention. The bacterium may be, for example, Escherichia coli, Agrobacterium tumefaciens, or Vibrio natriegens.

[0148] According to a preferred embodiment, the recombinant microorganism 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.

[0149] Preferably, the recombinant microorganism 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 recombinant microorganism 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.

[0150] In particular, yeast can be:

[0151] - 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

[0152] - 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

[0153] - a yeast of the genus Naumovozyma chosen from the group consisting of Naumovozyma castellii, Naumovozyma baii and Naumovozyma dairenensis, or

[0154] - 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 - 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

[0155] - a yeast of the genus Vanderwaltozyma chosen from the group consisting of Vanderwaltozyma polyspora, Vanderwaltozyma tropicalis, Vanderwaltozyma verrucispora and Vanderwaltozyma yarrowii, or

[0156] - 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

[0157] - a yeast of the genus Pichia chosen from the group consisting of Pichia occidentalis, Pichia Kudriavzevii, Pichia fermentons, Pichia manshurica and Pichia terricola, or

[0158] - a yeast of the genus Kluyveromyces chosen from the group consisting of Kluyveromyces lactis and Kluyveromyces marxianus.

[0159] In a more particularly preferred manner, the said yeast is Saccharomyces cerevisiae.

[0160] According to another particular embodiment, the recombinant microorganism 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 cells from Trichoderma, Acremonium, Aspergillus, Aureobasidium, 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.

[0161] All embodiments relating to other aspects of the invention are also considered in this aspect. Method for expressing at least two coding sequences of interest in a microorganism

[0162] According to another aspect, the present invention relates to a method for expressing at least two coding sequences of interest in a microorganism, comprising the introduction into said microorganism of a genetic construct according to the invention comprising a combination of at least two transcriptional promoters as described above, each of said promoters being operationally linked to one or more coding sequences of interest, or the introduction into said microorganism of a combination of at least two expression cassettes according to the invention each comprising at least one coding sequence of interest operationally linked to a transcriptional promoter of distinct sequence, said transcriptional promoters forming a combination of promoters according to the invention as defined above.

[0163] The present invention also relates to the use of a combination of at least two transcriptional promoters to express at least two coding sequences of interest in a microorganism, wherein said transcriptional promoters form a combination of promoters according to the invention as defined above and wherein each of said promoters is operationally linked to one or more coding sequences of interest.

[0164] The microorganism can be as defined above, preferably a bacterium or a yeast, and more particularly preferably a yeast.

[0165] Preferably, the coding sequences of interest are not functionally linked to said promoters in a natural genome and / or in said microorganism. More particularly preferred, the coding sequences of interest and the promoters are heterologous.

[0166] All embodiments relating to other aspects of the invention are also considered in this aspect.

[0167] The following examples are presented for illustrative purposes only and are not exhaustive.

[0168] EXAMPLES Example 1

[0169] Materials & Methods

[0170] Strains

[0171] 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 furacil, tryptophan, and leucine. The inventors modified this strain to also make it auxotrophic for histidine.

[0172] The clonings were carried out in a strain of Escherichia coli.

[0173] Genetic tools

[0174] The promoters ^TDH3 (SEQ ID NO: 17), and ^RLP18B (SEQ ID NO: 18) were recovered by PCR on genomic DNA from strain FY1679-28A.

[0175] 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), and the YFP gene optimized for expression in S. cerevisiae, were synthesized by Arurumolecular, Dundee, UK.

[0176] 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.

[0177] These vectors were then inserted into FY1679-28A yeasts auxotrophic for furacil, tryptophan, leucine, and histidine.

[0178] Growing conditions

[0179] For each strain, 12 clones were cultured in 24-well plates. 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 preculture grown under the same conditions. Fluorescence was measured.

[0180] Fluorescence measurements were performed at 24, 48, and 72 hours. For each strain, 100 µJ / L 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.

[0181] Results

[0182] The transcriptional promoter activity of natural promoters )RLP18B and )TDH3 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.

[0183] The results obtained with synthetic promoters A, B, C, D, and E are similar to those obtained for the strong promoter ^TDH3 (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.

[0184] Example 2

[0185] Materials & Methods

[0186] Strains

[0187] The yeasts used are the same as in example 1, namely the yeast FY1679-28A auxotrophic for uracil, tryptophan, leucine and histidine.

[0188] The clonings were carried out in a strain of Escherichia coli.

[0189] Genetic tools

[0190] The promoters ^TDH3 (SEQ ID NO: 17) and ^HTB2 (SEQ ID NO: 19) were recovered by PCR on genomic DNA from S. cerevisiae strains.

[0191] 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) as well as the YFP and Ruby2 genes optimized for expression in S. cerevisiae, were synthesized by Arurumolecular, Dundee, UK.

[0192] Expression cassettes, each containing one of the above promoters operationally linked to the YFP 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 (RLP18B, SEQ ID NO: 18) operationally linked to the Ruby2 gene.

[0193] These vectors were then inserted into FY1679-28A yeasts auxotrophic for uracil, tryptophan, leucine and histidine.

[0194] Growing conditions

[0195] 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.

[0196] Fluorescence measurement

[0197] 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 excitation and emission wavelengths of 530 nm and 610 nm, respectively, for Ruby2. Results

[0198] 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 Ruby2 protein placed under the control of the control promoter.

[0199] The results obtained with synthetic promoters 1 to 10 are similar to or better than those obtained for the strong promoter ^TDH3, 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.

[0200] Example 3

[0201] Materials & Methods

[0202] Strains

[0203] The yeasts used are the same as in example 1, namely Saccharomyces cerevisiae FY1679-28A auxotrophic for uracil, tryptophan, leucine and histidine.

[0204] The clonings were carried out in a strain of Escherichia coli.

[0205] Genetic tools

[0206] The p6 (SEQ ID NO: 7), p7 (SEQ ID NO: 8), and p1O (SEQ ID NO: 11) promoters, terminators, and the YFP and Ruby2 genes optimized for expression in S. cerevisiae were synthesized by Arurumolecular, Dundee, UK. The PPZ1 gene, partially optimized for S. cerevisiae, was synthesized by Twist Biosciences, South San Francisco, USA. The natural promoters (TDH3, REV1 (SEQ ID NO: 20)) were recovered by PCR on genomic DNA from strain Y1679-28A.

[0207] All of these genetic elements were cloned into a vector composed in particular of recombination sites to be integrated into the yeast genome and of an expression cassette of the LEU2 gene allowing to complement the auxotrophy for leucine in our yeast strains.

[0208] The yeast strains were transformed by heat shock. The integration of the constructs at the locus was verified by PCR.

[0209] Evaluation of the activity of promoters of a combination according to the invention in a genetic construct integrated into the genome of S. cerevisiae

[0210] For strains expressing both the YFP and Ruby2 genes, a population of 8 clones was cultured in 24-well plates. 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 leucine (CSM, Formedium, UK) at 30°C for 24 h with continuous shaking at 200 rpm. To promote a large number of generations (around 20 per cycle), three culture cycles were performed, diluting the previous culture by 10 minutes. 6After this final culture, the cells are fixed. To do this, the culture is centrifuged for 3 minutes at 5000 g. The cell pellet is resuspended in 200 µL of paraformaldehyde (Thermo Scientific Alfa Aesar, 15424389) and then incubated for 20 minutes at room temperature. The cells are then washed twice with 500 µL of buffer composed of µM KH₂PO₄ / K₂HPO₄ (Euromedex, 2028, PB0447-B) and µM Sorbitol (Euromedex, 6213). The cells are resuspended and stored in 500 µL of the same buffer at 4°C.

[0211] Fluorescence analysis was performed by flow cytometry (SONY SH800 cytometer, service provided by Hybrigenics). Channels FL2 and FL3 enabled the detection of fluorescence from the reporters YFP (Δex: 485 nm / Δem: 535 nm) and Ruby2 (Δex: 559 nm / Δem: 600 nm), respectively. Data were processed using the R programming language via the RStudio environment. YFP- and Ruby2-specific fluorescence regions were created ('gating') from singlets of controls expressing only the YFP or Ruby2 gene. Data analysis was then performed by successively applying Ruby2 gating followed by YFP gating.

[0212] Evaluation of the stability of genetic constructs using a combination of promoters according to the invention and integrated into the genome of S. cerevisiae

[0213] For strains expressing the Ruby2, PPZ1, and YFP genes, a population of 5 clones was cultured from a fresh colony subculture onto selective medium in a 96-well plate containing: minimum nitrogen base (Dutscher, Brumath, France) 6.7 g / L, glucose 40 g / L, and complete supplement mixture without leucine (CSM, Formedium, UK). To establish a population of clones, these cells were harvested and co-cultured in 750 µl of the same medium at 30°C for 48 hours with continuous shaking at 1000 RPM. To promote a high number of generations (around 10 per cycle), two culture cycles were performed, diluting the previous culture by 10⁻¹¹ 3After this final culture, the cells are fixed. To do this, the culture is centrifuged for 3 minutes at 5000 g. The cell pellet is resuspended in 200 µL of paraformaldehyde (Thermo Scientific Alfa Aesar, 15424389) and incubated for 20 minutes at room temperature. The cells are then washed twice with 500 µL of buffer composed of µM KH₂PO₄ / K₂HPO₄ (Euromedex, 2028, PB0447-B) and µM Sorbitol (Euromedex, 6213). The cells are resuspended and stored in 500 µL of the same buffer at 4°C.

[0214] Fluorescence analysis was performed by flow cytometry (SONY SH800 cytometer). Channels FL2 and FL3 allowed the detection of fluorescence from the reporters YFP (Δex: 485 nm / Δem: 535 nm) and Ruby2 (Δex: 559 nm / Δem: 600 nm), respectively. The data were processed using the R programming language via the RStudio environment. The creation of YFP- and Ruby2-specific fluorescence regions ('gating') was performed using singlets from controls expressing only the YFP or Ruby2 gene. Data analysis was then carried out by successively applying YFP gating followed by Ruby2 gating.

[0215] Results

[0216] Activity of promoters of a combination according to the invention in the same genetic construct integrated into the genome of S. cerevisiae

[0217] The aim of these complementary experiments is to demonstrate the functionality of a combination of two promoters according to the invention within the same genetic construct integrated into the yeast genome. To this end, a plasmid composed of two independent transcriptional units (TUs) was created. TU1 expresses the YFP gene and TU2 expresses the Ruby2 gene. The TUs are oriented in the same direction. The promoter combinations for each construct are summarized in Table 1.

[0218] Table 1: Promoter combinations present on the plasmids used

[0219] The YFP and Ruby2 genes encode fluorescent proteins. The fluorescence of both proteins was analyzed by flow cytometry. The results presented in Figure 5 show that for each promoter combination, 100% of cells expressing the Ruby2 protein also express the YFP protein. This indicates that both promoters in the combination, contained within the same genetic construct and integrated into the S. cerevisiae genome, are functional. The detection of fluorescent proteins after three successive subculturings, representing approximately 20 cell generations, indicates that the genetic construct containing the promoter combination is stable.

[0220] Stability of genetic constructs expressing heterologous proteins via the promoters of combinations

[0221] In synthetic biology, promoters are used to express heterologous enzymes that can impact cell fitness. To construct stable production strains of molecules of interest, it is necessary to have heterologous genetic constructs that are not susceptible to alteration by homologous recombination mechanisms induced by the host cell. Therefore, the stability of genetic constructs comprising a combination of two promoters according to the invention was compared to that comprising a pair of two identical natural promoters.

[0222] To achieve this, the cells were subjected to stress mimicking that which may occur during the implementation of heterologous metabolic pathways, namely stress induced by the constitutive expression of the PPZ1 gene. This gene, endogenous to S. cerevisiae, encodes a phosphatase involved in monovalent cation homeostasis (Posas F, et al (1993), FEBS Lett. 318:282-286) and the cell cycle (Velázquez D, et al (2020), Sci Rep.; 10(1): 15613). Briefly, its dysregulation leads to impaired mechanisms of adaptation to the environment and cell division. Thus, in order to induce continuous stress without significantly impacting cell viability, the cells were genetically modified with a UT where the expression of enQPPZl is dependent on the weak constitutive promoter PEVP. This UT is itself surrounded by two independent UTs oriented in the same direction, and expressing Ruby2 upstream and YFP downstream.The expression of these two reporter genes is ensured by a combination of promoters according to the invention or by two )TDH3 promoters. The constructs are summarized in Table 2.

[0223] Table 2: Promoters present on the plasmids used

[0224] Through the continuous expression of PPZ1, the aim is to introduce selective pressure on strains to identify homologous recombination events. This mechanism could occur at the level of promoters with identical or similar sequences, leading to the loss of TUT expressing PPZ1 and also the disappearance of the Ruby 2 gene.

[0225] At baseline (tO), nearly 99% of cells express both YFP and Ruby2, regardless of the promoter pair used. Each genetic construct thus appears stable in the S. cerevisiae strain. In contrast, after two subculturings of the strain containing the >TDH3 promoter upstream of the Ruby2 and YFP genes, nearly 3.7% of cells expressing YFP no longer express the Ruby2 protein. This result indicates that a homologous recombination event via the )TDH3 promoters may have occurred to interrupt PPZ1 expression, leading to the loss of the Ruby2 gene. Conversely, under the same experimental conditions, 100% of cells possessing P7 / P6 or P10 / P6 promoter combinations upstream of the Ruby2 and YFP genes express both fluorescent proteins, indicating that there has been no recombination event, particularly at the promoter level, impacting Ruby2 expression.These results therefore show that genetic constructs using promoter combinations according to the invention do not allow the emergence of homologous recombination events in the area concerned, unlike those duplicating the )TDH3 promoter.

Claims

DEMANDS 1. Recombinant microorganism comprising a combination of at least two heterologous transcriptional promoters, wherein said at least two heterologous transcriptional promoters each comprise a nucleotide sequence comprising 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, and the nucleotide sequences of said at least two heterologous transcriptional promoters do not include nucleotide sequences of 25 or more consecutive nucleotides identical to each other.

2. Recombinant microorganism according to claim 1, wherein each promoter of the combination has a sequence identity of 90% or less with the other promoters of the combination.

3. Recombinant microorganism according to claim 1 or 2, wherein the combination comprises between 3 and 80, preferably between 3 and 50, transcriptional promoters.

4. Recombinant microorganism according to any one of claims 1 to 3, wherein said at least two transcriptional promoters have constitutive transcriptional promoter activity when the microorganism is grown in a culture medium comprising glucose or ethanol as a carbon source.

5. Recombinant microorganism according to any one of claims 1 to 4, wherein said at least two transcriptional promoters comprise a nucleotide sequence selected from the group consisting of the nucleotide sequences SEQ ID NO: 2 to 16, and functional variants thereof, said functional variants comprising a sequence having at least 70% identity with one of the nucleotide sequences SEQ ID NO: 2 to 16.

6. Recombinant microorganism according to any one of claims 1 to 5, wherein said at least two transcriptional promoters are each operationally linked to a coding sequence, preferably a heterologous coding sequence.

7. Recombinant microorganism according to any one of claims 1 to 6, said microorganism being a bacterium or a yeast.

8. Recombinant microorganism according to any one of claims 1 to 6, said microorganism being a yeast of the genus Saccharomyces, Kazachstania, Naumovozyma, Nakaseomyces, Tetrapisispora, Vanderwaltozyma, Zygosaccharomyces, Schizosaccharomyces, Yarrow ia, Hansenula, Kluyveromyces, Pichia or Candida or a hybrid obtained from a strain belonging to one of these genera, preferably is a yeast of the species Saccharomyces cerevisiae.

9. Genetic construct comprising at least two transcriptional promoters as defined in any one of claims 1 to 5 and wherein said at least two transcriptional promoters are each operationally linked to a coding sequence.

10. Genetic construct according to claim 9, said construct being included in an expression vector.

11. Genetic construct according to claim 9, said construct being integrated into the genome of a recombinant microorganism.

12. Combination of at least two expression cassettes each comprising a coding sequence of interest operationally linked to a transcriptional promoter of distinct sequence, said transcriptional promoters being as defined in any one of claims 1 to 5.

13. Recombinant microorganism according to any one of claims 1 to 8, said microorganism comprising a genetic construct as defined in any one any of claims 9 to 11 or a combination of at least two expression cassettes as defined in claim 12.

14. A method for expressing at least two coding sequences of interest in a microorganism, comprising introducing into said microorganism a genetic construct as defined in claim 9 or 10 or a combination of at least two expression cassettes as defined in claim 12.

15. Use of a combination of at least two transcriptional promoters as defined in any one of claims 1 to 6 to express at least two coding sequences of interest in a microorganism, each of said promoters being operationally linked to one or more of said coding sequences of interest.

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