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FORMATE-INDUCIBLE PROMOTERS AND METHODS OF USE THEREOF

20230332166 · 2023-10-19

    Inventors

    Cpc classification

    International classification

    Abstract

    The invention provides nucleic acids capable of acting as inducible promoters in yeast species, in particular Yarrowia. The invention vectors, cells and associated methods of producing expression products from cells using the inducible promoters.

    Claims

    1. An isolated nucleic acid capable of acting as an inducible promoter in a non-methylotrophic yeast species, wherein expression from the promoter is induced by an inducing agent where the inducing agent is any one or more compound selected from the group consisting or comprising of: formate, formic acid, formaldehyde, methanol, ethanol, propanol, butanol and glycerol, and: wherein the nucleic acid: a) comprises a sequence that comprises or consists of the consensus sequence set out in SEQ ID NO: 1 GTG CG G CTCG G AA ATT C AC A W G G KCCGT-TY GTG CG G CTCG G AA AT, where: Y is a pyrimidine nucleotide, nucleobase or base; W is a Weak nucleotide, nucleobase or base, optionally an A nucleotide, nucleobase or base or a T nucleotide, nucleobase or base; K is a Keto nucleotide, nucleobase or base, optionally a G nucleotide, nucleobase or base or a T nucleotide, nucleobase or base; or any synthetic analogue or chemically modified nucleotide, nucleobase or base thereof; and/or b) comprises or consists of a portion of a sequence selected from a group comprising or consisting of: i) SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 9, SEQ ID NO: 25, SEQ ID NO: 11, SEQ ID NO: 27, SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 10, SEQ ID NO: 26, SEQ ID NO: 7, SEQ ID NO: 23, SEQ ID NO: 3, SEQ ID NO: 19, SEQ ID NO: 5, SEQ ID NO: 21, SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33; or comprises a portion of a sequence selected from a group comprising a sequence with at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 9, SEQ ID NO: 25, SEQ ID NO: 11, SEQ ID NO: 27, SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 10, SEQ ID NO: 26, SEQ ID NO: 7, SEQ ID NO: 23, SEQ ID NO: 3, SEQ ID NO: 19, SEQ ID NO: 5, SEQ ID NO: 21, SEQ ID NO: 4, SEQ ID NO: 20, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33; ii) SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 9, SEQ ID NO: 25, SEQ ID NO: 11, SEQ ID NO: 27, SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 10, SEQ ID NO: 26, SEQ ID NO: 7, SEQ ID NO: 23, SEQ ID NO: 3, SEQ ID NO: 19, SEQ ID NO: 5, SEQ ID NO: 21, SEQ ID NO: 4, SEQ ID NO: 20: or comprises a portion of a sequence selected from a group comprising a sequence with at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 9, SEQ ID NO: 25, SEQ ID NO: 11, SEQ ID NO: 27, SEQ ID NO: 2, SEQ ID NO: 18, SEQ ID NO: 10, SEQ ID NO: 26, SEQ ID NO: 7, SEQ ID NO: 23, SEQ ID NO: 3, SEQ ID NO: 19, SEQ ID NO: 5, SEQ ID NO: 21, SEQ ID NO: 4, SEQ ID NO: 20; iii) SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 9, SEQ ID NO: 25, or comprises a portion of a sequence selected from a group comprising a sequence with at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity toSEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 6, SEQ ID NO: 22, SEQ ID NO: 9, SEQ ID NO: 25; or iv) SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 6, SEQ ID NO: 22, or comprises a portion of a sequence selected from a group comprising a sequence with at least 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 8, SEQ ID NO: 24, SEQ ID NO: 6, SEQ ID NO: 22.

    2. The isolated nucleic acid according to claim 1 wherein expression from the promoter in the absence of the inducing agent is low or absent or expression from the promoter is induced in YNB media or in ACH +caa media.

    3. The isolated nucleic acid according to claim 1 wherein expression from the promoter is increased by at least 2-fold or at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 45 or at least 50-fold when the non-methylotrophic yeast species is cultured in YNB with 0.5% sodium format.

    4. The isolated nucleic acid of claim 1 wherein the portion of the sequence is between about 46 and 1500 bp in length, for example between 50 and 1500 bp in length, for example between 75 and 1500 bp in length, for example between 100 and 1500 bp in length, for example between 150 and 1400, 200 and 1300, 200 and 1200, 250 and 1100, 250 and 1000, 300 and 950, 350 and 900, 400 and 850, 450 and 800, 500 and 750, 550 and 700, 600 and 650 bp in length; and/or is at least 50, 46, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400 or at least 1500 bp in length.

    5. The isolated nucleic acid according to claim 1 wherein: (a) the nucleic acid is less than 1500 bp in length, optionally is about 46, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400 or about 1500 bp in length; (b) the nucleic acid comprises or consists of a sequence of a portion of a region of up to 1Kb or up to 1.5Kb directly upstream of the translation start codon of a FD-1 gene, or of a putative FDH gene identified in a non-methylotrophic organism, optionally wherein said portion is: about 46, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400 or about 1500 bp in length; and/or is between about 100 and 1500 bp in length, for example between 150 and 1400, 200 and 1300, 200 and 1200, 250 and 1100, 250 and 1000, 300 and 950, 350 and 900, 400 and 850, 450 and 800, 500 and 750, 550 and 700,600 and 650 bp in length; (c) expression from the inducible promoter is induced by formate; and/or (d) wherein the nucleic acid is flanked by one or more restriction enzyme digestion sites, optionally by one or more type II restriction enzyme digestion sites.

    6. (canceled)

    7. (canceled)

    8. (canceled)

    9. A nucleic acid construct comprising at least a first and a second nucleic acid sequence, wherein the first nucleic acid sequence comprises or consists of the isolated nucleic acid sequence of claim 1, optionally, wherein expression from the promoter is induced in YNB media or in ACH +caa media.

    10. The nucleic acid construct of claim 9, wherein the second nucleic acid sequence is a sequence capable of being transcribed into RNA, and wherein the first nucleic acid sequence is operably linked to the second nucleic acid sequence, optionally wherein the 3′ end of the first nucleic acid sequence is linked to the 5′ end of the second nucleic acid sequence by a sequence comprising or consisting the sequence CACA.

    11. The nucleic acid construct of claim 9 wherein the second nucleic acid sequence is transcribed into mRNA, optionally wherein the second nucleic acid sequence encodes a peptide or polypeptide.

    12. The nucleic acid construct of claim 9 wherein the second nucleic acid sequence is capable of being transcribed into an RNA sequence selected from the group consisting of or comprising: mRNA, rRNA, miRNA, siRNA, piRNA, snRNA, snoRNA, exRNA, scaRNA, lncRNA, gRNA, sgRNA, crRNA, and tracrRNA.

    13. The nucleic acid construct of claim 9, wherein the second nucleic acid sequence does not encode a formate dehydrogenase (FDH) gene, optionally does not encode a formate dehydrogenase gene from Yarrowia, optionally from Yarrowia lipolytica, wherein where: the first nucleic acid sequence comprises or consists of a portion of SEQ ID NO: 8 or 24, or comprises or consists of SEQ ID NO: 8 or 24, the second nucleic acid does not encode YALI0E14256 (SEQ ID NO:40); the first nucleic acid sequence comprises or consists of a portion of SEQ ID NO: 2 or 18, or comprises or consists of SEQ ID NO: 2 or 18, the second nucleic acid does not encode YALI0A21353 (SEQ ID NO:34); the first nucleic acid sequence comprises or consists of a portion of SEQ ID NO: 3 or 19, or comprises or consists of SEQ ID NO: 3 or 19, the second nucleic acid does not encode YALI0F15983 (SEQ ID NO:35); the first nucleic acid sequence comprises or consists of a portion of SEQ ID NO: 4 or 20, or comprises or consists of SEQ ID NO: 4 or 20, the second nucleic acid does not encode YALI0B22506 (SEQ ID NO:36); the first nucleic acid sequence comprises or consists of a portion of SEQ ID NO: 5 or 21, or comprises or consists of SEQ ID NO: 5 or 21, the second nucleic acid does not encode YALI0C08074 (SEQ ID NO:37); the first nucleic acid sequence comprises or consists of a portion of SEQ ID NO: 6 or 22, or comprises or consists of SEQ ID NO: 6 or 22, the second nucleic acid does not encode YALI0F13937 (SEQ ID NO:38); the first nucleic acid sequence comprises or consists of a portion of SEQ ID NO: 7 or 23, or comprises or consists of SEQ ID NO: 7 or 23, the second nucleic acid does not encode YALI0C14344 (SEQ ID NO:39); the first nucleic acid sequence comprises or consists of a portion of SEQ ID NO: 9 or 25, or comprises or consists of SEQ ID NO: 9 or 25, the second nucleic acid does not encode YALI0B19976 (SEQ ID NO:41); the first nucleic acid sequence comprises or consists of a portion of SEQ ID NO: 10 or 26, or comprises or consists of SEQ ID NO: 10 or 26, the second nucleic acid does not encode YALI0E15840 (SEQ ID NO:42); the first nucleic acid sequence comprises or consists of a portion of SEQ ID NO: I1 or 27, or comprises or consists of SEQ ID NO: 11 or 27, the second nucleic acid does not encode YALI0F28765 (SEQ ID NO:43); the first nucleic acid sequence comprises or consists of a portion of SEQ ID NO: 12 or 28, or comprises or consists of SEQ ID NO: 12 or 28, the second nucleic acid does not encode YALI0E19657g (SEQ ID NO:44); the first nucleic acid sequence comprises or consists of a portion of SEQ ID NO: 13 or 29, or comprises or consists of SEQ ID NO: 13 or 29, the second nucleic acid does not encode YALI0B21670g (SEQ ID NO:45); the first nucleic acid sequence comprises or consists of a portion of SEQ ID NO: 14 or 30, or comprises or consists of SEQ ID NO: 14 or 30, the second nucleic acid does not encode YALI0F29315g (SEQ ID NO:46); the first nucleic acid sequence comprises or consists of a portion of SEQ ID NO: 15 or 31, or comprises or consists of SEQ ID NO: 15 or 31, the second nucleic acid does not encode YALI0D25256g (SEQ ID NO:47); the first nucleic acid sequence comprises or consists of a portion of SEQ ID NO: 16 or 32, or comprises or consists of SEQ ID NO: 16 or 32, the second nucleic acid does not encode YALI0C11099g (SEQ ID NO:48); the first nucleic acid sequence comprises or consists of a portion of SEQ ID NO: 17 or 33, or comprises or consists of SEQ ID NO: 17 or 33, the second nucleic acid does not encode YALI0F09966g (SEQ ID NO:49).

    14. An expression cassette comprising the isolated nucleic acid of claim 1 or nucleic acid construct comprising at least a first and a second nucleic acid sequence, wherein the first nucleic acid sequence comprises or consists of the isolated nucleic acid sequence of claim 1, optionally, wherein expression from the promoter is induced in YNB media or in ACH +caa media.

    15. A vector comprising the isolated nucleic acid of claim 1, or nucleic acid construct of comprising at least a first and a second nucleic acid sequence, wherein the first nucleic acid sequence comprises or consists of the isolated nucleic acid sequence of claim 1, optionally wherein the vector is selected from a group comprising a plasmid or an artificial chromosome, optionally wherein the artificial chromosome is selected from a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), and a Human artificial chromosome (HAC), optionally, wherein expression from the promoter is induced in YNB media or in ACH +caa media.

    16. A cell comprising: i) the isolated nucleic acid of claim 1; ii) a nucleic acid construct comprising at least a first and a second nucleic acid sequence, wherein the first nucleic acid sequence comprises or consists of the isolated nucleic acid sequence of claim 1; iii) an expression cassette of comprising the isolated nucleic acid of claim 1 or nucleic acid construct comprising at least a first and a second nucleic acid sequence, wherein the first nucleic acid sequence comprises or consists of the isolated nucleic acid sequence of claim 1; and/or iv) a vector of comprising the isolated nucleic acid of claim 1, or nucleic acid construct of comprising at least a first and a second nucleic acid sequence, wherein the first nucleic acid sequence comprises or consists of the isolated nucleic acid sequence of claim 1, optionally wherein the vector is selected from a group comprising a plasmid or an artificial chromosome, optionally wherein the artificial chromosome is selected from a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), and a Human artificial chromosome (HAC); optionally wherein the cell is an engineered cell, and optionally, wherein expression from the promoter is induced in YNB media or in ACH +caa media.

    17. The cell of claim 16, wherein: (a) the yeast cell is a cell belonging to the species Yarrowia lipolytica: (b) the cell is a eukaryotic cell, optionally wherein the cell is a cell selected from a group comprising: a fungal cell; a plant cell; and an animal cell; optionally wherein the fungal cell is a yeast cell, optionally wherein the yeast cell is: not a methylotrophic yeast cell, optionally from a genus selected from a group consisting or comprising: Ashbya, Blastobotrys, Crytococcus, Cutaneotrichosporon, Dekkera, Kluveromyces, Rhodosporidium, Rhodotorula, Lipomyces, Saccharomyces, and Yarrowia; or a methylotrophic yeast cell, optionally from a genus selected from a group consisting or comprising: Candida, Hansenula, Komagataella, and Pichia; (c) the isolated nucleic acid, nucleic acid construct, expression cassette, or vector is episomally maintained by said cell and/or (d) the isolated nucleic acid, inducible promoter, nucleic acid construct, expression cassette, or vector is integrated into the genome of said cell.

    18. (canceled)

    19. (canceled)

    20. (canceled)

    21. (canceled)

    22. The cell of claim 17, wherein: (a) the isolated nucleic acid, inducible promoter, nucleic acid construct, expression cassette, or vector is integrated upstream of a second sequence located in the genome, and wherein following integration the isolated nucleic acid, inducible promoter, nucleic acid construct, epression cassete, or vector is capable of driving transcription of the second sequence; and/or (b) wherein following integration the isolated nucleic acid, inducible promoter, nucleic acid construct, expression cassette, or vector is capable of driving transcription of the second sequence the isolated nucleic acid, inducible promoter, nucleic acid, expression cassette, or vector is integrated into the genome of said cell at a different locus to the locus of the native promoter, optionally wherein where: the isolated nucleic acid has a sequence of 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 8 or 24 the isolated nucleic acid, inducible promoter, nucleic acid, expression cassette, or vector is integrated at a genomic locus that is different to the locus of native SEQ ID NO: 8 or 24, i.e., is not operably inserted upstream of the gene encoding SEQ ID NO: 40; the isolated nucleic acid has a sequence of 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ II) NO: 2 or 18 the isolated nucleic acid, inducible promoter, nucleic acid, expression cassette, or vector is integrated at a genomic locus that is different to the locus of native SEQ ID NO: 2 or 18, i.e., is not operably inserted upstream of the gene encoding SEQ ID NO: 34; the isolated nucleic acid has a sequence of 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 3 or 19 the isolated nucleic acid, inducible promoter, nucleic acid, expression cassette, or vector is integrated at a genomic locus that is different to the locus of native SEQ ID NO: 3 or 19, i.e., is not operably inserted upstream of the gene encoding SEQ ID NO: 35; the isolated nucleic acid has a sequence of 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 4 or 20 the isolated nucleic acid, inducible promoter, nucleic acid, expression cassette, or vector is integrated at a genomic locus that is different to the locus of native SEQ ID NO: 4 or 20, i.e., is not operably inserted upstream of the gene encoding SEQ ID NO: 36; the isolated nucleic acid has a sequence of 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 5 or 21 the isolated nucleic acid, inducible promoter, nucleic acid, expression cassette, or vector is integrated at a genomic locus that is different to the locus of native SEQ ID NO: 5 or 21, i.e., is not operably inserted upstream of the gene encoding SEQ ID NO: 37; the isolated nucleic acid has a sequence of 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 6 or 22 the isolated nucleic acid, inducible promoter, nucleic acid, expression cassette, or vector is integrated at a genomic locus that is different to the locus of native SEQ ID NO: 6 or 22, i.e., is not operably inserted upstream of the gene encoding SEQ ID NO: 38; the isolated nucleic acid has a sequence of 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 7 or 23 the isolated nucleic acid, inducible promoter, nucleic acid, expression cassette, or vector is integrated at a genomic locus that is different to the locus of native SEQ ID NO: 7 or 23, i.e., is not operably inserted upstream of the gene encoding SEQ ID NO: 39; the isolated nucleic acid has a sequence of 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 9 or 25 the isolated nucleic acid, inducible promoter, nucleic acid, expression cassette, or vector is integrated at a genomic locus that is different to the locus of native SEQ ID NO: 9 or 25, i.e., is not operably inserted upstream of the gene encoding SEQ ID NO: 41; the isolated nucleic acid has a sequence of 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 10 or 26 the isolated nucleic acid, inducible promoter, nucleic acid, expression cassette, or vector is integrated at a genomic locus that is different to the locus of native SEQ ID NO: 10 or 26, i.e., is not operably inserted upstream of the gene encoding SEQ ID NO: 42; the isolated nucleic acid has a sequence of 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 11 or 27 the isolated nucleic acid, inducible promoter, nucleic acid, expression cassette, or vector is integrated at a genomic locus that is different to the locus of native SEQ ID NO: 11 or 27, i.e., is not operably inserted upstream of the gene encoding SEQ ID NO: 43; the isolated nucleic acid has a sequence of 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 12 or 28 the isolated nucleic acid, inducible promoter, nucleic acid, expression cassette, or vector is integrated at a genomic locus that is different to the locus of native SEQ ID NO: 12 or 28, i.e., is not operably inserted upstream of the gene encoding SEQ ID NO: 44; the isolated nucleic acid has a sequence of 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 13 or 29 the isolated nucleic acid, inducible promoter, nucleic acid, expression cassette, or vector is integrated at a genomic locus that is different to the locus of native SEQ ID NO: 13 or 29, i.e., is not operably inserted upstream of the gene encoding SEQ ID NO: 45; the isolated nucleic acid has a sequence of 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 14 or 30 the isolated nucleic acid, inducible promoter, nucleic acid, expression cassette, or vector is integrated at a genomic locus that is different to the locus of native SEQ ID NO: 14 or 30, i.e., is not operably inserted upstream of the gene encoding SEQ ID NO: 46; the isolated nucleic acid has a sequence of 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 15 or 31 the isolated nucleic acid, inducible promoter, nucleic acid, expression cassette, or vector is integrated at a genomic locus that is different to the locus of native SEQ ID NO: 15 or 31, i.e., is not operably inserted upstream of the gene encoding SEQ ID NO: 47; the isolated nucleic acid has a sequence of 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 16 or 32 the isolated nucleic acid, inducible promoter, nucleic acid, expression cassette, or vector is integrated at a genomic locus that is different to the locus of native SEQ ID NO: 16 or 32, i.e., is not operably inserted upstream of the gene encoding SEQ ID NO: 48; or the isolated nucleic acid has a sequence of 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 17 or 33 the isolated nucleic acid, inducible promoter, nucleic acid, expression cassette, or vector is integrated at a genomic locus that is different to the locus of native SEQ ID NO: 17 or 33, i.e., is not operably inserted upstream of the gene encoding SEQ ID NO: 49.

    23. A method of producing an expression product of a gene or a secondary metabolite or animal feed, wherein the method comprises the use of the isolated nucleic acid of claim 1 and optionally an, inducible promoter, optionally wherein the isolated nucleic acid is in a nucleic acid construct, optionally wherein the isolated nucleic acid is in an expression cassette, a vector, and/or a cell.

    24. (canceled)

    25. (canceled)

    26. A kit comprising at least two of any of: (a) the isolated nucleic acid of claim 1; (b) a nucleic acid construct comprising at least a first and a second nuclei acid sequence, wherein the first nucleic acid sequence comprises or consists of the isolated nucleic acid sequence of claim 1, optionally, wherein expression from the promoter is induced in YNB media or in ACH +caa media; (b) an expression cassette comprising the isolated nucleic acid of claim 1 or nucleic acid construct comprising at least a first and a second nucleic acid sequence, wherein the first nucleic acid sequence comprises or consists of the isolated nucleic acid sequence of claim 1, optionally, wherein expression from the promoter is induced in YNB media or in ACH +caa media; (c) a vector comprising the nucleic acid of claim 1; (d) a cell comprising the nucleic acid of claim 1; an inducing agent, optionally wherein the inducing agent is formate, optionally, wherein the expression from the promoter is induced in YNB media or in ACH +caa media.

    27. (canceled)

    28. (canceled)

    29. A method of producing a vaccine comprising using the isolated nucleic acid of claim 1, optionally, inducible promoter, nucleic acid construct comprising the nucleic acid of claim 1, an expression cassette comprising the nucleic acid of claim 1, a vector comprising the nucleic acid of claim 1, or a cell comprising the nucleic acid of claim 1.

    30. A food product wherein the food product comprises a cell according to claim 16.

    Description

    FIGURE LEGENDS

    [0357] FIG. 1: Consensus motif of ten formate induced promoters of Yarrowia lipolytica. The consensus motif sequence was derived by aligning 1500 bp upstream of the 10 fdh genes identified herein using MUSCLE to identify conserved regions.

    [0358] FIG. 2: Multiple sequence alignment of all putative Yarrowia lipolytica FDH protein sequences with YALI0E14256g1_1.

    [0359] FIG. 3: Transcription levels of the FDH genes of Yarrowia lipolytica W29 measured by qPCR, normalised to actin (ACT1) expression. Yarrowia lipolytica was grown in YNB supplemented with 1% glucose and 0.5% yeast extract at 15 h at 28° C., before being washed and transferred into YNB with 0.5% of sodium formate for induction and grown for 6 h.

    [0360] FIG. 4: OD-normalized fluorescence of YL DSM 17984 transformed with empty vector (nc, p15021), pFDH-sfGFP (p15032) or pTEF sfGFP (p28003) grown in YNB (100 mg/L lysine, 260 mg/L leucine), with and without 1% of sodium formate. Cultivation in 30 mL of medium in shake flasks with n=3.

    [0361] FIG. 5a: Measured fluorescence values for three different strains of Y. lipolytica grown in YNB supplemented with or without 0.05%, 1%, or 2.5% sodium formate. Formate was added at the start of incubation (early), after 24 hours (late), or not at all (no). Strain S29008 carries a genomically integrated sfGFP controlled by a putative formate induced promoter. Strain S11082 acts as a negative control carrying no sfGFP. Strain S06003 (positive control) expresses sfGFP constitutively under a TEF promoter (genomically integrated).

    [0362] FIG. 5b: Measured fluorescence values for three different strains of Y. lipolytica grown in YPD supplemented with or without 0.05%, 1%, or 2.5% sodium formate. Formate was added at the start of incubation (early), after 24 hours (late), or not at all (no). Strain S29008 carries a genomically integrated sfGFP controlled by a putative formate induced promoter. Strain S11082 acts as a negative control carrying no sfGFP. Strain S06003 (positive control) expresses sfGFP constitutively under a TEF promoter (genomically integrated).

    [0363] FIG. 5c: Measured fluorescence values for strains of Y. lipolytica carrying different plasmids, grown in ACH media supplemented with 14 g/L casamino acids (CAA) and 0.05%, 1%, or 2.5% sodium formate. Formate was added at the start of incubation (early), after 24 hours (late), or not at all (no). Plasmid p28003 carries a sfGFP sequence controlled by a constitutive pTEF promoter. Plasmid p15032 uses p28003 as a backbone but has the pTEF promoter replaced by the putative formate induced promoter. Plasmid p15021 acts as a negative control carrying no sfGFP.

    [0364] FIG. 5d: Measured fluorescence values for strains of Y. lipolytica carrying different plasmids, grown in ACH media supplemented with 3.5 g/L casamino acids (CAA) and 0.05%, 1%, or 2.5% sodium formate. Formate was added at the start of incubation (early), after 24 hours (late), or not at all (no). Plasmid p28003 carries a sfGFP sequence controlled by a constitutive pTEF promoter. Plasmid p15032 uses p28003 as a backbone but has the pTEF promoter replaced by the putative formate induced promoter. Plasmid p15021 acts as a negative control carrying no sfGFP.

    [0365] The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

    [0366] Preferences and options for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features and parameters of the invention. For example, the invention provides an isolated nucleic acid capable of acting as an inducible promoter in a non-methylotrophic yeast species wherein the nucleic acid comprises a 150 bp region of SEQ ID NO: 3 and which is inducible by formate in YNB media. The invention also provides a nucleic acid construct comprising a nucleic acid capable of acting as an inducible promoter, wherein the nucleic acid comprises a sequence according to SEQ ID NO: 1 and a second nucleic acid, wherein the second nucleic acid encodes a protein involved in the production of phenolic compounds.

    EXAMPLES

    [0367] The invention will now be exemplified with the following non-limiting Examples.

    Example 1

    [0368] In order to identify formate-inducible promoters in the non-methylotrophic yeast Yarrowia lipolytica, formate dehydrogenase (FDH) genes encoded in the Yarrowia lipolytica E150 were first identified by homology to the Y. lipolytica FDH protein, YALI0E14256g1_1. 16 putative FDH proteins were identified in total (FIG. 2), corresponding to the 16 putative FDH genes described herein (SEQ ID NO: 18-33).

    [0369] The inducibility of the putative FDH genes with formate was then tested by qPCR. YALI0E14256g1_1 and the nine putative FDH with the highest % identity to YALI0E14256g1_1 were selected for testing. Y. lipolytica JMY2900 was cultured in YNB media supplemented with 1% glucose and 0.5% yeast extract for 15 h at 28° C. Cells were washed with distilled water and cultured in fresh liquid YNB media supplemented with 0.5% sodium formate at 28° C. with agitation at 160 rpm, before being harvested at 6 h post-inoculated. Cells were frozen in liquid nitrogen and stored at −80° C. RNA was subsequently extracted using the RNeasy Mini Kit (Qiagen), and 2 μg was treated with DNAse (Ambion; Life Science Technologies, Saint-Aubain, France). cDNA was synthesised using the Maxima First Strand cDNA synthesis kit (Thermo Fischer Scientific, Villebon sur Yvette, France). qPCR was then performed using the SYBRgreen mastermix (Thermo Fischer Scientific) with gene-specific primers designed using Primer3 software. Relative expression levels were calculated using ΔC.sub.T and ΔΔC.sub.T methods. Expression levels were normalised to actin.

    [0370] qPCR analysis of 10 putative FDH genes demonstrated increased expression in all ten genes tested when Y. lipolytica is cultured with 0.5% sodium formate, compared to culturing in the absence of formate. See FIG. 3 (induced=+0.5% sodium formate; not_induced=−0.5% sodium formate) and Table 1.

    TABLE-US-00003 TABLE 1 fold change expression of putative FDH genes upon induction with sodium formate Putative FDH gene Fold increase on induction Yali0F28765 8.1 Yali0F15983 4.2 Yali0C08074 3.7 Yali0A21353 7.9 Yali0E15840 5.7 Yali0F13937 38.6 Yali0B22506 2.9 Yali0B19976 11.4 Yali0E14256 23.9 Yali0C14344 4.9

    Example 2

    [0371] Expression from the putative FDH promoters was characterised by a promoter-GFP assay. A 1500 bp fragment directly upstream of the YALI0E14256 gene was amplified from the Y. lipolytica H222 genome and cloned into the p28003 backbone plasmid with sfGFP by SLiCE cloning (Messerschmidt et al., 2016), resulting in the plasmid pFDH-sfGFP, containing a fusion of the 1500 bp FDH promoter operably linked to the sfGFP gene. pFDH-sfGFP was transformed into Y. lipolytica s15028 for testing. A positive control plasmid, p28803 or pTEF-sfGFP, comprising the constitutive TEF promoter was also transformed into Y. lipolytica s15028 as a fluorescence positive control.

    [0372] Fluorescence signal produced by untransformed cells and cells transformed with pFDH-sfGFP or pTEF-sfGFP was tested by culturing the cells in a 24-well plate in YNB supplemented with 20 gL.sup.−1 glucose, 100 mgL.sup.−1 lysine and 260 mgL.sup.−1 leucine at 30° C. with agitation at 150 rpm. GFP expression was induced by the addition of sodium formate to a final concentration of 1% (w/v). Fluorescence was detected and normalised to the OD.sub.600 of the cultures.

    [0373] FIG. 4 shows that the addition of 1% (w/v) sodium formate induces expression of sfGFP from pFDH-sfGFP. No difference was observed in the fluorescence of untransfected cells. A small increase was observed in fluorescence produced by cells transformed with pTEF-sfGFP. These data show that FDH promoters are inducible to a high level by formate (FIG. 3, nc=untransformed cells; pFDH=pFDH-sfGFP; pTEF =pTEF-sfGFP; _ind=+sodium formate).

    Example 3

    [0374] To measure the inducible expression of GFP under the control of Y. lipolytica FDH promoters under different conditions, three different media for the cultivation of Y. lipolytica were compared: yeast extract peptone dextrose media (YPD; Carl Roth—Karlsruhe, Germany; 50 g/L); yeast nitrogen base without amino acids media (YNB; Carl Roth—Karlsruhe, Germany; 6.8 g/L); and ACH media (6.7 g/L YNB, 14 g/L CAS amino acids, 10 g/L glucose, all obtained from Carl Roth—Karlsruhe, Germany).

    [0375] Precultures were grown overnight in TPD, YNB, or ACH. Cells were pelleted by centrifugation before being resuspended in the corresponding media. The OD of each suspension was measured. The suspension was then diluted before 10 μl diluted cell suspension was inoculated into 180 μl of the corresponding media in the wells of a 96-well plate, to a final OD.sub.600=0.1. Inoculated plates were incubated in a Cytomat 2 tower shaker at 28° C. for 72 h with agitation at 1000 rpm. Expression was induced by addition of sodium formate to a final concentration of 0.05%, 1%, or 2.5% (w/v) at the start of plate incubation (early) or after 24 hours (late). A third group remained without formate addition (no). Four repeats were conducted per condition.

    [0376] Every 3 hours during incubation, each plate was transferred from the incubator to a PHERAstar FSX plate reader, where OD, fluorescence and fluorescence polarization at two different gain levels were measured. Plates were transferred by a four-axis Precise Flex 760 (Precise Automation) laboratory robot. An excitation wavelength of 485 nm was used, while emission was measured at 520 nm. “Late” addition of sodium formate was conducted at 24 h post-inoculation by pipetting 10 μL of sodium formate solution from a storage plate into each well using a CyBio Felix with a 96-channel head. Wells filled with media, were used as blanks. Into the respective blanks, formate was added along the corresponding conditions for each induction timepoint.

    [0377] As a result, we observed that no induction was observed in YPD while a clear induction was found in both YNB and ACH+caa when formate was added in the culture media. FDH allowed the transition from close to zero expression (with no addition of formate) to strong expression (when formate was added). As a control pTEF was used, which showed expression in any media regardless of the addition of formate. Some variations was observed in TEF expression when formate was added, which could be explained by general metabolic changes caused by the metabolism of this compound by FDHs genes, which affect redox state of the cell.