Methods and compositions

Abstract

The present invention relates to the biological production of dyes and staining biomaterials, and in particular to cells and methods for the production of natural blue pigments. The invention also provides a dyed cellulosic material and a production method thereof.

Description

Technical Field

[0001] This invention belongs to the field of bioproduction of dyes and dyeing biomaterials, and specifically relates to cells and methods for producing dyes and dyeing biomaterials. Background Technology

[0002] The textile industry is associated with severe water and other environmental pollution due to the emission of toxic chemicals as waste products during fabric dyeing and production. Public awareness and support for more sustainable production processes are rapidly increasing due to the climate crisis and ecological problems caused by unsustainable industries, including dyeing and related processes. Therefore, there is an urgent need to develop new, sustainable dye manufacturing processes that minimize environmental pollution and meet consumer demand for environmentally friendly products.

[0003] Indigoidine is a non-ribosomal peptide (NRP) with a deep blue color similar to the industrial dye indigo. It is naturally produced by several genera of bacteria. Furthermore, due to its molecular structure, indigoidine is a promising organic semiconductor with potential applications in bioelectronics. This pigment also exhibits antioxidant and antimicrobial activities. Therefore, indigoidine is an attractive natural blue dye that can be used in the textile industry as a replacement for polluting synthetic blue dyes; and it also has applications in the electronics and medical fields.

[0004] Natural blue pigment is produced by the condensation of two L-glutamine molecules catalyzed by non-ribosomal peptide synthase (NRPS). L-glutamine itself is produced by the condensation of glutamate and ammonia via glutamine synthase. α-ketoglutarate is converted to glutamate by glutamate dehydrogenase.

[0005] The low yield levels of nonribosomal peptides (NRPs) in wild-type NRP-producing strains, the structural complexity of NRPs, and the associated challenges of purifying NRPs from biological materials make the production of natural blue pigments in natural strains commercially unfeasible.

[0006] Synthetic biology approaches have been developed in this field to increase the production of natural blue pigments. For example, methods capable of expressing NRPS (such as...) have been developed. Streptomyces lavendulaeEngineered bacterial strains containing NRPS (BpsA) and a phosphoproteotransferase (PPTase) peptide that activates the function of the NRPS peptide were developed. Although L-glutamine condensation into native blue pigment was observed in these engineered bacterial strains, yield levels were limited, for example, by the availability of L-glutamine in the cells. While L-glutamine supplementation has been shown to increase native blue pigment production, the cost of exogenous L-glutamine makes this approach commercially unfeasible.

[0007] Therefore, engineered strains are needed to increase the yield of natural blue pigment. Summary of the Invention

[0008] The inventors have developed a robust platform for the production of natural blue pigment in recombinant yeast. They have surprisingly discovered that expressing heterologous nonribosomal peptide synthase (NRPS) peptides and heterologous phosphopantoylthioethylamine transferase (PPTase) peptides in yeast cells enables the production of natural blue pigment.

[0009] The inventors have further discovered that by overexpressing endogenous glutamate dehydrogenase and glutamine synthase peptides in recombinant yeast cells, the production of natural blue pigment can be increased without the need for supplemental exogenous L-glutamine.

[0010] The production of natural blue pigment can also be increased by changing the carbon and nitrogen sources present in the culture medium used to culture recombinant yeast cells, and by changing the carbon-nitrogen (C / N) ratio.

[0011] The inventors have also surprisingly discovered that naturally blue-stained bacterial cellulose can be produced by staining a bacterial membrane with a natural blue pigment extracted from recombinant yeast cells; and / or by co-culturing recombinant yeast cells with cellulose-producing bacteria. Attached Figure Description

[0012] Figure 1 —Produces natural blue pigment Yarrowia lipolytica Microscopic images of the strain. (A) Blue spots show the distribution of pigments within the cells. (B) From engineered... Yarrowia lipolytica Natural blue pigment extract obtained from the biomass of cells. (C) In engineered... Yarrowia lipolytica The strain biosynthesizes a natural blue pigment from α-ketoglutarate. Endogenous glutarate dehydrogenase and glutamine synthase produce L-glutamine, which is condensed by heterologously expressed BpsA (activated by heterologously expressed PPTase) to generate the natural blue pigment.

[0013] Figure 2 —(A) Natural blue pigment titer, (B) Growth curve, and (C) Production of natural blue pigment in flasks in rich and basic media. Yarrowia lipolytica pH changes during the strain's lifespan. (D) Cell morphology and (E) Condition of the flasks after 72 and 120 hours of incubation.

[0014] Figure 3 —The effect of carbon source on the yield of natural blue pigment. (A) Analytical grade substrate, (B) Lignocellulose hydrolysate, (C) Waste substrate. Cultured in deep-well plates for 120 hours.

[0015] Figure 4 —The effects of (A) nitrogen quality and (B) availability on the yield of natural blue pigment. Cultures were carried out in deep-well plates for 120 hours.

[0016] Figure 5 —The effect of stirring speed on (A) the biosynthesis of natural blue pigment and (B) the production of natural blue pigment Yarrowia lipolytica The effect of proof-of-concept strains on growth.

[0017] Figure 6 —The effects of (A) temperature and (B) water type on the yield of natural blue pigment. The concentration of natural blue pigment after 120 hours of incubation in (C) superbasic medium (SMm).

[0018] Figure 7 —(A) Natural blue pigment is produced during bioreactor culture in a culture medium containing urea and ammonium sulfate. Yarrowia lipolytica Natural blue pigment production, growth, and substrate consumption of the strain. (B) Natural blue pigment-producing strain (left) and wild-type Yersinia lipolytica strain (right) during cultivation in a bioreactor.

[0019] Figure 8 —(A) The biosynthetic pathway of natural blue pigment and (B) the effects of glutamine and glutamate supplementation on natural blue pigment yield after 120 hours of culture. Red rectangles indicate… Yarrowia lipolytica The inherent enzymatic reactions are represented by blue rectangles, while heterologous reactions are indicated by blue rectangles. YNB—a basic culture medium containing 20 g / L glucose.

[0020] Figure 9 —The effect of endogenous precursor supply on the production of natural blue pigment in Yersinia lipolytica.

[0021] Figure 10 —Producing natural blue pigment through co-cultivation Yarrowia lipolytica Blue bacterial cellulose (BC) fibers obtained from strains of bacteria and cellulose-producing bacteria.

[0022] Figure 11 —Production of colored BC films. (A) Inoculation to produce natural blue pigment.Yarrowia lipolytica Previous bacterial films. Film size can be adjusted by selecting the culture vessel. (B) Formation of colored bacterial cellulose biomaterial after shaking for 72 to 120 hours. Uncolored. Yarrowia lipolytica Cells appear as gray areas on the biomaterial. Microscopic images of stained bacterial cellulose films. (C) Microscopic top view of the biomaterial surface and (D) Outline view. White arrows highlight the filaments inside / near the bacterial cellulose network. Yarrowia lipolytica Natural blue pigments are deposited throughout the biomaterial in the form of blue crystals.

[0023] Figure 12 --use Yarrowia lipolytica Biomass for textile dyeing. (A) The effect of applying dye paste multiple times to cotton fabric. (B) and (C) Biomass containing natural blue pigments can be used as a robust dyeing method.

[0024] Figure 13 — Comparison between the natural protein (BpsA_X5) and the designed protein (BpsA_X5-UNC). (A) Amino acid sequence alignment; red areas indicate identical amino acids; blue areas show similar amino acids. (B) Three-dimensional structure of the BpsA protein. Detailed Implementation

[0025] The invention is defined in the claims.

[0026] A first aspect of the invention provides a recombinant yeast cell capable of producing a natural blue pigment, wherein the cell is capable of expressing: a) A heterologous nonribosomal peptide synthase (NRPS) polypeptide capable of converting glutamine into natural blue pigment; and b) Heterogeneous phosphopantoylthioethylamine transferase (PPTase) peptide.

[0027] As those skilled in the art will understand, recombinant yeast cells may be able to express NRPS and PPTase via corresponding RNA and / or DNA nucleic acids.

[0028] Therefore, those skilled in the art will understand that the cell comprises: a) Nucleic acids containing a nucleotide sequence encoding a heterologous NRPS polypeptide; and b) Nucleic acids containing a nucleotide sequence encoding a heterologous PPTase polypeptide; and Among them, NRPS peptide and PPTase peptide can be expressed from nucleic acids.

[0029] Nucleic acids can be either DNA or RNA.

[0030] Importantly, the nucleic acids are arranged such that NRPS and PPTase peptides can be expressed from said nucleic acids, for example, when the nucleic acid is DNA, the nucleic acid can be operatively associated with a suitable promoter, as discussed elsewhere herein.

[0031] Nucleic acid molecules containing sequences encoding NRPS polypeptides can be identical to nucleic acid molecules containing sequences encoding PPTases. i.e. Both polypeptides are expressed from the same nucleic acid molecule. For example, each sequence may be located on the same chromosome in the genome, or on the same vector (such as a plasmid). In other embodiments, the sequence encoding NRPS and the sequence encoding PPTase are located on different nucleic acid molecules. For example, each sequence may be located in a different region of the genome, in different vectors (such as plasmids), or both.

[0032] Therefore, in some embodiments of recombinant yeast cells, The NRPS peptide can be any NRPS peptide, as long as it is heterologous to the host yeast cell. In some embodiments, the NRPS peptide is a naturally occurring or wild-type peptide. In other embodiments, the NRPS peptide is a non-naturally occurring or engineered peptide that contains one or more mutations, substitutions, or deletions relative to a naturally occurring or wild-type peptide.

[0033] In some embodiments, the heterologous NRPS peptide is: a) BpsA; arbitrarily chosen: i) Streptomyces lavendulae BpsA; or ii) Synthesized BpsA; b)IndC; optionally Streptomyces chromofuscus IndC; or c)IndB; optionally Streptomyces chromofuscus IndB.

[0034] In some embodiments, the heterologous NRPS peptide: a) Contains or consists of the following: an amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; or an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; and / or b) Encoded by the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 7; or the same nucleotide sequence as SEQ ID NO: 5 or SEQ ID NO: 7 by at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0035] The PPTase peptide can be any PPTase peptide, as long as it is heterologous to the host yeast cell. In some embodiments, the PPTase peptide is a naturally occurring or wild-type peptide. In other embodiments, the PPTase peptide is a non-naturally occurring or engineered peptide that contains one or more mutations, substitutions, or deletions relative to a naturally occurring or wild-type peptide.

[0036] In some embodiments, heterologous PPTase peptides are E. Coli PPTase or Bacillus subtilis Yarrowia PPTase.

[0037] In some embodiments, heterologous PPTase peptides: a) Containing an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 17, or SEQ ID NO: 19; or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 17, or SEQ ID NO: 18; and / or b) Encoded by a nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 16 or SEQ ID NO: 18; or encoded by a nucleic acid having sequence identity with SEQ ID NO: 2, SEQ ID NO: 16 or SEQ ID NO: 18 of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.

[0038] In some embodiments, the recombinant yeast cells are further capable of expressing or overexpressing glutamine dehydrogenase (GDH) peptides and / or glutamine synthase (GS) peptides. Each of the glutamine dehydrogenase (GDH) peptide and / or glutamine synthase (GS) peptide can be endogenous or heterologous.

[0039] In some preferred embodiments, recombinant yeast cells overexpress endogenous GDH. In the same or different embodiments, recombinant yeast cells overexpress endogenous GS.

[0040] In some preferred embodiments, recombinant yeast cells overexpress heterologous GDH. In the same or different embodiments, recombinant yeast cells overexpress heterologous GS.

[0041] In some preferred embodiments, recombinant yeast cells overexpress endogenous GDH. In the same or different embodiments, recombinant yeast cells overexpress heterologous GS.

[0042] In some preferred embodiments, recombinant yeast cells overexpress heterologous GDH. In the same or different embodiments, recombinant yeast cells overexpress endogenous GS.

[0043] In some embodiments, the recombinant yeast cell comprises: a) engineered nucleic acids containing a nucleotide sequence encoding a GDH polypeptide; and / or b) Engineered nucleic acids containing nucleotide sequences encoding GS polypeptides; and Among them, GDH peptide and GS peptide can be expressed from nucleic acids.

[0044] As will be understood, a nucleic acid may be referred to as "engineered" if the nucleotide sequence contained within it has been altered or edited, for example, by introducing a foreign nucleotide sequence into the nucleic acid or by deleting a nucleotide sequence from the nucleic acid. In some embodiments, engineered nucleic acids are synthetic nucleic acids. In some embodiments, engineered nucleic acids are heterologous nucleic acids not naturally contained in yeast cells. Heterologous nucleic acids are known to those skilled in the art and include nucleic acids selected from or consisting of the group consisting of plasmids, phage particles, yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC).

[0045] As described above, any one or more proteins or peptides can be expressed from a single nucleic acid molecule (e.g., a single vector) or from separate vectors. For example, a nucleic acid molecule containing a sequence encoding a GDH peptide can be the same as a nucleic acid molecule containing a sequence encoding GS—that is, both peptides are expressed from the same nucleic acid molecule. For example, each sequence can be located on the same chromosome in the genome or on the same vector (such as a plasmid). In other embodiments, the sequence encoding GDH and the sequence encoding GS are located on different nucleic acid molecules. For example, each sequence can be located in different regions of the genome, in different vectors (such as plasmids), or both.

[0046] In some embodiments, a single nucleic acid molecule may contain sequences encoding any one or more of the following: GDH, GS, PPTase, and / or NRPS.

[0047] As also stated above, the nucleic acid containing the sequence encoding any one or more of these polypeptides must be able to express the polypeptide from the nucleic acid, that is, be able to transcribe and / or translate from the nucleic acid.

[0048] In some preferred embodiments, NRPS peptides, PPTase peptides, GDH peptides, and / or GS peptides are overexpressed in cells. Those skilled in the art will understand the meaning of "overexpression" and the ways in which this overexpression is achieved. For example, the peptide may be expressed from a strong promoter, as described herein; or it may be expressed from a high-copy plasmid.

[0049] In some embodiments, the GDH peptide and / or GS peptide are endogenous GDH and / or GS peptides—that is, naturally occurring GS and GDH peptides in recombinant yeast cells.

[0050] In some embodiments, the GDH polypeptide is encoded by an open reading frame (ORF) defined by YALI0E09603g or YALI0F17820g. The nucleic acid sequences of these ORFs can be found in many public databases such as GRYC (infra.fr), NCBI (nih.gov), Uniprot (uniport.org), and KEGG (genome.jp / kegg / ).

[0051] In some embodiments, the GDH peptide: a) Containing an amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21; or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 20 or SEQ ID NO: 21; and / or b) Encoded by a nucleotide sequence of SEQ ID NO: 11 or SEQ ID NO: 12; or encoded by a nucleic acid having sequence identity with SEQ ID NO: 11 or SEQ ID NO: 12 of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0052] In some embodiments, the GS peptide is encoded by an open reading frame defined by YALI0D13024g or YALI0F00506g.

[0053] The GS polypeptide may contain the amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23; or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 22 or SEQ ID NO: 23; and / or It may be encoded by a nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 10; or by a nucleic acid having a sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 9 or SEQ ID NO: 10.

[0054] As described above, in each case, the nucleic acid encoding the desired polypeptide must be able to express that polypeptide. For example, those skilled in the art will know that heterologous NRPS polypeptides, heterologous PPTase polypeptides, GDH polypeptides, and / or GS polypeptides must each be operatively linked to a promoter. In some cases, the same promoter can drive the expression of more than one polypeptide, but preferably and more generally, each polypeptide is expressed from a nucleic acid operatively linked to a separate promoter.

[0055] Those skilled in the art can select the most suitable promoter for a specific situation. For example, in some embodiments, the promoter is: a) Constitutive promoters; or b) Inducible promoters.

[0056] Those skilled in the art also know that different expression intensities can be obtained from different promoters. For example, those skilled in the art understand the concept of a “strong” promoter, and in some embodiments, the promoter is a strong promoter.

[0057] In some embodiments, the promoter driving the expression of any of the polypeptides described herein is selected from or grouped with the following: a) A constitutive promoter selected from the group consisting of or including the following: ATP1 promoter [SEQ ID NO: 24], ATP2 promoter [SEQ ID NO: 25], FBAin promoter [SEQ ID NO: 26], PGK1 promoter [SEQ ID NO: 27], GPM1 promoter [SEQ ID NO: 28], HHF1 promoter [SEQ ID NO: 29], CYC1 promoter [SEQ ID NO: 30], HHT1 promoter [SEQ ID NO: 31], HTB1 promoter [SEQ ID NO: 32], EXP1 promoter [SEQ ID NO: 33], TDH1 promoter [SEQ ID NO: 34], RPL25 promoter [SEQ ID NO: 35], TEF1 promoter [SEQ ID NO: 36], TEFin promoter [SEQ ID NO: 37], TEF2UAS promoter [SEQ ID NO: 38], CYC1 promoter [SEQ ID NO: 39], HHT1 promoter [SEQ ID NO: 30], HHT1 promoter [SEQ ID NO: 31], HTB1 promoter [SEQ ID NO: 32], EXP1 promoter [SEQ ID NO: 33], TDH1 promoter [SEQ ID NO: 34], RPL25 promoter [SEQ ID NO: 35], TEF1 promoter [SEQ ID NO: 36], TEFin promoter [SEQ ID NO: 37], TEF2UAS promoter [SEQ ID NO: 38], CYC1 promoter [SEQ ID NO: 39], CYC1 promoter [SEQ ID NO: 30], CYC1 promoter [SEQ ID NO: 30], CYC1 promoter [SEQ ID NO: 36], CYC1 promoter [SEQ ID NO: 37], CYC2UAS promoter [SEQ ID NO: 38], CYC2UAS promoter [SEQ ID NO: 39], CYC2UAS promoter [SEQ ID NO: 30], CYC2UAS promoter [ 38], TEF4UAS bootloader [SEQ ID NO: 39] and TEF8UAS bootloader [SEQ ID NO: 40], pTEF bootloader [SEQ ID NO: 13 and 14], GAP bootloader [SEQ ID NO: 57], or b) Inducible promoters selected from the group consisting of or including the following: pPOX2 promoter [SEQ ID NO: 41], pXPR2 promoter [SEQ ID NO: 42], pFBP1 promoter [SEQ ID NO: 43], pMDH1a promoter [SEQ ID NO: 44], ACL2 promoter [SEQ ID NO: 45], XPR2 promoter [SEQ ID NO: 58], POT1 promoter [SEQ ID NO: 46], LIP2 promoter [SEQ ID NO: 47], ICL promoter [SEQ ID NO: 48], YAT1 promoter [SEQ ID NO: 49], CTR1 promoter [SEQ ID NO: 50], CTR2 promoter [SEQ ID NO: 51], pYALI0B18194 promoter [SEQ ID NO: 52], pYALI0C11165 promoter [SEQ ID NO: 52], pYALI0C11165 promoter [SEQ ID NO: 52], pYALI0B18194 ...

[53] , pYALI0C15004 promoter [SEQ ID NO: 54], pYALI0E14256 promoter [SEQ ID NO: 55] and pYALI0F13937 promoter [SEQ ID NO: 56].

[0058] These promoters are described in “A DNA assembly toolkit to unlock the CRISPR / Cas9 potential for metabolic engineering” (version 1), published on April 4, 2023, by Tigran Yuzbashev, Evgeniya Yuzbasheva, Olga Melkina, Davina Patel, Dmitrii Bubnov, Heiko Dietz, and Rodrigo Ledesma-Amaro. See https: / / www.researchsquare.com / article / rs-2738543 / v1 for details.

[0059] In some preferred embodiments, the promoter is a pTEF promoter; preferably, the pTEF promoter has the nucleotide sequence of SEQ ID NO: 13 or SEQ ID NO: 14.

[0060] Those skilled in the art will also understand that an appropriate terminator sequence is required for proper protein expression. Therefore, the nucleotide sequence encoding a heterologous NRPS peptide, a heterologous PPTase peptide, a GDH peptide, and / or a GS peptide is operatively linked to a terminator sequence. In some embodiments, the terminator sequence is a tLIP2 terminator; for example, a tLIP2 terminator having the nucleotide sequence of SEQ ID NO: 15.

[0061] Those skilled in the art will understand that nucleic acids expressing desired proteins (such as NRPS, PPTase, GS, and GDH described herein) can be expressed at chromosomal locations or extrachromosomally, for example, from plasmids.

[0062] In some embodiments, nucleic acids comprising nucleotide sequences encoding NRPS peptides, PPTases, GDH peptides, and / or GS peptides are integrated into the genome of a cell. Methods for integrating nucleic acids into the genome of a cell are known to those skilled in the art. For example, nucleic acids can be appropriately integrated into the genome of a cell via homologous recombination-targeted integration or random genomic integration.

[0063] In some embodiments, nucleic acids comprising nucleotide sequences encoding NRPS peptides, PPTases, GDH peptides, and / or GS peptides are maintained in free form, for example, on one or more plasmids.

[0064] In some embodiments, the endogenous form of any one or more of these genes has been deleted from the host recombinant yeast cell. By "deletion," we mean both complete gene deletion and functional loss of function, where the gene is still present but carries one or more mutations / insertions / deletions, meaning that no functional GDH and / or GS proteins are produced.

[0065] For example, in some embodiments, the recombinant yeast cells contain the deletion of the endogenous chromosomal GDH and / or GS genes.

[0066] In the same or different embodiments, the recombinant yeast cells contain the deletion of at least one gene selected from the group consisting of or composed of open reading frames selected from the group consisting of: YALI0E09603g, YALI0F17820g, YALI0D13024g, YALI0F00506g, YALI0E18634g, YALI0B00572g and / or YALI0F01606g or any combination thereof.

[0067] Codon optimization is the process of modifying protein-coding sequences to take full advantage of codon bias in a given organism. In some embodiments, the nucleotide sequences encoding NRPS peptides and / or PPTase peptides and / or, when present, encoding GDH and / or GS, are codon-optimized for expression in yeast, for example, having been specifically optimized for expression in yeast. Yarrowia Rhodosporidium Chinese expression.

[0068] The recombinant yeast cells of the present invention can belong to any genus or species. Preferably, the recombinant yeast cells are oil-producing yeast cells.

[0069] In some embodiments, oil-producing yeast cells are not Rhodosporidium toruloides Cells, for example, not for Yarrowia cell.

[0070] Preferably, yeast cells are Yarrowia Cells. In some embodiments, Yarrowia bubula Cells can be selected from the following or a group consisting of the following: Yarrowia deformans cell, Yarrowia lipolytica Yarrowia porcina cell, Yarrowia yakushimensis cell, Yarrowia parophonii Yarrowia galli cell, Yarrowia oslonensis cell, Yarrowia alimentaria Yarrowia hollandica cell, Yarrowia phangngaensis cell, Yarrowia Yarrowia lipolytica cell, Yarrowia lipolytica cell, Candidalipolytica Cells and Yarrowia lipolytica cell; Preferably, recombination Candida Cells are lipolytica cell.

[0071] As is known in the art, Komagaeibacter The previous category name was Escherichia Gluconacetobacter Therefore, the classification names used in this article... Acetobacter and Sarcina They are interchangeable.

[0072] The aforementioned recombinant yeast cells are capable of producing natural blue pigment. Therefore, it is evident that the present invention also provides a method for producing natural blue pigment using the recombinant yeast cells of the present invention.

[0073] Therefore, the present invention also provides a method for producing natural blue pigment, the method comprising culturing the recombinant yeast cells of the present invention.

[0074] For example, methods for producing natural blue pigments include The recombinant yeast cells of the present invention express heterologous nonribosomal peptide synthase (NRPS) polypeptides and heterologous phosphopantoylthioethylamine transferase (PPTase) polypeptides capable of converting glutamine into natural blue pigment. The expression of glutamine leads to the conversion of glutamine into a natural blue pigment, thereby producing a natural blue pigment.

[0075] It is evident that this method requires culturing cells under conditions suitable for expressing the NRPS peptide and PPTase peptide.

[0076] Recombinant yeast cells can be cultured in any culture medium. In some embodiments, the cells are cultured in a culture medium selected from: a) Enriched culture medium, optionally YPD or YP4D; b) Basic culture medium, optionally YNB containing glucose or YNB containing glycerol; or c) Superbasic culture medium; optionally, wherein the superbasic culture medium is SM1, SM2, SM3, SM4 or SM5.

[0077] For example, in some embodiments, the culture medium is selected from or grouped with the following: YP4G medium, YNB glucose medium and YNB glycerol medium, optionally YNB glucose medium or YNB glycerol medium.

[0078] Those skilled in the art will readily understand what these yeast culture media are, but in some embodiments, they are briefly described below: A) YP4G medium contains: [or YP4D medium containing dextrose instead of the following glucose] Yeast extract: 10 g / L Peptone: 20 g / L Glucose: 40 g / L B) YNB glucose medium contains: -YNB without amino acids: 6.8 g / L - Phosphate buffer 500mM pH 6.8: 100 mL / L - Glucose: 20 g / L C) YNB glycerol medium contains: -YNB without amino acids: 6.8 g / L - Phosphate buffer 500mM pH 6.8: 100 mL / L - Glycerin: 20 g / L D) SM1 superbasic culture medium contains: - 18 g / L urea - 20 g / L glycerin E) SM2 superbasic culture medium contains: - 18 g / L urea - 25 g / L crude glycerol (which is waste (crude) glycerol and is produced as a byproduct of biodiesel and soap production processes); composition: 38 to 96% glycerol (80% in this study), up to 50% methanol, 13% soap, 2% water, 2 to 3% salt. F) SM3 superbasic culture medium contains: - 18 g / L urea - 25 g / L crude glycerol - 0.17 g / L YNB G) SM4 superbasic culture medium contains: - 18 g / L urea - 25 g / L crude glycerol - 1.2 g / L K2PO4 H) SM5 superbasic culture medium contains: - 18 g / L urea - 25 g / L crude glycerol - 1 g / L MnSO4 I) YNB culture medium contains: - YNB without amino acids: 6.8 g / L - Phosphate buffer 500mM pH 6.8: 100 mL / L J) YPD medium contains: 10 g / L yeast extract 20 g / L peptone 20 g / L glucose Typically, a culture medium contains: a) a carbon source; b) a nitrogen source; and / or c) water. Suitable carbon sources, nitrogen sources, and water for culture media are known to those skilled in the art.

[0079] The inventors have surprisingly discovered that the carbon-to-nitrogen ratio (C / N) affects the synthesis of natural blue pigments. Therefore, in some embodiments, the recombinant yeast cells of this invention are cultured in a medium with a C / N ratio of: a) Between 2 and 160, 2 and 150, 2 and 140, 2 and 130, 2 and 120, 2 and 110, 2 and 100, 2 and 90, 2 and 80, 2 and 70, 2 and 60, 2 and 50, 2 and 40, 2 and 30, 2 and 20, 2 and 10, etc. 2 and John 9, John 2 and John 8, John 2 and John 7, John 2 and John 6, John 2 and John 5, John 2 and John 4, John 2 and John 3, John 4 and John 160, John 4 and John 150, John 4 and John 140, John 4 and John 130, John 4 and John 140, John 4 and John 110, John 4 and John 100, John 4 and John 90, John 4 and John 80, John 4 and John 70, John 4 and John 60, John 4 and John 50, John 4 and John 40, John 4 and John 30, John 4 and John 20, John 4 and John 10, John 4 and John 9, John 4 and John 8, John 4 and John 7, John 4 and John 6, John 4 and John 5, John 6 and John 160, John 6 and John 150, John 6 and John 140, John 6 and John 130, John 6 and John 160, John 6 and John 110, John 6 and John 100, John 6 and John 90, John 6 and John 80, John 6 and John 70, John 6 and John 60, John 6 and John 50, John 6 and John 40, John 6 and John 30, John 6 and John 20, John 6 and John 10, John 6 and John 9, John 6 and John 8, John 6 and John 7, John 8 and John 160, John 8 and John 150, John 8 and John 140, John 8 and John 130, John 8 and John 180, John 8 and John 110, John 8 and John 100, John 8 and John 90, John 8 and John 80, John 8 and John 70, John 8 and John 60, John 8 and John 50, John 8 and John 40, John 8 and John 30, John 8 and John 20, John 8 and John 10, John 8 and John 9, John 8 and John 8, John 8 and John 7, John 8 and John 6, John 8 and John 5, John 8 and John 4, John 8 and John 3, John 10 and John 160, John 10 With about 150, about 10 with about 140, about 10 with about 130, about 10 with about 1100, about 10 with about 110, about 10 with about 100, about 10 with about 90, about 10 with about 80, about 10 with about 70, about 10 with about 60, about 10 with about 50, about 10 with about 40, about 10 with about 30, about 10 with about 20, about 20 with about 160, Approximately 20 and approximately 150, approximately 20 and approximately 140, approximately 20 and approximately 130, approximately 20 and approximately 1200, approximately 20 and approximately 110, approximately 20 and approximately 100, approximately 20 and approximately 90, approximately 20 and approximately 80, approximately 20 and approximately 70, approximately 20 and approximately 60, approximately 20 and approximately 50, approximately 20 and approximately 40, approximately 20 and approximately 30, approximately 30 and approximately 160, approximately 30 and approximately 150, about 30 and about 140, about 30 and about 130, about 30 and about 1300, about 30 and about 110, about 30 and about 100, about 30 and about 90, about 30 and about 80, about 30 and about 70, about 30 and about 60, about 30 and about 50, about 30 and about 40, about 40 and about 160, about 40 and about 150, about 40 and about 140,Between approximately 40 and approximately 130, approximately 40 and approximately 1400, approximately 40 and approximately 110, approximately 40 and approximately 100, approximately 40 and approximately 90, approximately 40 and approximately 80, approximately 40 and approximately 70, approximately 40 and approximately 60, approximately 40 and approximately 50, approximately 50 and approximately 160, approximately 50 and approximately 150, approximately 50 and approximately 140, approximately 50 and approximately 130, approximately 50 and approximately 150, approximately 50 and approximately 110, approximately 50 and approximately 100, approximately 50 and approximately 90, approximately 50 and approximately 80, approximately 50 and approximately 70, or approximately 50 and approximately 60; b) Between 2 and 160, 2 and 150, 2 and 140, 2 and 130, 2 and 120, 2 and 110, 2 and 100, 2 and 90, 2 and 80, 2 and 70, 2 and 60, 2 and 50, 2 and 40, 2 and 30, 2 and 20, 2 and 10, 2 and 9, 2 and 8, 2 and 7, 2 and 6, 2 and 5, 2 and 4, 2 and 3, 4 and 160, 4 and 150, 4 and 140, 4 and 130, 4 and 140, 4 and 110, 4 and 100, 4 and 90, 4 and 80, 4 and 70, 4 and 60, 4 and 50, 4 and 40, 4 and 30, 4 and 20, 4 and 10, 4 and 9, 4 and 8, 4 and 7 4 and 6, 4 and 5, 6 and 160, 6 and 150, 6 and 140, 6 and 130, 6 and 160, 6 and 110, 6 and 100, 6 and 90, 6 and 80, 6 and 70, 6 and 60, 6 and 50, 6 and 40, 6 and 30, 6 and 20, 6 and 10, 6 and 9, 6 and 8, 6 and 7, 8 and 160, 8 and 150, 8 and 140, 8 and 130, 8 and 180, 8 and 110, 8 and 100, 8 and 90, 8 and 80, 8 and 70, 8 and 60, 8 and 50, 8 and 40, 8 and 30, 8 and 20, 8 and 10, 8 and 9, 8 and 8, 8 and 7, 8 and 6, 8 and 5, 8 and 4 8 and 3, 10 and 160, 10 and 150, 10 and 140, 10 and 130, 10 and 1100, 10 and 110, 10 and 100, 10 and 90, 10 and 80, 10 and 70, 10 and 60, 10 and 50, 10 and 40, 10 and 30, 10 and 20, 20 and 160, 20 and 150, 20 and 140, 20 and 130, 20 and 1200, 20 and 110, 20 and 100, 20 and 90, 20 and 80, 20 and 70, 20 and 60, 20 and 50, 20 and 40, 20 and 30, 30 and 160, 30 and 150, 30 and 140, 30 Between 130, 30 and 1300, 30 and 110, 30 and 100, 30 and 90, 30 and 80, 30 and 70, 30 and 60, 30 and 50, 30 and 40, 40 and 160, 40 and 150, 40 and 140, 40 and 130, 40 and 1400, 40 and 110, 40 and 100, 40 and 90, 40 and 80, 40 and 70, 40 and 60, 40 and 50, 50 and 160, 50 and 150, 50 and 140, 50 and 130, 50 and 150, 50 and 110, 50 and 100, 50 and 90, 50 and 80, 50 and 70 or 50 and 60; c) At least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150 or more; d) Less than approximately 160, less than approximately 150, less than approximately 140, less than approximately 130, less than approximately 120, less than approximately 110, less than approximately, less than approximately 100, less than approximately 90, less than approximately 80, less than approximately 70, less than approximately 60, less than approximately 50, less than approximately 40, less than approximately 30, less than approximately 20, less than approximately 10, less than approximately 9, less than approximately 8, less than approximately 7, less than approximately 6, less than approximately 5, less than approximately 4, less than approximately 3; and / or e) At least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150 or more; f) Less than 160, less than 150, less than 140, less than 130, less than 120, less than 110, less than approximately, less than 100, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3; g) Approximately 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, approximately 8, approximately 9, approximately 10, approximately 20, approximately 30, approximately 40, approximately 50, approximately 60, approximately 70, approximately 80, approximately 90, approximately 100, approximately 110, approximately 120, approximately 130, approximately 140, approximately 150; and / or h) 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160.

[0080] Preferably, the C / N ratio is between the following values: a) about 4 and about 20; and / or b) 4 and 20.

[0081] Or: c) 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about or 20; and / or d) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0082] The carbon source can be any carbon source. Preferably, the carbon source is not derived from food ingredients, but from waste products, or from raw materials specifically grown for the purpose of producing natural blue pigment.

[0083] In some embodiments, the carbon source is selected from or the group consisting of glucose, fructose, mannose, citrate, acetate, rapeseed oil, sunflower oil, palm oil, waste cooking oil, rapeseed oil and sunflower oil, lignocellulose hydrolysate, glycerol and / or waste glycerol. In some embodiments, the lignocellulose hydrolysate is selected from or the group consisting of hydrolyzed flaxseed bran, chili peppers, vegetable mixtures and urban trimmings or any combination thereof.

[0084] In a preferred embodiment, the carbon source is a lignocellulose hydrolysis product.

[0085] The water can be any kind of water. Similar to the raw materials, there are certain environmental and cost advantages if the water used to cultivate the yeast does not need to be purified or refined.

[0086] In some embodiments, the water is selected from the group consisting of seawater, tap water, and distilled water; or any combination thereof.

[0087] The nitrogen source can be any nitrogen source. In some embodiments, the nitrogen source is selected from or grouped with the following: urine, synthetic urine, urea, ammonium chloride, peptone, and ammonium sulfate, or any combination thereof. In some embodiments, the synthetic urine contains 18.018 g / L urea, 1.96 g / L creatine, 1.44 g / L citric acid, 0.28 g / L glycine, 18.76 g / L sodium nitrate, 1.36 g / L hippuric acid, and 0.48 g / L cysteine.

[0088] In some embodiments, the nitrogen source is ammonium sulfate.

[0089] Yeast can be cultured in solid (agar) medium or in liquid medium. Preferably, the method for producing natural blue pigment includes culturing the recombinant yeast cells of the present invention in a liquid medium. Preferably, the liquid culture is cultured in a shaking incubator under agitation (such as shaking). The inventors have found that increasing the agitation speed during culture affects the yield of natural blue pigment.

[0090] In some embodiments, the culture includes stirring a liquid culture medium during cell growth; for example, the liquid culture medium is stirred at the following speeds: a) Between approximately 150 and approximately 2000 RPM, approximately 150 and approximately 250 RPM, approximately 150 and approximately 200 RPM, approximately 200 and approximately 250 RPM, approximately 150 and approximately 800 RPM, approximately 200 and approximately 800 RPM, approximately 250 and approximately 800 RPM, approximately 800 and approximately 2000 RPM, approximately 1000 and approximately 1800 RPM, approximately 1200 and approximately 1400 RPM; b) Between 150 and 2000 RPM, 150 and 250 RPM, 150 and 200 RPM, 200 and 250 RPM, 150 and 800 RPM, 200 and 800 RPM, 250 and 800 RPM, 800 and 2000 RPM, 1000 and 1800 RPM, and 1200 and 1400 RPM; c) At least 150 RPM, at least 200 RPM, at least 250 RPM, at least 800 RPM, at least 1000 RPM, at least 1200 RPM, at least 1400 RPM, at least 1600 RPM, at least 1800 RPM; d) Less than 2000 RPM, less than 1800 RPM, less than 1600 RPM, less than 1400 RPM, less than 1200 RPM, less than 1000 RPM, less than 800 RPM, less than 250 RPM, or less than 200 RPM; c) Approximately 150 RPM, approximately 200 RPM, approximately 250 RPM, approximately 800 RPM, approximately 1000 RPM, approximately 1200 RPM, approximately 1400 RPM, approximately 1600 RPM, approximately 1800 RPM, or approximately 2000 RPM; or d) 150 RPM, 200 RPM, 250 RPM, 800 RPM, 1000 RPM, 1200 RPM, 1400 RPM, 1600 RPM, 1800 RPM or 2000 RPM.

[0091] The inventors have also discovered that supplementing the culture medium with glutamine and / or glutamate increases the yield of natural blue pigment. Therefore, in some embodiments, the culture medium is supplemented with glutamine and / or glutamate, for example, supplemented with: Glutamine, its concentration is: i) Between approximately 1 mm and approximately 100 mm, approximately 2 mm and approximately 90 mm, approximately 3 mm and approximately 80 mm, approximately 4 mm and approximately 70 mm, approximately 5 mm and approximately 60 mm, approximately 6 mm and approximately 50 mm, approximately 7 mm and approximately 40 mm, approximately 8 mm and approximately 30 mm, approximately 9 mm and approximately 20 mm, approximately 10 mm and approximately 15 mm, approximately 1 mm and approximately 10 mm, approximately 2 mm and approximately 9 mm, approximately 3 mm and approximately 8 mm, approximately 4 mm and approximately 7 mm, approximately 5 mm and approximately 6 mm Between mM, approximately 10mM and approximately 100mM, approximately 20mM and approximately 90mM, approximately 30mM and approximately 80mM, approximately 40mM and approximately 70mM, approximately 50mM and approximately 60mM, approximately 50mM and approximately 150mM, approximately 60mM and approximately 140mM, approximately 70mM and approximately 130mM, approximately 80mM and approximately 120mM, or approximately 90mM and approximately 110mM glutamine; ii) Between 1mM and 100mM, 2mM and 90mM, 3mM and 80mM, 4mM and 70mM, 5mM and 60mM, 6mM and 50mM, 7mM and 40mM, 8mM and 30mM, 9mM and 20mM, 10mM and 15mM, 1mM and 10mM, 2mM and 9mM, 3mM and 8mM, 4mM and 7mM, 5mM and 6mM, 10 Between mM and 100mM, 20mM and 90mM, 30mM and 80mM, 40mM and 70mM, 50mM and 60mM, 50mM and 150mM, 60mM and 140mM, 70mM and 130mM, 80mM and 120mM, or 90mM and 110mM glutamine; iii) At least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, at least 10 mM, at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 110 mM, at least 120 mM, at least 130 mM, at least 140 mM, at least 150 mM or more glutamine; iv) Less than 150mM, less than 140mM, less than 130mM, less than 120mM, less than 120mM, less than 110mM, less than 100mM, less than 90mM, less than 80mM, less than 70mM, less than 60mM, less than 50mM, less than 40mM, less than 30mM, less than 20mM, less than 10mM, less than 9mM, less than 8mM, less than 7mM, less than 6mM, less than 5mM, less than 4mM, less than 3mM, less than 2mM or less glutamine; v) Approximately 1 mM, approximately 2 mM, approximately 3 mM, approximately 4 mM, approximately 5 mM, approximately 6 mM, approximately 7 mM, approximately 8 mM, approximately 9 mM, approximately 10 mM, approximately 20 mM, approximately 30 mM, approximately 40 mM, approximately 50 mM, approximately 60 mM, approximately 70 mM, approximately 80 mM, approximately 90 mM, approximately 100 mM, approximately 110 mM, approximately 120 mM, approximately 130 mM, approximately 140 mM, or approximately 150 mM glutamine; and / or vi) 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM or 150mM glutamine; and / or It supplements with glutamic acid, at the following concentration: i) Between approximately 1 mm and approximately 100 mm, approximately 2 mm and approximately 90 mm, approximately 3 mm and approximately 80 mm, approximately 4 mm and approximately 70 mm, approximately 5 mm and approximately 60 mm, approximately 6 mm and approximately 50 mm, approximately 7 mm and approximately 40 mm, approximately 8 mm and approximately 30 mm, approximately 9 mm and approximately 20 mm, approximately 10 mm and approximately 15 mm, approximately 1 mm and approximately 10 mm, approximately 2 mm and approximately 9 mm, approximately 3 mm and approximately 8 mm, approximately 4 mm and approximately 7 mm, approximately 5 mm and approximately 6 mm Between mM, approximately 10mM and approximately 100mM, approximately 20mM and approximately 90mM, approximately 30mM and approximately 80mM, approximately 40mM and approximately 70mM, approximately 50mM and approximately 60mM, approximately 50mM and approximately 150mM, approximately 60mM and approximately 140mM, approximately 70mM and approximately 130mM, approximately 80mM and approximately 120mM, or approximately 90mM and approximately 110mM glutamate; ii) Glutamate between 1mM and 100mM, 2mM and 90mM, 3mM and 80mM, 4mM and 70mM, 5mM and 60mM, 6mM and 50mM, 7mM and 40mM, 8mM and 30mM, 9mM and 20mM, 10mM and 15mM, 1mM and 10mM, 2mM and 9mM, 3mM and 8mM, 4mM and 7mM, 5mM and 6mM, 10mM and 100mM, 20mM and 90mM, 30mM and 80mM, 40mM and 70mM, 50mM and 60mM, 50mM and 150mM, 60mM and 140mM, 70mM and 130mM, 80mM and 120mM, or 90mM and 110mM; iii) At least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, at least 10 mM, at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 110 mM, at least 120 mM, at least 130 mM, at least 140 mM, at least 150 mM or more glutamate; iv) Less than 150mM, less than 140mM, less than 130mM, less than 120mM, less than 120mM, less than 110mM, less than 100mM, less than 90mM, less than 80mM, less than 70mM, less than 60mM, less than 50mM, less than 40mM, less than 30mM, less than 20mM, less than 10mM, less than 9mM, less than 8mM, less than 7mM, less than 6mM, less than 5mM, less than 4mM, less than 3mM, less than 2mM or less glutamate; v) Approximately 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, or 150 mM glutamate; and / or vi) 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM or 150mM glutamate.

[0092] In some embodiments of the recombinant yeast cells of the present invention, the yeast cells express endogenous glutamate dehydrogenase (GDH) peptides and / or endogenous glutamine synthase (GS) peptides. In some embodiments, GDH and GS may be heterologous to the yeast cells. In some preferred embodiments, the expression of GDH and / or GS is overexpression, for example via a strong promoter or via expression from a multicopy plasmid.

[0093] Methods for producing natural blue pigment may include steps related to the extraction or purification of natural blue pigment from cells or cell cultures. For example, in some embodiments, the extraction and / or purification of natural blue pigment from cells or cell cultures includes cell lysis. Cell lysis can be performed in any manner. In some embodiments, cells are lysed by homogenization in DMSO. Such methods are known and, in some embodiments, involve the use of glass beads, cell disruption at a rotation speed of 8,000 rpm, and centrifugation.

[0094] Yeast cells can be collected from the culture by centrifugation or filtration, and in some embodiments, they are dried before being lysed by grinding.

[0095] Other methods and uses of the recombinant yeast cells of the present invention will be apparent to those skilled in the art and constitute a part of the present invention.

[0096] For example, this invention provides the use of the recombinant yeast cells of this invention in a method for producing natural blue pigment. This method can be the method for producing natural blue pigment described herein.

[0097] The present invention also provides a natural blue pigment, which is obtained or available from the recombinant yeast cells of the present invention.

[0098] The present invention also provides a natural blue pigment, which is obtained or can be obtained by the method of producing natural blue pigment of the present invention.

[0099] As described elsewhere in this document, the inventors have discovered that the natural blue pigment produced by the recombinant yeast cells of the present invention can be used to produce naturally blue pigment-stained bacterial cellulose through co-culture of cellulose-producing bacteria and the recombinant yeast cells of the present invention or through a stepwise separation culture process.

[0100] For example, the present invention provides a method for producing bacterial cellulose or bacterial cellulose films stained with natural blue pigment, wherein the method comprises co-culturing the recombinant yeast cells of the present invention with cellulose-producing second cells.

[0101] The second cell is a bacterial cell, and: a) It can produce bacterial cellulose; b) Express all of bcsA, bcsD, bscC, and bscD; c) Belongs to a genus selected from the group consisting of or including the following: Agrobacterium Azotobacter , Rhizobium , Pseudomonas , Salmonella Alcaligenes , Komagaeibacter , rhaeticus ,Komagaeibacter xylinus Komagaeibacter , hansenii , Komagaeibacter medellinensis , Komagaeibacter europaeus ​ and ​ ; d) Selected from the following or a group consisting of the following: ​ ​ ; ​ , ​ ​ , ​ , ​ (Komagaeibacter europaeus) , Maltaceaeibacter maltaceti , Letty Komagata Komagaeibacter pomaceti , Obedient to Komagaeibacter oboediens or sucrose Komagaeibacter saccharivoans ; e) Selected from or consisting of the following items: i) Selected from the group consisting of or including the following items Gerolactobacillus Strains: Gerolactobacillus iGEM; Gerolactobacillus AF1; Gerolactobacillus LMG22126; or ii) Xylomycin CGMCC 2995; and / or f) is Gerolactobacillus iGEM ​​cells.

[0102] In some embodiments, a method for producing bacterial cellulose or a cellulose film stained with natural blue pigment includes culturing recombinant yeast cells and cellulose-producing second cells under conditions that allow cellulose-producing second cells to produce cellulose or a cellulose film. In some embodiments, the resulting cellulose may be considered a film, and therefore in some embodiments, the conditions allow the cellulose-producing second cells to produce a film.

[0103] Exemplary conditions for the production of bacterial cellulose and bacterial cellulose films are described in WO 2023 / 285800, the entire contents of which are hereby incorporated by reference. Specifically, pages 13, lines 21-28; Example 1, page 88; Example 2, page 91, lines 37-92, lines 15, are hereby incorporated by reference.

[0104] For example, in some embodiments, conditions that allow the cellulose-producing second cell to produce cellulose or a cellulose film include culturing the cell at the following pH and in the following culture medium: a) pH is: i) pH between 3 and 7, optionally between 3.25 and 6.75, 3.5 and 6.5, 3.5 and 6.25, 3.75 and 6, 4 and 5.75, 4.25 and 5.5, 4.5 and 5.25; pH 5.8; and / or ii) At least 3 but less than or equal to pH 7, for example, at least 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 5.8, 6, 6.25, 6.5, 6.75, but less than or equal to pH 7; And / or b) The culture medium is: i) HS culture medium; ii) YPD medium; or iii) Coconut water culture medium.

[0105] The present invention also provides a method for producing bacterial cellulose dyed with natural blue pigment or a thin film dyed with natural blue pigment, the method comprising: a) Culturing cellulose-producing second cells to form bacterial cellulose and / or cellulose films; and subsequently b) Contact the cellulose or cellulose film produced by the cellulose-producing second cell in (a) with the recombinant yeast cell of the present invention and as described elsewhere herein.

[0106] The preference for cellulose-producing second cells is as described elsewhere in this article.

[0107] Our use of the term "contact" includes incubating the recombinant yeast cells of the present invention with cellulose or a film. As described herein, cellulose or a film can be incubated together with the recombinant yeast cells of the present invention under suitable conditions for culturing recombinant yeast cells.

[0108] Preferences for culture conditions for producing bacterial cellulose or cellulose films are described elsewhere in this article.

[0109] In some embodiments of the method for producing cellulose or films dyed with natural blue pigment, the cellulose or film may be sterilized, for example, wherein sterilization is selected from or grouped with the following: autoclaving, heating and drying, or any combination thereof.

[0110] Those skilled in the art will understand that the natural blue pigment produced by the recombinant yeast cells of the present invention can be used to dye textiles and other materials, including bacterial cellulose / films that can themselves be used as textiles. Therefore, in some embodiments, the present invention provides a method for producing textiles or threads dyed with natural blue pigment, the method comprising: a) Production of natural blue pigment according to the method of the present invention; as well as b) Contact the textile or thread with the natural blue pigment obtained in (a).

[0111] In some embodiments, as described elsewhere herein, the natural blue pigment produced by recombinant yeast cells is extracted and purified, for example, by cell lysis. Cell lysis can be performed in any manner. In some embodiments, cells are lysed by homogenization in DMSO. Such methods are known, and in some embodiments, involve the use of glass beads, cell disruption at 8,000 rpm, and centrifugation.

[0112] Yeast cells can be collected from the culture by centrifugation or filtration, and in some embodiments, they are dried before being lysed by grinding.

[0113] In some embodiments, contacting the textile or thread with the natural blue pigment obtained in (a) includes immersing the textile or thread in the natural blue pigment. In some embodiments, immersing the textile or thread in the dyeing mixture for the following durations: a) Between approximately 4 hours and approximately 12 hours, approximately 5 hours and approximately 11 hours, approximately 6 hours and approximately 10 hours, approximately 7 hours and approximately 9 hours; b) Between 4 hours and 12 hours, 5 hours and 11 hours, 6 hours and 10 hours, 7 hours and 9 hours; c) At least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 12 hours; d) Less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours; e) Approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours; and / or f) 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours.

[0114] In some embodiments, the textiles or threads are then subjected to the following treatment: c) Autoclaving; and / or d) Washing.

[0115] The present invention also provides bacterial cellulose stained with natural blue pigment, which is obtained or available from any of the methods of the present invention.

[0116] The present invention also provides a garment comprising bacterial cellulose dyed with the natural blue pigment of the present invention, a film dyed with the natural blue pigment, or a textile or thread dyed with the natural blue pigment.

[0117] The present invention also provides an expression construct comprising nucleic acid, the nucleic acid comprising: a) For example, the nucleotide sequence encoding the NRPS polypeptide described herein; b) For example, the nucleotide sequence encoding the PPTase polypeptide described herein; c) For example, the nucleotide sequence encoding the GDH polypeptide described herein; and / or d) For example, the nucleotide sequence encoding the GS polypeptide described herein; The nucleotide sequences encoding NRPS peptides, PPTase peptides, GDH peptides, and / or GS peptides are operatively linked to the promoter.

[0118] Expression constructs, including any associated sequences (such as promoters) or homologous regions required to guide homologous recombination integration into the genome, can be provided in the form of PCR products. Right now It is amplified from the template vector.

[0119] Preferences for nucleic acids, NRPS, PPTase, GDH, and GS, as well as any associated promoters and terminators, are described elsewhere in this article.

[0120] The present invention also provides a vector comprising the expression construct of the present invention. The vector can be any vector, for example, selected from or consisting of plasmids, phage particles, YAC, and BAC.

[0121] The present invention also provides an engineered nonribosomal peptide synthase (NRPS) polypeptide that can convert glutamine into a natural blue pigment, wherein the engineered NRPS: a) Includes: i) The amino acid sequence of SEQ ID NO: 8 or the amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 8; ii) An amino acid subsequence of SEQ ID NO: 8 capable of converting glutamine into natural blue pigment, or an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 8; or iii) It has at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical amino acid sequences; and / or b) Encoded as follows: i) The nucleotide sequence of SEQ ID NO: 7 or the nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 7; ii) A nucleotide subsequence of SEQ ID NO: 7 or a nucleotide subsequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 7; or iii) The same nucleic acid sequence as at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0122] The present invention also provides a nucleic acid comprising a nucleotide sequence encoding the engineered nonribosomal peptide synthase (NRPS) polypeptide of the present invention. This nucleic acid is operatively linked to a promoter and / or terminator as defined herein.

[0123] This invention provides a cell comprising the expression construct, nucleic acid, and / or vector of this invention. The cell can be any cell, such as any cell described herein. In some embodiments, the cell is a cell such as *Escherichia coli* and is used for cloning purposes. In some embodiments, the cell is a recombinant yeast cell as described herein and is used for the preparation of a natural blue pigment.

[0124] The present invention also provides a plurality of cells of the present invention.

[0125] In some embodiments, the plurality of cells includes a first plurality of cells that are recombinant yeast cells and a second plurality of cells that are cellulose-producing cells.

[0126] It will be apparent to those skilled in the art that many of the methods and compositions of the present invention are suitable for provision in the form of a kit or component kit. Therefore, the present invention also provides a kit comprising: a) The recombinant yeast cells of the present invention; b) The culture medium as described herein; c) The second cellulose-producing cell of the present invention; d) The natural blue pigment of this invention; e) Bacterial cellulose of the present invention; f) The thin film of the present invention; g) The textiles or threads of the present invention; h) The clothing of the present invention; i) The expression construct of the present invention; j) The carrier of the present invention; k) The engineered nonribosomal peptide synthase (NRPS) of the present invention; l) The nucleic acid of the present invention; and / or m) Multiple cells of the present invention.

[0127] The present invention also provides embodiments numbered as follows: 1. A recombinant yeast cell capable of producing a natural blue pigment, wherein the cell expresses or is able to express: a) A heterologous nonribosomal peptide synthase (NRPS) polypeptide capable of converting glutamine into natural blue pigment; and b) Heterogeneous phosphopantoylthioethylamine transferase (PPTase) peptide.

[0128] 2. The recombinant yeast cell according to Example 1, wherein the cell comprises: a) Nucleic acids containing a nucleotide sequence encoding a heterologous NRPS polypeptide; and b) Nucleic acids containing a nucleotide sequence encoding a heterologous PPTase polypeptide; and Among them, NRPS peptide and PPTase peptide can be expressed from nucleic acids.

[0129] 3. The recombinant yeast cell according to Example 1, wherein the cell contains nucleic acid, the nucleic acid comprising: a) The nucleotide sequence encoding the heterologous NRPS polypeptide; and b) The nucleotide sequence encoding the heterologous PPTase polypeptide; and Among them, NRPS peptide and PPTase peptide can be expressed from nucleic acids.

[0130] 4. The recombinant yeast cells according to any one of Examples 1 to 3, wherein the heterologous NRPS polypeptide is: a) BpsA; arbitrarily chosen: i) Streptomyces lavender BpsA; or ii) Synthesized BpsA; b)IndC; optionally Brown Streptomyces IndC; or c)IndB; optionally Brown Streptomyces IndB.

[0131] 5. Recombinant yeast cells according to any one of Examples 1 to 4, wherein the heterologous NRPS polypeptide: a) Contains or consists of the following: an amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; or an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; and / or b) Encoded by the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 7; or the same nucleotide sequence as SEQ ID NO: 5 or SEQ ID NO: 7 by at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0132] 6. The recombinant yeast cells according to any one of Examples 1 to 5, wherein the heterologous PPTase polypeptide is the large intestine bacilli PPTase or Bacillus subtilis PPTase.

[0133] 7. Recombinant yeast cells according to any one of Examples 1 to 6, wherein the heterologous PPTase polypeptide: a) Containing an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 17, or SEQ ID NO: 19; or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 17, or SEQ ID NO: 18; and / or b) Encoded by a nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 16 or SEQ ID NO: 18; or encoded by a nucleic acid having sequence identity with SEQ ID NO: 2, SEQ ID NO: 16 or SEQ ID NO: 18 of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.

[0134] 8. Recombinant yeast cells according to any one of Examples 1 to 7, wherein the cells further express or overexpress, or are able to express or overexpress glutamine dehydrogenase (GDH) peptides and / or glutamine synthase (GS) peptides.

[0135] 9. The recombinant yeast cell according to Example 8, wherein the cell comprises: a) engineered nucleic acids containing a nucleotide sequence encoding a GDH polypeptide; and / or b) Engineered nucleic acids containing nucleotide sequences encoding GS polypeptides; and Among them, GDH peptide and GS peptide can be expressed from nucleic acids.

[0136] 10. The recombinant yeast cell according to Example 8, wherein the cell contains engineered nucleic acid comprising: a) The nucleotide sequence encoding the GDH polypeptide; and / or b) The nucleotide sequence encoding the GS polypeptide; and Among them, GDH peptide and GS peptide can be expressed from nucleic acids.

[0137] 11. The recombinant yeast cells according to any one of Examples 1 to 8, wherein the GDH peptide and / or GS peptide are endogenous GDH and / or GS peptides.

[0138] 12. The recombinant yeast cells according to Example 11, wherein the GDH polypeptide is encoded by an open reading frame defined by YALI0E09603g or YALI0F17820g.

[0139] 13. Recombinant yeast cells according to any one of Examples 11 to 12, wherein the GDH polypeptide: a) Containing an amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21; or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 20 or SEQ ID NO: 21; and / or b) Encoded by a nucleotide sequence of SEQ ID NO: 11 or SEQ ID NO: 12; or encoded by a nucleic acid having sequence identity with SEQ ID NO: 11 or SEQ ID NO: 12 of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0140] 14. The recombinant yeast cells according to any one of Examples 11 to 13, wherein the GS polypeptide is encoded by an open reading frame defined by YALI0D13024g or YALI0F00506g.

[0141] 15. Recombinant yeast cells according to any one of Examples 11 to 14, wherein the GS polypeptide: a) Containing an amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23; or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 22 or SEQ ID NO: 23; and / or b) Encoded by a nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 10; or encoded by a nucleic acid having sequence identity with SEQ ID NO: 9 or SEQ ID NO: 10 having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence.

[0142] 16. The recombinant yeast cell according to any one of Examples 2 to 15, wherein the nucleotide sequences encoding the heterologous NRPS polypeptide, the heterologous PPTase polypeptide, the GDH polypeptide and / or the GS polypeptide are each operatively linked to a promoter, and optionally each is operatively linked to a separate promoter.

[0143] 17. The recombinant yeast cell according to Example 16, wherein the promoter is: a) Constitutive promoters; or b) Inducible promoters; Optional The promoter is a strong promoter.

[0144] 18. The recombinant yeast cell according to any one of Examples 16 or 17, wherein the promoter is selected from the group consisting of or including the following: a) A constitutive promoter selected from the group consisting of or including the following: ATP1 promoter [SEQ ID NO: 24], ATP2 promoter [SEQ ID NO: 25], FBAin promoter [SEQ ID NO: 26], PGK1 promoter [SEQ ID NO: 27], GPM1 promoter [SEQ ID NO: 28], HHF1 promoter [SEQ ID NO: 29], CYC1 promoter [SEQ ID NO: 30], HHT1 promoter [SEQ ID NO: 31], HTB1 promoter [SEQ ID NO: 32], EXP1 promoter [SEQ ID NO: 33], TDH1 promoter [SEQ ID NO: 34], RPL25 promoter [SEQ ID NO: 35], TEF1 promoter [SEQ ID NO: 36], TEFin promoter [SEQ ID NO: 37], TEF2UAS promoter [SEQ ID NO: 38], CYC1 promoter [SEQ ID NO: 39], HHT1 promoter [SEQ ID NO: 30], HHT1 promoter [SEQ ID NO: 31], HTB1 promoter [SEQ ID NO: 32], EXP1 promoter [SEQ ID NO: 33], TDH1 promoter [SEQ ID NO: 34], RPL25 promoter [SEQ ID NO: 35], TEF1 promoter [SEQ ID NO: 36], TEFin promoter [SEQ ID NO: 37], TEF2UAS promoter [SEQ ID NO: 38], CYC1 promoter [SEQ ID NO: 39], CYC1 promoter [SEQ ID NO: 30], CYC1 promoter [SEQ ID NO: 30], CYC1 promoter [SEQ ID NO: 36], CYC1 promoter [SEQ ID NO: 37], CYC2UAS promoter [SEQ ID NO: 38], CYC2UAS promoter [SEQ ID NO: 39], CYC2UAS promoter [SEQ ID NO: 30], CYC2UAS promoter [ 38], TEF4UAS bootloader [SEQ ID NO: 39] and TEF8UAS bootloader [SEQ ID NO: 40], pTEF bootloader [SEQ ID NO: 13 and 14], GAP bootloader [SEQ ID NO: 57], or b) Inducible promoters selected from the group consisting of or including the following: pPOX2 promoter [SEQ ID NO: 41], pXPR2 promoter [SEQ ID NO: 42], pFBP1 promoter [SEQ ID NO: 43], pMDH1a promoter [SEQ ID NO: 44], ACL2 promoter [SEQ ID NO: 45], XPR2 promoter [SEQ ID NO: 58], POT1 promoter [SEQ ID NO: 46], LIP2 promoter [SEQ ID NO: 47], ICL promoter [SEQ ID NO: 48], YAT1 promoter [SEQ ID NO: 49], CTR1 promoter [SEQ ID NO: 50], CTR2 promoter [SEQ ID NO: 51], pYALI0B18194 promoter [SEQ ID NO: 52], pYALI0C11165 promoter [SEQ ID NO: 52], pYALI0C11165 promoter [SEQ ID NO: 52], pYALI0B18194 ...

[53] , pYALI0C15004 promoter [SEQ ID NO: 54], pYALI0E14256 promoter [SEQ ID NO: 55] and pYALI0F13937 promoter [SEQ ID NO: 56].

[0145] 19. Recombinant yeast cells according to any one of Examples 16 to 18, wherein the promoter is the pTEF promoter; optionally The pTEF promoter has a nucleotide sequence encoded by either SEQ ID NO: 13 or SEQ ID NO: 14.

[0146] 20. A recombinant yeast cell according to any one of Examples 2 to 15, wherein the nucleotide sequence encoding a heterologous NRPS polypeptide, a heterologous PPTase polypeptide, a GDH polypeptide, and / or a GS polypeptide is operatively linked to a terminator sequence.

[0147] 21. The recombinant yeast cells according to Example 20, wherein the terminator sequence is the tLIP2 terminator; optionally The tLIP2 terminator has the nucleotide sequence of SEQ ID NO: 15.

[0148] 22. A recombinant yeast cell according to any one of Examples 2 to 21, wherein a nucleic acid comprising a nucleotide sequence encoding an NRPS polypeptide and / or a PPTase polypeptide is integrated into the cell’s genome.

[0149] 23. The recombinant yeast cell according to any one of Examples 8 to 22, wherein a nucleic acid comprising a nucleotide sequence encoding a GDH polypeptide and / or a GS polypeptide is integrated into the cell’s genome.

[0150] 24. The recombinant yeast cell according to any one of Examples 2 to 23, comprising the deletion of the endogenous chromosome GDH and / or GS genes.

[0151] 25. The recombinant yeast cell according to any one of Examples 2 to 24, comprising the deletion of at least one gene selected from the group consisting of or composed of open reading frames selected from the group consisting of: YALI0E09603g, YALI0F17820g, YALI0D13024g, YALI0F00506g, YALI0E18634g, YALI0B00572g and / or YALI0F01606g or any combination thereof.

[0152] 26. Recombinant yeast cells according to any one of Examples 2 to 25, wherein the nucleotide sequences encoding NRPS polypeptides and / or PPTase polypeptides and / or, when present, the nucleotide sequences encoding GDH and / or GS are codon-optimized for expression in yeast, optionally optimized for expression in... Yersinia Chinese expression.

[0153] 27. The recombinant yeast cell according to any one of the foregoing embodiments, wherein the yeast cell is an oil-producing yeast cell.

[0154] 28. The recombinant yeast cells according to Example 27, wherein the oil-producing yeast cells are not... genus Rhodotorula cell, Optional The cells are not Rhodotorula buergerianum cell.

[0155] 29. The recombinant yeast cell according to any one of the foregoing embodiments, wherein the yeast cell is... Yersinia Cells, optionally selected from the group consisting of or including the following: Yogurtia spp. cell, Deformed Yersinia cell, Yarrowia lipolytica cell, Yeast rice cell, Yarrowia Yakushima cell, Parofonymycosis cell, Chicken jellyfish yeast cell, Yeast Oslo cell, Yeast in food cell, Yarrowia hollandiensis Cells and Yarrowia pangaensis cell; Preferably Among them, restructuring Yersinia Cells are Yarrowia lipolytica cell.

[0156] 30. A method for producing natural blue pigment, the method comprising: The heterologous nonribosomal peptide synthase (NRPS) polypeptide and the heterologous phosphopantoylthioethylamine transferase (PPTase) polypeptide, which can convert glutamine into natural blue pigment, are expressed in recombinant yeast cells according to any one of Examples 1 to 29. The expression of glutamine leads to the conversion of glutamine into a natural blue pigment, thereby producing a natural blue pigment.

[0157] 31. The method according to Example 30, wherein the method comprises culturing cells under conditions suitable for expressing the NRPS polypeptide and the PPTase polypeptide.

[0158] 32. The method according to Example 31, wherein the cells are cultured in a culture medium, optionally of: a) Enriched culture medium, optionally YPD or YP4D; b) Basic culture medium, optionally YNB containing glucose or YNB containing glycerol; or c) Superbasic culture medium; optionally, wherein the superbasic culture medium is SM1, SM2, SM3, SM4 or SM5.

[0159] 33. The method according to any one of Examples 30 to 32, wherein the culture medium comprises: a) Carbon source; b) Nitrogen source; and / or c) Water.

[0160] 34. The method according to any one of Examples 32 or 33, wherein the carbon-nitrogen (C / N) ratio of the culture medium is: a) Between 2 and 160, 2 and 150, 2 and 140, 2 and 130, 2 and 120, 2 and 110, 2 and 100, 2 and 90, 2 and 80, 2 and 70, 2 and 60, 2 and 50, 2 and 40, 2 and 30, 2 and 20, 2 and 10, etc. 2 and John 9, John 2 and John 8, John 2 and John 7, John 2 and John 6, John 2 and John 5, John 2 and John 4, John 2 and John 3, John 4 and John 160, John 4 and John 150, John 4 and John 140, John 4 and John 130, John 4 and John 140, John 4 and John 110, John 4 and John 100, John 4 and John 90, John 4 and John 80, John 4 and John 70, John 4 and John 60, John 4 and John 50, John 4 and John 40, John 4 and John 30, John 4 and John 20, John 4 and John 10, John 4 and John 9, John 4 and John 8, John 4 and John 7, John 4 and John 6, John 4 and John 5, John 6 and John 160, John 6 and John 150, John 6 and John 140, John 6 and John 130, John 6 and John 160, John 6 and John 110, John 6 and John 100, John 6 and John 90, John 6 and John 80, John 6 and John 70, John 6 and John 60, John 6 and John 50, John 6 and John 40, John 6 and John 30, John 6 and John 20, John 6 and John 10, John 6 and John 9, John 6 and John 8, John 6 and John 7, John 8 and John 160, John 8 and John 150, John 8 and John 140, John 8 and John 130, John 8 and John 180, John 8 and John 110, John 8 and John 100, John 8 and John 90, John 8 and John 80, John 8 and John 70, John 8 and John 60, John 8 and John 50, John 8 and John 40, John 8 and John 30, John 8 and John 20, John 8 and John 10, John 8 and John 9, John 8 and John 8, John 8 and John 7, John 8 and John 6, John 8 and John 5, John 8 and John 4, John 8 and John 3, John 10 and John 160, John 10 With about 150, about 10 with about 140, about 10 with about 130, about 10 with about 1100, about 10 with about 110, about 10 with about 100, about 10 with about 90, about 10 with about 80, about 10 with about 70, about 10 with about 60, about 10 with about 50, about 10 with about 40, about 10 with about 30, about 10 with about 20, about 20 with about 160, Approximately 20 and approximately 150, approximately 20 and approximately 140, approximately 20 and approximately 130, approximately 20 and approximately 1200, approximately 20 and approximately 110, approximately 20 and approximately 100, approximately 20 and approximately 90, approximately 20 and approximately 80, approximately 20 and approximately 70, approximately 20 and approximately 60, approximately 20 and approximately 50, approximately 20 and approximately 40, approximately 20 and approximately 30, approximately 30 and approximately 160, approximately 30 and approximately 150, about 30 and about 140, about 30 and about 130, about 30 and about 1300, about 30 and about 110, about 30 and about 100, about 30 and about 90, about 30 and about 80, about 30 and about 70, about 30 and about 60, about 30 and about 50, about 30 and about 40, about 40 and about 160, about 40 and about 150, about 40 and about 140,Between approximately 40 and approximately 130, approximately 40 and approximately 1400, approximately 40 and approximately 110, approximately 40 and approximately 100, approximately 40 and approximately 90, approximately 40 and approximately 80, approximately 40 and approximately 70, approximately 40 and approximately 60, approximately 40 and approximately 50, approximately 50 and approximately 160, approximately 50 and approximately 150, approximately 50 and approximately 140, approximately 50 and approximately 130, approximately 50 and approximately 150, approximately 50 and approximately 110, approximately 50 and approximately 100, approximately 50 and approximately 90, approximately 50 and approximately 80, approximately 50 and approximately 70, or approximately 50 and approximately 60; b) Between 2 and 160, 2 and 150, 2 and 140, 2 and 130, 2 and 120, 2 and 110, 2 and 100, 2 and 90, 2 and 80, 2 and 70, 2 and 60, 2 and 50, 2 and 40, 2 and 30, 2 and 20, 2 and 10, 2 and 9, 2 and 8, 2 and 7, 2 and 6, 2 and 5, 2 and 4, 2 and 3, 4 and 160, 4 and 150, 4 and 140, 4 and 130, 4 and 140, 4 and 110, 4 and 100, 4 and 90, 4 and 80, 4 and 70, 4 and 60, 4 and 50, 4 and 40, 4 and 30, 4 and 20, 4 and 10, 4 and 9, 4 and 8, 4 and 7 4 and 6, 4 and 5, 6 and 160, 6 and 150, 6 and 140, 6 and 130, 6 and 160, 6 and 110, 6 and 100, 6 and 90, 6 and 80, 6 and 70, 6 and 60, 6 and 50, 6 and 40, 6 and 30, 6 and 20, 6 and 10, 6 and 9, 6 and 8, 6 and 7, 8 and 160, 8 and 150, 8 and 140, 8 and 130, 8 and 180, 8 and 110, 8 and 100, 8 and 90, 8 and 80, 8 and 70, 8 and 60, 8 and 50, 8 and 40, 8 and 30, 8 and 20, 8 and 10, 8 and 9, 8 and 8, 8 and 7, 8 and 6, 8 and 5, 8 and 4 8 and 3, 10 and 160, 10 and 150, 10 and 140, 10 and 130, 10 and 1100, 10 and 110, 10 and 100, 10 and 90, 10 and 80, 10 and 70, 10 and 60, 10 and 50, 10 and 40, 10 and 30, 10 and 20, 20 and 160, 20 and 150, 20 and 140, 20 and 130, 20 and 1200, 20 and 110, 20 and 100, 20 and 90, 20 and 80, 20 and 70, 20 and 60, 20 and 50, 20 and 40, 20 and 30, 30 and 160, 30 and 150, 30 and 140, 30 Between 130, 30 and 1300, 30 and 110, 30 and 100, 30 and 90, 30 and 80, 30 and 70, 30 and 60, 30 and 50, 30 and 40, 40 and 160, 40 and 150, 40 and 140, 40 and 130, 40 and 1400, 40 and 110, 40 and 100, 40 and 90, 40 and 80, 40 and 70, 40 and 60, 40 and 50, 50 and 160, 50 and 150, 50 and 140, 50 and 130, 50 and 150, 50 and 110, 50 and 100, 50 and 90, 50 and 80, 50 and 70 or 50 and 60; c) At least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150 or more; d) Less than approximately 160, less than approximately 150, less than approximately 140, less than approximately 130, less than approximately 120, less than approximately 110, less than approximately, less than approximately 100, less than approximately 90, less than approximately 80, less than approximately 70, less than approximately 60, less than approximately 50, less than approximately 40, less than approximately 30, less than approximately 20, less than approximately 10, less than approximately 9, less than approximately 8, less than approximately 7, less than approximately 6, less than approximately 5, less than approximately 4, less than approximately 3; and / or e) At least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150 or more; f) Less than 160, less than 150, less than 140, less than 130, less than 120, less than 110, less than approximately, less than 100, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3; g) Approximately 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, approximately 8, approximately 9, approximately 10, approximately 20, approximately 30, approximately 40, approximately 50, approximately 60, approximately 70, approximately 80, approximately 90, approximately 100, approximately 110, approximately 120, approximately 130, approximately 140, approximately 150; and / or h) 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160.

[0161] 35. The method according to any one of Examples 32 to 34, wherein the C / N ratio is between the following values: a) about 4 and about 20; and / or b) 4 and 20.

[0162] 36. The method according to Example 35, wherein the C / N ratio is: a) 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about or 20; and / or b) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0163] 37. The method according to any one of Examples 32 to 36, wherein the culture medium is selected from the group consisting of or including YP4G medium, YNB glucose medium and YNB glycerol medium, optionally YNB glucose medium or YNB glycerol medium.

[0164] 38. The method according to any one of Examples 32 to 36, wherein the carbon source is selected from the group consisting of or including glucose, fructose, mannose, citrate, acetate, rapeseed oil, sunflower oil, palm oil, waste cooking oil, rapeseed oil and sunflower oil, lignocellulose hydrolysate, glycerol and / or waste glycerol; Optional The lignocellulose hydrolysate is selected from the group consisting of or including the following: hydrolyzed flaxseed, chili peppers, vegetable mixtures and urban trimmings or any combination thereof.

[0165] 39. The method according to any one of Examples 33 to 37, wherein the carbon source is a lignocellulose hydrolysate.

[0166] 40. The method according to any one of Examples 33 to 37, wherein the water is selected from the group consisting of seawater, tap water, and distilled water; or any combination thereof.

[0167] 41. The method according to any one of Examples 33 to 40, wherein the nitrogen source is selected from the group consisting of or including: urine, synthetic urine, urea, ammonium chloride, peptone and ammonium sulfate or any combination thereof.

[0168] 42. The method according to any one of Examples 33 to 41, wherein the nitrogen source is ammonium sulfate.

[0169] 43. The method according to any one of Examples 32 to 42, wherein the culture medium is a liquid culture medium.

[0170] 44. The method according to Example 43, wherein the culture includes stirring the liquid culture medium during cell growth; optionally, wherein the liquid culture medium is stirred at the following rotational speeds: a) Between approximately 150 and approximately 2000 RPM, approximately 150 and approximately 250 RPM, approximately 150 and approximately 200 RPM, approximately 200 and approximately 250 RPM, approximately 150 and approximately 800 RPM, approximately 200 and approximately 800 RPM, approximately 250 and approximately 800 RPM, approximately 800 and approximately 2000 RPM, approximately 1000 and approximately 1800 RPM, approximately 1200 and approximately 1400 RPM; b) Between 150 and 2000 RPM, 150 and 250 RPM, 150 and 200 RPM, 200 and 250 RPM, 150 and 800 RPM, 200 and 800 RPM, 250 and 800 RPM, 800 and 2000 RPM, 1000 and 1800 RPM, and 1200 and 1400 RPM; c) At least 150 RPM, at least 200 RPM, at least 250 RPM, at least 800 RPM, at least 1000 RPM, at least 1200 RPM, at least 1400 RPM, at least 1600 RPM, at least 1800 RPM; d) Less than 2000 RPM, less than 1800 RPM, less than 1600 RPM, less than 1400 RPM, less than 1200 RPM, less than 1000 RPM, less than 800 RPM, less than 250 RPM, or less than 200 RPM; c) Approximately 150 RPM, approximately 200 RPM, approximately 250 RPM, approximately 800 RPM, approximately 1000 RPM, approximately 1200 RPM, approximately 1400 RPM, approximately 1600 RPM, approximately 1800 RPM, or approximately 2000 RPM; or d) 150 RPM, 200 RPM, 250 RPM, 800 RPM, 1000 RPM, 1200 RPM, 1400 RPM, 1600 RPM, 1800 RPM or 2000 RPM.

[0171] 45. The method according to any one of Examples 32 to 44, wherein the culture medium is supplemented with glutamine and / or glutamate.

[0172] 46. ​​The method according to any one of Examples 32 to 45, wherein: a) The culture medium is supplemented with glutamine, and the concentration of glutamine is: i) Between approximately 1 mm and approximately 100 mm, approximately 2 mm and approximately 90 mm, approximately 3 mm and approximately 80 mm, approximately 4 mm and approximately 70 mm, approximately 5 mm and approximately 60 mm, approximately 6 mm and approximately 50 mm, approximately 7 mm and approximately 40 mm, approximately 8 mm and approximately 30 mm, approximately 9 mm and approximately 20 mm, approximately 10 mm and approximately 15 mm, approximately 1 mm and approximately 10 mm, approximately 2 mm and approximately 9 mm, approximately 3 mm and approximately 8 mm, approximately 4 mm and approximately 7 mm, approximately 5 mm and approximately 6 mm Between mM, approximately 10mM and approximately 100mM, approximately 20mM and approximately 90mM, approximately 30mM and approximately 80mM, approximately 40mM and approximately 70mM, approximately 50mM and approximately 60mM, approximately 50mM and approximately 150mM, approximately 60mM and approximately 140mM, approximately 70mM and approximately 130mM, approximately 80mM and approximately 120mM, or approximately 90mM and approximately 110mM glutamine; ii) Between 1mM and 100mM, 2mM and 90mM, 3mM and 80mM, 4mM and 70mM, 5mM and 60mM, 6mM and 50mM, 7mM and 40mM, 8mM and 30mM, 9mM and 20mM, 10mM and 15mM, 1mM and 10mM, 2mM and 9mM, 3mM and 8mM, 4mM and 7mM, 5mM and 6mM, 10 Between mM and 100mM, 20mM and 90mM, 30mM and 80mM, 40mM and 70mM, 50mM and 60mM, 50mM and 150mM, 60mM and 140mM, 70mM and 130mM, 80mM and 120mM, or 90mM and 110mM glutamine; iii) At least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, at least 10 mM, at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 110 mM, at least 120 mM, at least 130 mM, at least 140 mM, at least 150 mM or more glutamine; iv) Less than 150mM, less than 140mM, less than 130mM, less than 120mM, less than 120mM, less than 110mM, less than 100mM, less than 90mM, less than 80mM, less than 70mM, less than 60mM, less than 50mM, less than 40mM, less than 30mM, less than 20mM, less than 10mM, less than 9mM, less than 8mM, less than 7mM, less than 6mM, less than 5mM, less than 4mM, less than 3mM, less than 2mM or less glutamine; v) Approximately 1 mM, approximately 2 mM, approximately 3 mM, approximately 4 mM, approximately 5 mM, approximately 6 mM, approximately 7 mM, approximately 8 mM, approximately 9 mM, approximately 10 mM, approximately 20 mM, approximately 30 mM, approximately 40 mM, approximately 50 mM, approximately 60 mM, approximately 70 mM, approximately 80 mM, approximately 90 mM, approximately 100 mM, approximately 110 mM, approximately 120 mM, approximately 130 mM, approximately 140 mM, or approximately 150 mM glutamine; and / or vi) 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM or 150mM glutamine; And / or b) The culture medium is supplemented with glutamic acid, and the concentration of this glutamic acid is: i) Between approximately 1 mm and approximately 100 mm, approximately 2 mm and approximately 90 mm, approximately 3 mm and approximately 80 mm, approximately 4 mm and approximately 70 mm, approximately 5 mm and approximately 60 mm, approximately 6 mm and approximately 50 mm, approximately 7 mm and approximately 40 mm, approximately 8 mm and approximately 30 mm, approximately 9 mm and approximately 20 mm, approximately 10 mm and approximately 15 mm, approximately 1 mm and approximately 10 mm, approximately 2 mm and approximately 9 mm, approximately 3 mm and approximately 8 mm, approximately 4 mm and approximately 7 mm, approximately 5 mm and approximately 6 mm Between mM, approximately 10mM and approximately 100mM, approximately 20mM and approximately 90mM, approximately 30mM and approximately 80mM, approximately 40mM and approximately 70mM, approximately 50mM and approximately 60mM, approximately 50mM and approximately 150mM, approximately 60mM and approximately 140mM, approximately 70mM and approximately 130mM, approximately 80mM and approximately 120mM, or approximately 90mM and approximately 110mM glutamate; ii) Glutamate between 1mM and 100mM, 2mM and 90mM, 3mM and 80mM, 4mM and 70mM, 5mM and 60mM, 6mM and 50mM, 7mM and 40mM, 8mM and 30mM, 9mM and 20mM, 10mM and 15mM, 1mM and 10mM, 2mM and 9mM, 3mM and 8mM, 4mM and 7mM, 5mM and 6mM, 10mM and 100mM, 20mM and 90mM, 30mM and 80mM, 40mM and 70mM, 50mM and 60mM, 50mM and 150mM, 60mM and 140mM, 70mM and 130mM, 80mM and 120mM, or 90mM and 110mM; iii) At least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, at least 10 mM, at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 110 mM, at least 120 mM, at least 130 mM, at least 140 mM, at least 150 mM or more glutamate; iv) Less than 150mM, less than 140mM, less than 130mM, less than 120mM, less than 120mM, less than 110mM, less than 100mM, less than 90mM, less than 80mM, less than 70mM, less than 60mM, less than 50mM, less than 40mM, less than 30mM, less than 20mM, less than 10mM, less than 9mM, less than 8mM, less than 7mM, less than 6mM, less than 5mM, less than 4mM, less than 3mM, less than 2mM or less glutamate; v) Approximately 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, or 150 mM glutamate; and / or vi) 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM or 150mM glutamate.

[0173] 47. The method according to any one of Examples 30 to 46, wherein the method further comprises: Endogenous glutamate dehydrogenase (GDH) polypeptide and / or endogenous glutamine synthase (GS) polypeptide are expressed in cells according to any one of Examples 1 to 29.

[0174] 48. The method according to any one of Examples 30 to 47, wherein the NRPS peptide, PPTase peptide, GDH peptide and / or GS peptide are overexpressed in cells.

[0175] 49. The method according to any one of Examples 30 to 48, wherein: a) The NRPS polypeptide is the NRPS polypeptide according to any one of Examples 4 to 5; b) The PPTase polypeptide is the PPTase polypeptide according to any one of Examples 6 to 7; c) The GDH polypeptide is the GDH polypeptide according to any one of Examples 11 to 13; and / or d) The GS polypeptide is the GS polypeptide according to any one of Examples 11, 14 to 15.

[0176] 50. The method according to any one of Examples 30 to 49, wherein the cells comprise nucleic acids according to any one of Examples 2 to 7, 9 to 23; and NRPS peptides, PPTase peptides, GDH peptides and / or GS peptides are overexpressed from nucleic acids.

[0177] 51. The method according to any one of Examples 30 to 50, wherein the method further comprises extracting and / or purifying the natural blue pigment from cells; optionally Extraction and / or purification of natural blue pigments from cells includes cell lysis; optionally The cells are lysed by homogenization in DMSO.

[0178] 52. Use of recombinant yeast cells according to any one of Examples 1 to 30 in a method for producing natural blue pigment.

[0179] 53. The use according to Example 52, wherein the method is the method according to any one of Examples 30 to 51.

[0180] 54. A natural blue pigment, which is obtained or can be obtained from cells according to any one of Examples 1 to 30 or by any one of Examples 31 to 51.

[0181] 55. A method for producing bacterial cellulose stained with natural blue pigment, the method comprising co-culturing recombinant yeast cells according to any one of Examples 1 to 30 with cellulose-producing second cells.

[0182] 56. The method according to Example 55, wherein the method comprises culturing recombinant yeast cells and cellulose-producing second cells under conditions that allow cellulose-producing second cells to produce cellulose; optionally The conditions allow for the formation of a thin film by the second cellulose-producing cell.

[0183] 57. The method according to Example 56, wherein the conditions allowing the cellulose-producing second cell to produce cellulose include culturing the cells in the following pH and culture medium: a) pH is: i) pH between 3 and 7, optionally between 3.25 and 6.75, 3.5 and 6.5, 3.5 and 6.25, 3.75 and 6, 4 and 5.75, 4.25 and 5.5, 4.5 and 5.25; pH 5.8; and / or ii) At least 3 but less than or equal to pH 7, for example, at least 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, 5, 5.25, 5.5, 5.75, 5.8, 6, 6.25, 6.5, 6.75, but less than or equal to pH 7; And / or b) The culture medium is: i) HS culture medium; ii) YPD medium; or iii) Coconut water culture medium.

[0184] 58. The method according to any one of Examples 55 to 57, wherein the cellulose-producing second cell is a bacterial cell.

[0185] 59. The method according to Example 58, wherein the bacterial cells: a) It can produce bacterial cellulose; b) Express all of bcsA, bcsD, bscC, and bscD; c) Belongs to a genus selected from the group consisting of or including the following: genus *Columella* , Escherichia coli , Portugal Gluconobacterium , Acetobacter , octopus , Agrobacterium , Nitrogen-fixing bacteria , Rhizobium , Pseudomonas , salmonella genus and Alcaligenes ; d) Selected from the following or a group consisting of the following: Gerolactobacillus ; Xylomycin , Hans's Colt Bacteroides , Medellin's Corydalis , European coccidioidomyces , Maltaceae , Lettuce-like bacteria , Obedience to Corydalis or Sucrose-bearing bacillus ; e) Selected from or consisting of the following items: i) Selected from the group consisting of or including the following items Gerolactobacillus Strains: Gerolactobacillus iGEM; Gerolactobacillus AF1; GerolactobacillusLMG22126; or ii) Xylomycin CGMCC 2995; And / or f) is Gerolactobacillus iGEM ​​cells.

[0186] 60. A method for producing bacterial cellulose for staining with natural blue pigment, the method comprising: a) Culturing cellulose-producing second cells to form bacterial cellulose; and subsequently b) Contact the cellulose produced by the cellulose-producing second cell in (a) with the recombinant yeast cell according to any one of Examples 1 to 30; Optionally, the second cell producing cellulose is a bacterial cell; optionally, the bacterial cell is: a) It can produce bacterial cellulose; b) Express all of bcsA, bcsD, bscC, and bscD; c) Belongs to a genus selected from the group consisting of or including the following: genus *Columella* , Escherichia coli , Portugal Gluconobacterium , Acetobacter , octopus , Agrobacterium , Nitrogen-fixing bacteria , Rhizobium , Pseudomonas , salmonella genus and Alcaligenes ; d) Selected from the following or a group consisting of the following: Gerolactobacillus ; Xylomycin , Hans's Colt Bacteroides , Medellin's Corydalis , European coccidioidomyces , Maltaceae , Lettuce-like bacteria , Obedience to Corydalis or Sucrose-bearing bacillus ; e) Selected from or consisting of the following items: i) Selected from the group consisting of or including the following items Gerolactobacillus Strains: Gerolactobacillus iGEM; Gerolactobacillus AF1; Gerolactobacillus LMG22126; or ii) Xylomycin CGMCC 2995; And / or f) is Gerolactobacillus iGEM ​​cells.

[0187] 61. A method for producing a thin film stained with natural blue pigment, the method comprising co-culturing recombinant yeast cells according to any one of Examples 1 to 30 with cellulose-producing second cells, Optionally, the second cell producing cellulose is a bacterial cell; optionally, the bacterial cell is: a) It can produce bacterial cellulose; b) Express all of bcsA, bcsD, bscC, and bscD; c) Belongs to a genus selected from the group consisting of or including the following: genus *Columella* , Escherichia coli , Portugal Gluconobacterium , Acetobacter , octopus , Agrobacterium , Nitrogen-fixing bacteria , Rhizobium , Pseudomonas , salmonella genus and Alcaligenes ; d) Selected from the following or a group consisting of the following: Gerolactobacillus ; Xylomycin , Hans's Colt Bacteroides , Medellin's Corydalis , European coccidioidomyces , Maltaceae , Lettuce-like bacteria , Obedience to Columbiformis or Sucrose-bearing bacillus ; e) Selected from or consisting of the following items: i) Selected from the group consisting of or including the following items Gerolactobacillus Strains: Gerolactobacillus iGEM; Gerolactobacillus AF1; Gerolactobacillus LMG22126; or ii) Xylomycin CGMCC 2995; And / or f) is Gerolactobacillus iGEM ​​cells.

[0188] 62. A method for producing a thin film dyed with natural blue pigment, the method comprising: a) Culturing cellulose-producing second cells to form a cellulose film; and subsequently b) Contact the resulting film with the cells according to any one of Examples 1 to 29. Optionally, the second cell producing cellulose is a bacterial cell; optionally, the bacterial cell is: a) It can produce bacterial cellulose; b) Express all of bcsA, bcsD, bscC, and bscD; c) Belongs to a genus selected from the group consisting of or including the following: genus *Columella* , Escherichia coli , Portugal Gluconobacterium , Acetobacter , octopus , Agrobacterium , Nitrogen-fixing bacteria , Rhizobium , Pseudomonas , salmonella genus and Alcaligenes ; d) Selected from the following or a group consisting of the following: Gerolactobacillus ; Xylomycin , Hans's Colt Bacteroides , Medellin's Corydalis , European coccidioidomyces , Maltaceae , Lettuce-like bacteria , Obedience to Corydalis or Sucrose-bearing bacillus ; e) Selected from or consisting of the following items: i) Selected from the group consisting of or including the following items Gerolactobacillus Strains: Gerolactobacillus iGEM; Gerolactobacillus AF1; Gerolactobacillus LMG22126; or ii) Xylomycin CGMCC 2995; And / or f) is Gerolactobacillus iGEM ​​cells.

[0189] 63. The method according to any one of Examples 55 to 62, wherein the cellulose-producing second cell is cultured under the conditions described in Example 56.

[0190] 64. The method according to any one of Examples 55 to 63, wherein contacting cellulose or contacting the film comprises incubating the recombinant yeast cells according to Examples 1 to 30 together with cellulose or the film.

[0191] 65. The method according to Example 64, wherein incubating the recombinant yeast cells according to Examples 1 to 30 together with cellulose or a film comprises culturing the recombinant yeast cells according to any one of Examples 31 to 54.

[0192] 66. The method according to any one of Examples 55 to 65, wherein the method further comprises sterilizing the cellulose or film, optionally wherein the sterilization is selected from the group consisting of autoclaving, heating and drying, or any combination thereof.

[0193] 67. A method for producing textiles or threads dyed with natural blue pigment, the method comprising: a) Producing natural blue pigment according to any one of Examples 31 to 54; ;as well as b) Contact the textile or thread with the natural blue pigment obtained in (a).

[0194] 68. The method according to Example 67, wherein producing natural blue pigment according to (s) further comprises: i) Optionally by centrifugation or filtration Collect yeast cells; ii) dried yeast cells; and / or iii) Lysis of dried yeast cells; Optionally, lysing yeast cells includes grinding the yeast cells. In some embodiments, cells are lysed by homogenization in DMSO. Such methods are known, and in some embodiments, involve using glass beads, cell disruption at 8,000 rpm, and centrifugation.

[0195] 69. The method according to any one of Examples 67 to 68, wherein b) contacting the textile or thread with the natural blue pigment obtained in (a) comprises immersing the textile or thread in the natural blue pigment.

[0196] 70. The method according to Example 69, wherein the textile or thread is immersed in the dyeing mixture for the following duration: a) Between approximately 4 hours and approximately 12 hours, approximately 5 hours and approximately 11 hours, approximately 6 hours and approximately 10 hours, approximately 7 hours and approximately 9 hours; b) Between 4 hours and 12 hours, 5 hours and 11 hours, 6 hours and 10 hours, 7 hours and 9 hours; c) At least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 12 hours; d) Less than 12 hours, less than 11 hours, less than 10 hours, less than 9 hours, less than 8 hours, less than 7 hours, less than 6 hours, less than 5 hours; e) Approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours; and / or f) 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours.

[0197] 71. The method according to any one of Examples 67 to 70, wherein the method further comprises, after step b), the following: c) Autoclaving the textiles or yarns; and / or d) Wash textiles or yarn.

[0198] 72. A bacterial cellulose dyed with a natural blue pigment, which is obtained or can be obtained from the method according to any one of Examples 55 to 66.

[0199] 73. A film dyed with a natural blue pigment, obtained by the method according to any one of Examples 55 to 66.

[0200] 74. A textile or thread dyed with a natural blue pigment, obtained from the method according to any one of Examples 67 to 71.

[0201] 75. A garment comprising bacterial cellulose dyed with natural blue pigment according to Example 72, a film dyed with natural blue pigment according to Example 73, or a textile or thread dyed with natural blue pigment according to Example 74.

[0202] 76. An expression construct comprising a nucleic acid, the nucleic acid comprising: a) The nucleotide sequence encoding the NRPS polypeptide; b) The nucleotide sequence encoding the PPTase polypeptide; c) The nucleotide sequence encoding the GDH polypeptide; and / or d) The nucleotide sequence encoding the GS polypeptide; The nucleotide sequences encoding NRPS peptides, PPTase peptides, GDH peptides, and / or GS peptides are operatively linked to the promoter.

[0203] 77. The expression construct according to Example 76, wherein: a) The NRPS polypeptide is the NRPS polypeptide according to any one of Examples 4 to 5; b) The PPTase polypeptide is the PPTase polypeptide according to any one of Examples 6 to 7; c) The GDH polypeptide is the GDH polypeptide according to any one of Examples 11 to 13; and / or d) The GS polypeptide is the GS polypeptide according to any one of Examples 11, 14 to 15.

[0204] 78. The expression construct according to any one of Examples 76 to 77, wherein the promoter is the promoter according to any one of Examples 17 to 19.

[0205] 79. The expression construct according to any one of Examples 76 to 78, wherein the nucleotide sequence encoding a heterologous NRPS polypeptide, a heterologous PPTase polypeptide, a GDH polypeptide, and / or a GS polypeptide is operatively linked to a terminator sequence.

[0206] 80. The expression construct according to Example 79, wherein the terminating subsequence is the terminating subsequence according to Example 21.

[0207] 81. A carrier comprising the expression construct described in any one of Examples 76 to 80.

[0208] 82. The vector according to Example 81, wherein the vector is selected from the group consisting of plasmids, phages, YAC and BAC.

[0209] 83. An engineered nonribosomal peptide synthase (NRPS) polypeptide that converts glutamine into a natural blue pigment, wherein the engineered NRPS: a) Includes: i) The amino acid sequence of SEQ ID NO: 8 or the amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 8; ii) An amino acid subsequence of SEQ ID NO: 8 capable of converting glutamine into natural blue pigment, or an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 8; or iii) It has at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical amino acid sequences; and / or b) Encoded as follows: i) The nucleotide sequence of SEQ ID NO: 7 or the nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 7; ii) A nucleotide subsequence of SEQ ID NO: 7 or a nucleotide subsequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 7; or iii) The same nucleic acid sequence as at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0210] 84. A nucleic acid comprising a nucleotide sequence encoding an engineered nonribosomal peptide synthase (NRPS) polypeptide according to Example 85.

[0211] 85. The nucleic acid according to Example 84, wherein the nucleotide sequence encoding the engineered NRPS polypeptide is operatively linked to a promoter.

[0212] 86. The nucleic acid according to Example 85, wherein the promoter is the promoter according to any one of Examples 17 to 19.

[0213] 87. The nucleic acid according to Examples 85 and 86, wherein the nucleotide sequence encoding the engineered NRPS polypeptide is operatively linked to a terminator sequence.

[0214] 88. The nucleic acid according to Example 87, wherein the terminator sequence is the terminator sequence according to Example 21.

[0215] 89. A vector comprising the expression construct described in any one of Examples 84 to 88.

[0216] 90. The vector according to Example 89, wherein the vector is selected from the group consisting of plasmids, phages, YAC and BAC.

[0217] 91. A cell comprising an expression construct according to any one of Examples 76 to 80, a nucleic acid according to any one of Examples 84 to 88, or a vector according to any one of Examples 81 to 82, 89 to 90.

[0218] 92. The cell according to Example 91, wherein the cell is the cell according to any one of Examples 1 to 29.

[0219] 93. A plurality of cells, wherein each of the plurality of cells is a cell as described in Example 92.

[0220] 94. A plurality of cells, comprising a first plurality of cells according to Example 93 and a second plurality of cells according to any one of Examples 55 to 63.

[0221] 95. A reagent kit comprising: a) Cells according to any one of Examples 1 to 29; b) The culture medium according to any one of Examples 32 to 43, 45 to 46; c) The second cell according to any one of Examples 55 to 63; d) The natural blue pigment according to Example 54; e) Bacterial cellulose according to any one of Examples 55 to 66, 72; f) The film according to any one of Examples 61 to 66, 73; g) The textile or thread according to any one of Examples 67 to 71, 74; h) The garment according to Example 75; i) The expression construct according to any one of Examples 76 to 80; j) The carrier according to any one of Examples 81 to 82, 89 to 90; k) The engineered nonribosomal peptide synthase (NRPS) according to Example 83; l) Nucleic acid according to any one of Examples 84 to 88; m) cells according to any one of Examples 91 to 92; and / or n) Multiple cells according to any one of Examples 93 to 94.

[0222] The enumeration or discussion of obviously previously disclosed documents in this specification is not necessarily an admission that such documents are part of the prior art or common general knowledge.

[0223] Unless the context otherwise indicates, preferences and options for a given aspect, feature, or parameter of the invention should be considered 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 Yarrowia lipolytica Cells that express synthetic BpsA and GDH peptides encoded by YALI0F17820g.

[0224] The sequences mentioned in this article >SEQ_ID_NO:_1_PPTase(wt) MKIYGIYMDRPLSQEENERFMTFISPEKREKCRRFYHKEDAHRTLLGDVLVRSVISRQYQLDKSDIRFSTQEYGKPCIPDLPDAHFNISHSGRWVIGAFDSQPIGIDIEKTKPISLEIAKRFFSKTEYSDLLAKDKDEQTDYFYHLWSMKESFIKQEGKGLSLPLDSFSVRLHQDGQVSIELPDSHSPCYIKTYEVDPGYKMAVCAAHPDFPEDITMVSYEELL >SEQ_ID_NO:_2_PPTase(codon_opt) ATGGGCATCTACGGAATCTACATGGACCGACCTCTGTCTCAAGAAGAGAACGAGCGATTCATGACCTTCATCTCTCCCGAGAAGCGAGAGAAGTGCCGACGATTCTACCACAAGGAAGATGCCCACCGAACTCTGCTGGGCGACGTGCTGGTGCGATCTGTGATCTCTCGACAGTACCAGCTGGACAAGTCTGACATCCGATTCTCTACCCAAGAGTACGGCAAGCCCTGCATTCCCGACCTGCCTGACGCTCACTTCAACATCTCTCACTCTGGCCGATGGGTGATCGGCGCCTTCGACTCTCAGCCCATCGGCATCGACATCGAAAAGACCAAGCCTATCTCTCTCGAGATCGCCAAGCGATTCTTCAGCAAGACCGAGTACTCTGACCTGCTGGCCAAGGACAAGGACGAGCAGACCGACTACTTCTACCACCTGTGGTCTATGAAGGAATCTTTCATCAAGCAAGAAGGCAAGGGCCTGTCTCTGCCCCTGGACTCTTTCTCTGTGCGACTGCACCAGGACGGCCAGGTGTCTATCGAGCTGCCCGACTCTCACTCGCCCTGCTACATCAAGACCTACGAGGTGGACCCCGGCTACAAGATGGCCGTGTGCGCTGCTCACCCTGACTTCCCCGAGGACATTACCATGGTGTCTTACGAGGAACTGCTGTAA >SEQ_ID_NO:_3_PPTase(codon_opt) MGIYGIYMDRPLSQEENERFMTFISPEKREKCRRFYHKEDAHRTLLGDVLVRSVISRQYQLDKSDIRFSTQEYGKPCIPDLPDAHFNISHSGRWVIGAFDSQPIGIDIEKTKPISLEIAKRFFSKTEYSDLLAKDKDEQTDYFYHLWSMKESFIKQEGKGLSLPLDSFSVRLHQDGQVSIELPDSHSPCYIKTYEVDPGYKMAVCAAHPDFPEDITMVSYEELL >SEQ_ID_NO:_4_BpsA(wt) >SEQ_ID_NO:_5_BpsA_V1254M (BpsA_X5)(codon_opt) >SEQ_ID_NO:_6_BpsA_V1254M (BpsA_X5)(codon_opt) >SEQ ID NO: 59 BpsA_X5 >SEQ_ID_NO:_7_BpsA_X5-UNC >SEQ_ID_NO:_8_BpsA_X5-UNC >SEQ_ID_NO:_9_YALI0D13024g (Wild type) >SEQ_ID_NO:_10_YALI0F00506g (wild type) >SEQ_ID_NO:_11_YALI0E09603g (Wild type) >SEQ_ID_NO:_12_YALI0F17820g (Wild Type) >SEQ_ID_NO:_13_pTEF(V1) Accgggttggcggcgtatttgtgtcccaaaaaacagccccaattgccccaattgaccccaaattgacccagtagcgggcccaaccccggcgagagcccccttcaccccacatatcaaacctcccccggttcccacacttgccgttaagggcgtagggtactgcagtctggaatctacgcttgttcagactttgtactagtttctttgtctggccatccgggtaacccatgccggacgcaaaatagactactgaaaatttttttgctttgtggttgggactttagccaagggtataaaagaccaccgtccccgaattacctttcctcttcttttctctctctccttgtcaactcacacccgaaatcgttaagcatttccttctgagtataagaatcattcaa >SEQ_ID_NO:_14_pTEF(V2) cgatagagaccgggttggcggcgtatttgtgtcccaaaaaacagccccaattgccccaattgaccccaaattgacccagtagcggacccaaccccggcgagagcccccttcaccccacatatcaaacctcccccggttcccacacttgccgttaagggcgtagggtactgcagtctggaatctacgcttgttcagactttgtactagtttctttgtctggccatccgggtaacccatgccggacgcaaaatagactactgaaaatttttttgctttgtggttgggactttagccaagggtataaaagaccaccgtccccgaattacctttcctcttcttttctctctctccttgtcaactcacacccgaag >SEQ_ID_NO:_15_tLIP2 Gtgtctgtggtatctaagctatttatcactctttacaacttctacctcaactatctactttaataaatgaatatcgtttattctctatgattactgtatatgcgttcctctaagacaaatcg >SEQ_ID_NO:_16_Ec_PPTase_DNA Atgaaaactacgcatacctccctcccctttgccggacatacgctgcattttgttgagttcgatccggcgaatttttgtgagcaggatttactctggctgccgcactacgcacaactgcaacacgctggacgtaaacgtaaaacagagcatttagccggacggatcgctgctgtttatgctttgcgggaatatggctataaatgtgtgcccgcaatcggcgagctacgccaacctgtctggcctgcggaggtatacggcagtattagccactgtgggactacggcattagccgtggtatctcgtcaaccgattggcattgatatagaagaaattttttctgtacaaaccgcaagagaattgacagacaacattattacaccagcggaacacgagcgactcgcagactgcggtttagccttttctctggcgctgacactggcattttccgccaaagagagcgcatttaaggcaagtgagatccaaactgatgcaggttttctggactatcagataattagctggaataaacagcaggtcatcattcatcgtgagaatgagatgtttgctgtgcactggcagataaaagaaaagatagtcataacgctgtgccaacacgattaa >SEQ_ID_NO:_17_Ec_PPTase_AA MKTTHTSLPFAGHTLHFVEFDPANFCEQDLLWLPHYAQLQHAGRKRKTEHLAGRIAAVYALREYGYKCVPAIGELRQPVWPAEVYGSISHCGTTALAVVSRQPIGIDIEEIFSVQTARELTDNIITPAEHERLADCGLAFSLALTLAFSAKESAFKASEIQTDAGFLDYQIISWNKQQVIIHRENEMFAVHWQIKEKIVITLCQHD >SEQ_ID_NO:_18_Bs_PPTase_DNA Atgatttacggcattgggctggacattaccgagcttaaacggatcgcctctatggctgggcgccagaaaaggtttgccgagcggattttgacgcgaagcgagcttgaccaatactatgagctttcagagaaaagaaaaaacgaatttctcgcgggcagattcgcggcaaaagaagcgttctcgaaagcatttggcaccggcattgggaggcagctcagctttcaggacattgaaattaggaaagaccaaaatggcaagccctatatcatttgtacgaaactgagccaggccgccgttcacgtatcgatcactcatacaaaagaatacgctgccgcgcaggttgtgattgaaaggttgtcaagctag >SEQ_ID_NO:_19_Bs_PPTase_AA MIYGIGLDITELKRIASMAGRQKRFAERILTRSELDQYYELSEKRKNEFLAGRFAAKEAFSKAFGTGIGRQLSFQDIEIRKDQNGKPYIICTKLSQAAVHVSITHTKEYAAAQVVIERLSS >SEQ_ID_NO:_20_YALI0E09603g_AA MDAIDVKGFIPENLIENETKWFYEQLSIDDSFFATESIDNIVSYIHTLYSAKIAAYARTDKKLDIQLVREDDAHAFYIDTSNPGTTNLEGPQYETRIDEKYLSSPNGGRFATNDSIPGDGALRCYFVYKCDFIEPNPKEGETDLTKISDKTFYEKATEYTRAIYSEIVNQVVQREGPVIEMFEIDGSRERRVIIGYKQETTPGYFSALSDLYHFYGLTSTRKYVEQFSNGVTIISMYLVPAFPQTANTADEIKAMKRYPPIENSIHQIVKEASLLFCLPNNAFKHHFARGDMSLQESIYAHCAFIFVQHFLNRLGSEYTTLQQMLGTGKEHVEILEKLKRRLRQETFTRDYLFELINNQLDVVKQMYLQFADVHYIQSKSEGDSFLPTLSYQRLQTQSVLSTDELKKLIRKKAANDHEAMVMEAFLTFNTHVLKTNFYTPTKVALSFRLSPDFLPESEYPQPLYGMFLVVGQEFRFFHLRFADIARGG IRIVKSRNREAYSINARSMFDENYNLANTQQRKNKDIPEGGSKGVILLNNEHQDKAEIAFHKYIDSVIDLLLKGDTPGIKEPIVDLHGSPEILFMGPDENTAGLVNWATMHAKQRGAPWWKSFFTGKSPSLGGIPHDEYGMTSLSVREYVKGIYRKLEIEQPTVRRQQTGGPDGDLGSNEILLSAEKYTTVIDGAGVLYDPNGLDREELLSLAKRRVMISEYDASKLSPEGYRVLDENDVTLPSGEVVSNGTQFRNTYHLRCESVDMFVPCGGRPEAIDINNVSQLFVDGKPKIKWLVEGANLFITQQAKLRLEEAGVVVYKDASANKGGVTSSSLEVLASLAFDDESFAKDMCIRDGVVPEFYKAYVKEVQSIIQNNARLEFEAIWREHEKTGKPRSILSDELSIAINDLSGELKNSALWDDVEFRNSVLHEALPKLLVNEIGLDVMLKRVPESYLKAIFGSYLAGRFVYEKGANPGQFAFFEYMAEKTKKQ >SEQ_ID_NO:_21_YALI0F17820g MNYPAEPEFQQAYDELYNSIHDSTLFDKHPEFEKVIPVVSVPERIIQFRVVWEDDQGKLQVNRGYRVQFNSALGPYKGGLRFHPSVNLSILKFLGYEQIFKNALTGLNIGGGKGGADFDPKGKSDAEIRRFCYAFMGELHRHIGADTDVPAGDIGVGGREVGFLFGAYKKYKNTWEGVLTGKGLTWGGSLIRPEATGFGLVYYVEKMIEYATSGKESFKGKRVAISGSGNVAQYAALKVIELGGNVVSMSDSKGALVLVSDFTEGFTPAEIEQIADIKLQHKELASLYPSATLPETKFKYIEDARPWCHVGKVDVALPCATQNEVSGEEAKSLVAAGCKFIAEGSNMGCESDAIEVFEAERMARPNGIWYGPGKAANAGGVAVSGLEMAQNSQRISWTKEEVDQKLKDIMVDCFNTCVETASTYSSEKVDGLPSLVKGANIAGFLKVSAAMKDHGDFW >SEQ_ID_NO:_22_YALI0D13024g MNWESELENDTAVKVAGIDIDGIVRGKSISKAKFLSVISKGFGFCGVIFGWDMHDKNYTKELTVSNKDNGYRDLLAIIDLSSFRRLPWENNIPFFLVHFKDSVTQEEIAPCPRSLLTAVTGLYFKDKMKAMAGAELEFYNFCLDRKDLPDSFAKLPPISTGMFGYSVQRPALNGDYFQAVWDTALKIDAPLEGWHTETGPGVLEAIAAFDEVNKLADKTSLFKLMVKSIAPQYKVIPCFMAKPQQGMPGNSGHLHVSLVDQESGKNLFARDQPDPNPEWPDVEYLSDLGRHFLAGVLDGLPDIMPFAPTINSYKRLVENFWAPVTVSWGLEHRIASIRLIAPPTGSASATRFEIRTPGADVQPHFALAAILALGHRGIAKKMPLTVPPMGDSSPDKFERLPRDLMRATEHFMRPDSLARELFGDKFVEHYGETRLHECREFMESVTAWEVDRYIETV >SEQ_ID_NO:_23_YALI0F00506g MVLSKYLDLPQHGAVLAEYIWIDAHFNIRSKCKTLDKKPTSIEDLPEWNFDGSSTDQAPGHDSDIYLRPAAIYPDPFRRGDNIIVLAECWNNDGTPNKFNHRHEKALKLMSAHEKEVIWFGIEQEYTMFDESDNPVGWPKGGFPAPQGPYYCGVGTGKVFARDVVEAHYRACLYSGINISGIN AEVMPSQWEYQVGPCEGISMADELWMSRYLLHRVAEEFGIKISFHPKPLQGDWNGAGCHTNVSTKSMREPGGMKHIEAAIEKLAARHKEHIAVYGEDNDMRLTGRHETGISGSFSSGVANRGCSIRIPRSVAKEGYGYFEDRRPASNIDPYLVTGIMTETICGSIPDADMVEETKRGEEEGF composition >SEQ ID NO: 24 ATP1 promoter Tttttttgtattcgggttaggtgttctgctttggataatagggttggggttaatggaatgacgcgctgccccctaactaggtttagggttgctccgattaggacaaacttgctccatgtgtaaactgaacaagaacaaatgattgaggcagcaacaggtgtgctgatcgtggtttataaaacggtggcatcaatttgaaggctattttcatcttttaatcttcagtattcaccaattatactcttttattccttcggaaaatgttacacaggcgggattcgaactggtgtggtgggtttctgaggctgattctttatggaggtacatttcccggcgtcgatcagaagaagaaatgcgaccagtaatgtcgcagtagtgccttttccgtaacccaaccttttaaatccccccatatcttccgtaatcgcccccaatcacctattttcgcttacccagggcacgaccccagtttctgagttcccggctaataagctttagggttagggcgagttgggagtggttgggaggtaaccaagtctcaaaattggcatatggtgaagtgtcaaaatttgggagcgatgtaaaagcctcgcattttgtgtggtatttccggtcccagcaccacgtagtgcagcatatcacagcacggcatgtatatcagggaccggttgataggaaacgcctaattgggagccccccccacaacacaaaacagacccaagggagaggcaaaaaaatatataagaccagccggcccctcccaaaggtcttgcttcccacacacacaacaaatactaaac >SEQ ID NO: 25 ATP2 promoter Gtggccggcagcattatggacggtgtaaacggccggggaaattctcgtcgcggcaattccacgtgccccaaacctcttgctccctaccccagaaactgaccactcacggctaaaaggccccgggtgagaaagtgtatgcacatccgggctcggtcgggacagttagggagcggtatatttgggggttgctaggggcgaattgacaaagaagagatatatttgcaatctgcgcgctgttgtggctctgaatccaccttctccgaaccaattgcgtgtcgggaacgtgcacaaaaacagcccgccataaaatggaccctcaactggccacgaaaataccgtgcactgcagagtctggcgaaatttgggtttcgggcgtaaagagcaatttgaaatggcgcattgggggattcgggtcattgtggacgttgtagacactcaatggagaagctgaaatatcagccgagctcggagcgcaccatagagtatttctaggtgccgaaaaacaacaattttggccggcaacggcgcgattgcaactgtctccaaccccgagatagctcccaaggttgcaaaaggttccttgtttacatgcattctcagatatgtgcagtgccccaaaatacgctggcaaagtcccaactcgcccgctgattggctgcgtttgctcccagctccattttgctacaaccttttcatctcgtgtcgcttcctctctttttctcctcgttagcctctagccagagaacacactgttacacacacagagaagctcctctttttaacaggcacgattgaacac >SEQ ID NO: 26 FBAin promoter Aacaattgccccggagaacacggccaggccgcctagatgacaaattcaacaactcacagctgactttctgccattgccactaggggggggcctttttatatggccaagccaagctctccacgtcggttgggctgcacccaacaataaatgggtagggttgcaccaacaaagggatgggatggggggtagaagatacgaggataacggggctcaatggcacaaataagaacgaatactgccattaagactcgtgatccagcgactgacaccattgcatcatctaagggcctcaaaactacctcggaactgctgcgctgatctggacaccacagaggttccgagcactttaggttgcaccaaatgtcccaccaggtgcaggcagaaaacgctggaacagcgtgtacagtttgtcttagcaaaaagtgaaggcgctgaggtcgagcagggtggtgtgacttgttatagcctttagagctgcgaaagcgcgtatggatttggctcatcaggccagattgagggtctgtggacacatgtcatgttagtgtacttcaatcgccccctggatatagccccgacaataggccgtggcctcatttttttgccttccgcacatttccattgctcggtacccacaccttgcttctcctgcacttgccaaccttaatactggtttacattgaccaacatcttacaagcggggggcttgtctagggtatatataaacagtggctctcccaatcggttgccagtctcttttttcctttctttccccacagattcgaaatctaaactacacatcacac >SEQ ID NO: 27 PGK1 promoter Tgtgcacacttggcgaacggtttagccccaaagttggactgccaccaaggataccttggcaacaagggtgtaaggtgttattatagcgacttggcagacagtgacgagtcatacattctccgtataatatcgtgtatgtccagacgatagtcgtactcgtactcgttactgtaactactgtgcgagtactcgtgcatgtatcgtaggtattgtatgttcgagtacatacacatacgataccaaacactgcccactgttctgtcatgttagatcatggccaatccacgtgacttgcatgcaggtttggcattgaatattcagcgtggctactacaagtagtacatactgtatcaatacgattgtacatacggtactcaccctttgctacagtatgtacatacaagggcgcacatggcagaataccatgggagaattggcccgcatggagttcagatgagccctaacaacgcccctgttcggcttcagaagcaattggcttttggaaattatttggcgagtgaacaatggcgtgtatggagccgtattcgtgctggtgcttgttgaatcagcccattgcgcgaaattgttggctctcacaactcaaccctctcttttaccctgtcgtgaccagacgctactgtagcgcttgtcggtcggaccacaccaaaactgggcctgtattgcattgtactcagatgtaagcaccaagagctgggatccacgtgatcgcccccacacaagacgcgtccatctgtctattgctcattctccccggcgctctccgatctcttccgacgaa >SEQ ID NO: 28 GPM1 promoter AGCAGGCACCCTTGACAACCTTTACAGTATGTACAGTAGCGACAGTATCTTCCATACTTCACTTTACAGTAAATTAAAGAATACACCAAAACTCGTTCTCAAGCTCTGTCAAACAGCTCCAAAAAATATAAAATATATATATATATATATCGCGATACCTCATTAATTCTCACGTGACACAGATTATTAACGTCTCGTACCAACCACAGATTACGACCCATTCGCAGTCACAGTTCACTAGGGTTTGGGTTGCATCCGTTGAGAGTGGTTTGTTTTTAACCTTCTCCATGTGCTCACTCAGGTTTTGGGTTCAGATCAAATCAAGGCGTGAACCACTGTTTGAGGACAAATGTGACACAACCAACCAGTGTCAGGGGCAAGTCCGTGACAAAGGGGAAGATACAATGCAATTACTGACAGTTACGGACTGCCTCGATGCCCTAACCTTGCCCCAAAATAAGACAACTGTCCTCGTTTAAGCGCAACCCTATTCAGCGTCACGTCATAATAGCGTTTGGATAGCACTAGTCTATGAGGAGCGTTTTATGTTGCGGTGAGGGCGATTGGTGCTCATATGGGTTCAATTGAGGTGGTGGAACGAGCTTAGTCTTCAATTGAGGTGCGAGCGACACAATTGGGTGTCACGTGGCCTAATTGACCTCGGATCGTGGAGTCCCCAGTTATACAGCAACCACGAGGTGCATGAGTAGGAGACGTCACCAGACAATAGGGTTTTTTTGGACTGGAGAGGGTAGGGCAAAAGCGCTCAACGGGCTGTTTGGGGAGCTATGGGGGAGGAATTGGCGATATTTGTGAGGTTGACGGCTCCGATTTGCGTGTTTTGTCGCTTCTGCATCTCCCCATACCCATATCTTCCCTCCCCACCTCTTTCCACGATAATTTTACGGATCAGCAATAAGGTTCCTTCTCCTAGTTTCCACGTCCATATATATCTATGCTGCGTCGTCCTTTTCGTGACATCACCAAAACACATACAAAA。 >SEQ ID NO: 29 HHF1 promoter Tttttttgtattcgggttaggtgttctgctttggataatagggttggggttaatggaatgacgcgctgccccctaactaggtttagggttgctccgattaggacaaacttgctccatgtgtaaactgaacaagaacaaatgattgaggcagcaacaggtgtgctgatcgtggtttataaaacggtggcatcaatttgaaggctattttcatcttttaatcttcagtattcaccaattatactcttttattccttcggaaaatgttacacaggcgggattcgaactggtgtggtgggtttctgaggctgattctttatggaggtacatttcccggcgtcgatcagaagaagaaatgcgaccagtaatgtcgcagtagtgccttttccgtaacccaaccttttaaatccccccatatcttccgtaatcgcccccaatcacctattttcgcttacccagggcacgaccccagtttctgagttcccggctaataagctttagggttagggcgagttgggagtggttgggaggtaaccaagtctcaaaattggcatatggtgaagtgtcaaaatttgggagcgatgtaaaagcctcgcattttgtgtggtatttccggtcccagcaccacgtagtgcagcatatcacagcacggcatgtatatcagggaccggttgataggaaacgcctaattgggagccccccccacaacacaaaacagacccaagggagaggcaaaaaaatatataagaccagccggcccctcccaaaggtcttgcttcccacacacacaacaaatactaaac >SEQ ID NO: 30 CYC1 promoter Tgtgtgttgttgttgttgttagataaaagtgttagtgaggagtggaaataaaagtgattttatttcgcagattgtgcacttgaattggttgtggaatgaaaagggtagaaagagggggacacgagcaggtatatataagtttggggggcgtgaggaagggtggcgcacggtagcttgggggttcaatgttatttcctgtttggtgcacgtggaatgtaatgttgggagggggttaccgccaggagtctgtgtgttacgtgttgatgcgtggtgttgctatcaactgctatcaggtttctttgtctcgtcatttcctatttgggacagtggtgggaatacttttgatgcagctctaaatgccatagtatgcgtggcgctgtaatttgggagaaatggagcctattgtgctcgcgttgggagtttgtgcgtcgtgctgtaaaatttacactcctctgcatatcagagaacggctgttagcgattaaggttctatccaaagtcacctggctagcacgcccaaaactcccaattgtgcatacaccatggccacgcacgtcgagtctaacccacatcgtgcacaaatccattcacaggacaaaagttattataaataaccccccctccccatcacatcaccttctttcgatacacacctttatcatccgcctcaaatcgagataaaagttacacactttcaaccaacacacacagaacac >SEQ ID NO: 31 HHT1 promoter Gtggccggcagcattatggacggtgtaaacggccggggaaattctcgtcgcggcaattccacgtgccccaaacctcttgctccctaccccagaaactgaccactcacggctaaaaggccccgggtgagaaagtgtatgcacatccgggctcggtcgggacagttagggagcggtatatttgggggttgctaggggcgaattgacaaagaagagatatatttgcaatctgcgcgctgttgtggctctgaatccaccttctccgaaccaattgcgtgtcgggaacgtgcacaaaaacagcccgccataaaatggaccctcaactggccacgaaaataccgtgcactgcagagtctggcgaaatttgggtttcgggcgtaaagagcaatttgaaatggcgcattgggggattcgggtcattgtggacgttgtagacactcaatggagaagctgaaatatcagccgagctcggagcgcaccatagagtatttctaggtgccgaaaaacaacaattttggccggcaacggcgcgattgcaactgtctccaaccccgagatagctcccaaggttgcaaaaggttccttgtttacatgcattctcagatatgtgcagtgccccaaaatacgctggcaaagtcccaactcgcccgctgattggctgcgtttgctcccagctccattttgctacaaccttttcatctcgtgtcgcttcctctctttttctcctcgttagcctctagccagagaacacactgttacacacacagagaagctcctctttttaacaggcacgattgaacac >SEQ ID NO: 32 HTB1 promoter Tgtgtgttgttgttgttgttagataaaagtgttagtgaggagtggaaataaaagtgattttatttcgcagattgtgcacttgaattggttgtggaatgaaaagggtagaaagagggggacacgagcaggtatatataagtttggggggcgtgaggaagggtggcgcacggtagcttgggggttcaatgttatttcctgtttggtgcacgtggaatgtaatgttgggagggggttaccgccaggagtctgtgtgttacgtgttgatgcgtggtgttgctatcaactgctatcaggtttctttgtctcgtcatttcctatttgggacagtggtgggaatacttttgatgcagctctaaatgccatagtatgcgtggcgctgtaatttgggagaaatggagcctattgtgctcgcgttgggagtttgtgcgtcgtgctgtaaaatttacactcctctgcatatcagagaacggctgttagcgattaaggttctatccaaagtcacctggctagcacgcccaaaactcccaattgtgcatacaccatggccacgcacgtcgagtctaacccacatcgtgcacaaatccattcacaggacaaaagttattataaataaccccccctccccatcacatcaccttctttcgatacacacctttatcatccgcctcaaatcgagataaaagttacacactttcaaccaacacacacagaacac >SEQ ID NO: 33 EXP1 promoter >SEQ ID NO: 34 TDH1 promoter Tgtgtgttgttgttgttgttagataaaagtgttagtgaggagtggaaataaaagtgattttatttcgcagattgtgcacttgaattggttgtggaatgaaaagggtagaaagagggggacacgagcaggtatatataagtttggggggcgtgaggaagggtggcgcacggtagcttgggggttcaatgttatttcctgtttggtgcacgtggaatgtaatgttgggagggggttaccgccaggagtctgtgtgttacgtgttgatgcgtggtgttgctatcaactgctatcaggtttctttgtctcgtcatttcctatttgggacagtggtgggaatacttttgatgcagctctaaatgccatagtatgcgtggcgctgtaatttgggagaaatggagcctattgtgctcgcgttgggagtttgtgcgtcgtgctgtaaaatttacactcctctgcatatcagagaacggctgttagcgattaaggttctatccaaagtcacctggctagcacgcccaaaactcccaattgtgcatacaccatggccacgcacgtcgagtctaacccacatcgtgcacaaatccattcacaggacaaaagttattataaataaccccccctccccatcacatcaccttctttcgatacacacctttatcatccgcctcaaatcgagataaaagttacacactttcaaccaacacacacagaacac >SEQ ID NO: 35 RPL25 promoter Accgggttggcggcgtatttgtgtcccaaaaaacagccccaattgccccaattgaccccaaattgacccagtagcgggcccaaccccggcgagagcccccttcaccccacatatcaaacctcccccggttcccacacttgccgttaagggcgtagggtactgcagtctggaatctacgcttgttcagactttgtactagtttctttgtctggccatccgggtaacccatgccggacgcaaaatagactactgaaaatttttttgctttgtggttgggactttagccaagggtataaaagaccaccgtccccgaattacctttcctcttcttttctctctctccttgtcaactcacacccgaaatcgttaagcatttccttctgagtataagaatcattcaa >SEQ ID NO: 36 TEF1 promoter Accgggttggcggcgtatttgtgtcccaaaaaacagccccaattgccccaattgaccccaaattgacccagtagcgggcccaaccccggcgagagcccccttcaccccacatatcaaacctcccccggttcccacacttgccgttaagggcgtagggtactgcagtctggaatctacgcttgttcagactttgtactagtttctttgtctggccatccgggtaacccatgccggacgcaaaatagactactgaaaatttttttgctttgtggttgggactttagccaagggtataaaagaccaccgtccccgaattacctttcctcttcttttctctctctccttgtcaactcacacccgaaatcgttaagcatttccttctgagtataagaatcattcaa >SEQ ID NO: 37 TEFin promoter Gaccgggttggcggcgtatttgtgtcccaaaaaacagccccaattgccccaattgaccccaaattgacccagtagcgggcccaaccccggcgagagcccccttcaccccacatatcaaacctcccccggttcccacacttgccgttaagggcgtagggtactgcagtctggaatctacgcttgttcagactttgtactagtttctttgtctggccatccgggtaacccatgccggacgcaaaatagactactgaaaatttttttgctttgtggttgggactttagccaagggtataaaagaccaccgtccccgaattacctttcctcttcttttctctctctccttgtcaactcacacccgaaatcgttaagcatttccttctgagtataagaatcattcaaaatggtgagtttcagaggcagcagcaattgccacgggctttgagcacacggccgggtgtggtcccattcccatcgacacaagacgccacgtcatccgaccagcactttttgcagtactaacc >SEQ ID NO: 38 TEF2UAS promoter Ctgaggtgtctcacaagtgccgtgcagtcccgcccccacttgcttctctttgtgtgtagtgtacgtacattatcgagaccgttgttcccgcccacctcgatccgggtcgacctgaggtgtctcacaagtgccgtgcagtcccgcccccacttgcttctctttgtgtgtagtgtacgtacattatcgagaccgttgttcccgcccacctcgatccgggcatgcctgcagcctagaagcttttgtggttgggactttagccaagggtataaaagaccaccgtccccgaattacctttcctcttcttttctctctctccttgtcaactcacacccgaaatcgttaagcatttccttctgagtataagaatcattcaaa >SEQ ID NO: 39 TEF4UAS promoter Ctgaggtgtctcacaagtgccgtgcagtcccgcccccacttgcttctctttgtgtgtagtgtacgtacattatcgagaccgttgttcccgcccacctcgatccggtctagactgaggtgtctcacaagtgccgtgcagtcccgcccccacttgcttctctttgtgtgtagtgtacgtacattatcgagaccgttgttcccgcccacctcgatccggggatccctgaggtgtctcacaagtgccgtgcagtcccgcccccacttgcttctctttgtgtgtagtgtacgtacattatcgagaccgttgttcccgcccacctcgatccgggtcgacctgaggtgtctcacaagtgccgtgcagtcccgcccccacttgcttctctttgtgtgtagtgtacgtacattatcgagaccgttgttcccgcccacctcgatccgggcatgcctgcagcctagaagcttttgtggttgggactttagccaagggtataaaagaccaccgtccccgaattacctttcctcttcttttctctctctccttgtcaactcacacccgaaatcgttaagcatttccttctgagtataagaatcattcaaa >SEQ ID NO: 40 TEF8UAS promoter >SEQ ID NO: 57 GAP promoter GGTTGAAATGAATCGGCCGACGCTCGGTAGTCGGAAAGAGCCGGGACCGGCCGGCGAGCATAAACCGGACGCAGTAGGATGTCCTGCACGGGTCTTTTTGTGGGGTGTGGAGAAAGGGGTGCTTGGAGATGGAAGCCGGTAGAACCGGGCTGCTTGGGGGGATTTGGGGCCGCTGGGCTCCAAAGAGGGGTAGGCATTTCGTTGGGGTTACGTAATTGCGGCATTTGGGTCCTGCGCGCATGTCCCATTGGTCAGAATTAGTCCGGATAGGAGACTTATCAGCCAATCACAGCGCCGGATCCACCTGTAGGTTGGGTTGGGTGGGAGCACCCCTCCACAGAGTAGAGTCAAACAGCAGCAGCAACATGATAGTTGGGGGTGTGCGTGTTAAAGGAAAAAAAAAGAAGCTTGGGTTATATTCCCGCTCTATTTAGAGGTTGCGGGATAGACGCCGACGGAGGGCAATGGCGCCATGGAACCTTGCGGATATCGATACGCCGCGGCGGACTGCGTCCGAACCAGCTCCAGCAGCGTTTTTTCCGGGCCATTGAGCCGACTGCGACCCCGCCAACGTGTCTTGGCCCACGCACTCATGTCATGTTGGTGTTGGGAGGCCACTTTTTAAGTAGCACAAGGCACCTAGCTCGCAGCAAGGTGTCCGAACCAAAGAAGCGGCTGCAGTGGTGCAAACGGGGCGGAAACGGCGGGAAAAAGCCACGGGGGCACGAATTGAGGCACGCCCTCGAATTTGAGACGAGTCACGGCCCCATTCGCCCGCGCAATGGCTCGCCAACGCCCGGTCTTTTGCACCACATCAGGTTACCCCAAGCCAAACCTTTGTGTTAAAAAGCTTAACATATTATACCGAACGTAGGTTTGGGCGGGCTTGCTCCGTCTGTCCAAGGCAACATTTATATAAGGGTCTGCATCGCCGGCTCAATTGAATCTTTTTTCTTCTTCTCTTCTCTATATTCATTCTTGAATTAAACACACATCAACA。 Inducible promoters >SEQ ID NO: 41 pPOX2 >SEQ ID NO: 42 pXPR2 GGTTCTGGCCGTACAGACCTCGGCCGACAATTATGATGACAGTTAGAGCAGCAACGCGTGGAGAGTTTGGGTTTTGGGTTACGTACGTAGAGCCGTTTGATAGATGGTACATCCACCGGCTAGCGGAACACAGTGTCAAGACAAGCCTGCAACACAGTCATAATATTTGCGATATTCAGGCGTATCAGGTACAATCTGAGGTGTCTCACAAGTGCCGTGCAGTCCCGCCCCCACTTGCTTCTCTTTGTGTGTAGTGTACGTACATTATCGAGACCGTTGTTCCCGCCCACCTCGATCCGGGGTCCTATGCATCCCTGAAACATTGATTGGAAATTAACATATGAGCTGCGTGCTTTTTGCATTCAAGGGCGCAGCTTATCTTGTATCCTTAATTACACATGACCTCTTGAGCGCCACGGTACATTCCTGGCGTCAGTTCGGTGGAGCGGACACTTTTCTCTCCTTTGTCTGACATGTTGGTTAAGTTGTAGTCCAGGGACACAAGGGGTTCCAACGGCAGTGGCAGCCTACCCCACGCTACCCACCACTGGCCCTGGTCTAACTTCGACGATCGGCATCAGGGTTCATGGATAGGCGGTGTGATTTACGATGTGATGGACAATGTTAGAGAGATCCCACTACTTGTAGTCAGGCCATCTTTTACGTACGCACTGTACCATGATGTCAATGGAGTATGATGAACCGACTTTGAGAGACTCACATCTGCACAACACCATGTTTCAGCGGAATCCGACTTCCAACCCAAACCCAAGCCCCTGTCAGATATCGTGAGAAGGCACGGCACCAACTAATGCACACACTCCACCTGTATTGCACCAAGATAATGAGGGCATCGTCTTGGCGCGTCTTGGCGAGAGCCGTGTTTCGTGACGCAATCAGAGCAGTTTCTGGATAGTATCTTGTCCAGAAACACGATATAAACCCCATCGACGGGCCCGTTGAAGAGCACCAACCCACTATCCAATCCTCCAATCCAACA。 >SEQ ID NO: 43 pFBP1 TCACCCTACAAAAAGTCGCGTTCGGTGCTACAATACTCACTGGAGGTTGCTTTTGCGGGGATAGACTATGAACCAGGTCAGACCATCCATTCAAGTGAGACCAAATCAGGTGTCTGGAGCATACGGGCGGGGTAATGGCGTCATTTCAGCCCAGTATTGAAGTCGAGTGTACACGTGGTTATGTGGTTGTATGGAGTGTGAGTGGGGAGCGAGTTGGAGCAATCTACAAGTAGCACATGGTCAAGTAAGTGCAAGTAAAGGTATGAACTAAGATGTGTACGATACTTAAAGATCCTTCAGTAGTCAACACAGCAGTAGCAATCCAAGCACTCTCAAACCGCCTCTATCGCCACCTCCATCGCCACCTCCATCGCCTCCACCGCTCCATCACCACCACCTCCACTCCACCCCCGCCACGTGATAATCCAAAACCGCGTGAAATAGTAAAGGTTGAAAAAACAGACAACTGAGGTAATTGAGCTTAGGTGTCGTGTGATCTGGTGAGCCAATGCGGCGACGGAAAACAATCGCAAAACCCCGGTTGCCACAGACCACATGCTACGTCACGTACACAGTCACGTGACCCCCTCACTCCCGCCGTTTTCTCGCAAAACACGTTGCTACAACCATCACAGCCGAGCTTTACATTTTGGACATACATGTCGCAGGCCGACTCGGAGATTTTTAGCTTTGCGAGAGGGGCGTTAAATGTGGCTCGATGGAGGTGTTTGGGGGTCACATGGGCCGTGTGGCGGGGCTCAGTGTATTGTACAAACGACCAGCTAGAAAATGGTATAAATATCTCTGCTACCGCCGGCTTATTTCCCCACACAAACCCCACAATATGTGGTGTTCCGGACGGTTCCGAACCACGGCAATTTCCCGGCCAACCTTAACCACTTAAACCTTCGGCACGGGGGTACAAGACACTTGGAGCGTGTCGTCATCTCCCCTTCTGCCCCAATCCCCCCCTCTCTCAACAAGTCCATCACATTTCACA。 >SEQ ID NO: 44 pMDH1a acatgcgtccagtgtaccccggataacaccattagtgtggctaatagcgaggatggggacttggagggatgggttcgcagggatggactcggagggatggacgcgaatggcgtggagggctcggatggcgcggagggttcggagatgggtccagggccaaaaatccggtttaaaaaggtggatatggtcgattgtaggctcaatggctagatcgatacattggtttgtgtgttgtgagtgggtccgacccgatacatgtgtgaacatgatgtaaccgtggtgtatatggtatagcttatagacgatacggaaatatcggagatagagcgaaggggccattgacgggaggacaccgggagaatcggtggtgacatgggataattgcaggttgacaaccgtcgccaacgcttttgcccgcccattatctcctctaaacagctcaattaaatgtctctgttttttcgagtccacgccaaagtggtccttctgttcataaacttcacatgcacatctgagcggatgtgtgggagatgttatcgccgaggcagttcagaacggcgaataatgatcggaatggcgcgggaaaaaggcgaaatgaattcgattctcgggaagagtccggttatcgacccagatgtctctccgctcctccatttgcgcccccatctccctctccatcctgcattgcgccattaactctaaaacccattgagttgtgtctgcacgctggcctctcatttgccatttaaacccgtccagtcccacccagcgaccttccccaactccacatcacatcaaa >SEQ ID NO: 45 ACL2 GCACTTTACCTGCACCTCCTCTCCTGCAGCTCGCTGAGAGTACAGAAGCTATTTACGCACATTTTTTTTTAACGGCGGCGATTTGGGGCTGAGAAAAATAGAGTTGCGACGAACCCGAAGGATAAGACGTTAGCGAGAGATAGCGAAAAGGGGGTTTGGAGCGGTGCAGAAAATCGGAGGAAATTAGGGAAAATCGGGGAAAATGACGACAATTCGAACCGAGGCCAATTGAGCACGCCGAAGCAGTTTCAACGCAATCGGCCGCGATTCGGACCAGTTGGGCCGCTCTTAGGCAGTCACCAACCAGATGTTCTGCACGGATTTCCCCGATTGTGGAAATGAAATCGACGCCGGCGTCATGGCAATCGACAGTTGGGGGAGAGATAAATCGGTGGTCACGTGTACACCCCCCACGAGCGTCTCCCGTGCCACTTTCTCCCCAGCGACCGTTATCTCAACTCACTTCCCCTCTCTCAACAGTTAACCCAACCCTCAACCAAATTGACGTGGTTAGCGGTTTGGCAGGCGACAAGCGAAATTGACTTTTCCAACAAACTCAGCACGCCACCACGCCCGTTCGCCATGCATGTTGTACCCCTCACCGAGGCCACCAGGATTGGGTCAAAGTGAGGGGGAGTATCAACAAGAAGGAAAAAGGGTCTTTTGGGAGCTCCAAAATAGTTGGGGAGGCGGACCAACAGAGACATATATAGACTCGAAACCAATGTCACAAATCCATCTTTACCAAATAAAGTTAGGGACACAAAACCGCTCTCCAAACAGGATATAATTGTCCCACCACCACCACACACACACACCACCCAACCCAACATTTAGCCTCCGTCTGCTACAACTTAATCAGGCACGACTCACTCTTCTTTCTACGTGACACAAACAGAGCGACACCTACGGAGCGACACCCCCCTCGACCGACACAGAAACAGCGTCGATCACACACACACACACACACACACCGAATCCGACACTCACCAGATCAATC。 >SEQ ID NO: 46 POT1 CACAATACCCCACAGTGTGCATATCAAACCTACCGGTTGTTGCTCTCTCCAGCCTTACTAAGAAGGAGGCGACGTGGCAGTGGCTCGCGGGAGGATCGGCGGGAAACTCCGGGATATCCGTCGAGAGTTTACACGTGAATGGGCAGCGCAATCCGTTGACGACGATACGACTGGCAAAGTAGCGACGATACCTGCCAGACAGGTGACATGTGCAGGCCGCACTAACAAGGAAACGGGCGCTGGGGGGGGCGGGCTTCTAGACTTTGCCCTTGAACAGGAATCTAGTGGGGGCTTGTCTTTCCGCCAATGGGGGAGCGCCTGTTGAGCGACCGTGCATGCTGGAACGCCAAGTGTATGTACAGCTGGTGGTCTCGCAGCGGTATGTGACGGGACTTACATCTCTCGTTTTTTCATGACCACGTTTTCACAGGCTCGGAGGTACGTTAAAGTTTTGAAGGCTGCATCTGAACCGAGGTATGGGGGAGTTTGAAGAGCAACAGTGTTGGGGCTGAGGGGGCCAAGATCGGGGCAAGCAGAGGTCTTAGATCAATTGTGGGGATCCCAAAGGGCTCGTTATCACCTTTTTCCACCCAATTCGGGTCCCAATTGATCCACTACTGGCTTGCCCAAGTTACCCCAGAAATGCCGCCCCGGATTTCTCCAAAAACCTAATAAGCTTCATGGAACTTGGTGGAAGTGACTTTCTACAGAGTGGAGAGAACCGTGGACACGTGGCAATGGCGCTGACCGTGTCCCCGAGCCGAATCGACGTGAGGGGAGAACGGAGTATCTGCGGTCATGTGACCTTCCAGAGCGGCGTCGCCAGTGTGCACGCGGTGACCCCCAGTTTGGGTCTCTGTCACACGCATACTACCTCGGCTCTCCACATGCTGAACTTTATCTTTCGTGGGGATCATACCGAAAGTTGCAACTACCAGGTGTATATAAAGCCTGGTAGACTCCCCCCACTTTGGACCTCATCCAACCAAGACACACAAAA。 >SEQ ID NO: 47 LIP2 promoter AAACTTCTCCGAGTCTGTGCCTTCAGGTGGGCATAGTTGATGGGTGTTTTGAAGTTAATAGTGGGGAAGAACTATGGCAAACAAGCAGATGCAGGCACCTTGTAACTGCAGACCGGTTCTTGTCTACCGACTCCGCTGCACCTGTGCCGCGGTACATGTCGTCACAGGCTGCGGGGTTCGGAGGCCCCCTTGCAACCTCCTTTGATAGTTGCTATGGCCCCAAAGAGTTATACGAGATAGACCCACAGATCTACTTGACTGTTGTCACAGAACCTGCTAGGTTTGCTTATTGTACCCGCTTTGTAGCTACTGTACAACGACAACGTCAAAAATTGAGACGCGAACAAACTCCAGATGCAGAACCCAAACCTCTCTCTCAGAGTTTCGAGTGCTTCTACCTCACAGTAAAGTGGAGGTGGACCTGCAAGGGAATTCAGTCACAAGGCCCCGAATGTCTCCGAAACTCCAATCGGACCGTTTAAACAGACTAATATCACGTCATTGATTGATATTAGCATCCGGCAAGAGCCGCAAGGTTATCTCCTCACCAATGAGCCTGTTGTACGGCTCATTCCGCATCTGCGGCTGATTCAGTTTCGAGTGGGGATGGTAGACTTCATTGCAGCATTCCTAACCTTCTACTTGGTCCGTGGAGATGTCATGGACATCGATTTTGGGCTGAGAAGCCTTTTGACGATGTTGATATCACTGACCGCTAATTTACTCTGGCAGTTTCTCCGGCTCTCGAGGCATCGTCGATCACCAAACACTATCTGCTAGTCTAAATGTCCGACACGACAGCTTTTGATCGCCGTGAACGGCGCAGACCTCATGCACCATGCACCAGGGCCAAATCAATTACGGGTCGCTTAGCGTTGCAGTCGGGGCATTATGGTGGAAGTTCCGATACGGCACAGACACATTCCATAGTGGGGGGATTGGATTATAAAAGGGCCATAGAAAGCCCTCAATTGATACCCAAGTACCAGCTCTCCTCACT。 >SEQ ID NO: 48 ICL1 promoter CCTTTTGCCAGTATATCCACCGCAGCACCCACCATGAGCGACATCTGATATCGTGCCGCGACCACTACCCCAAATAAGCTCCAACTAATATGCCGAGGCAGGTGGGAAACTATGCACTCCAGTTGACGCTGTAGAAGCACATGGAAGGTGCGGAGGCGGTGGCAACGAGGGGCATGAGCCATCAACGAGGAACCACAGACAAGGCAAGGGGGGAAACGCGACCGGAATCTCTCGCGGTCACGTGACCCGCCCGGGTTCACTCGTCCATGTTGTGTCTCTGGTGTCTTCGGCCGACTCGCATTGGTTAAACTTCCACCACCGCAATCACGTCCCACTGGCCAAACTTTTTCTGCTTTCTCTGACTTTTTCTGGCCAAAAGGCAACGTCGGAAAGGGTCGGGAGGATTCGGAACCGACGAAAATCGGCCGGCTCCAGCGGGGGTAGTTCGGCAGTCCTGGTGGGAGCTCTAGGGGAGCTGTGGTCTGTGTAGGGCGCGGGTCCGGGTTTGTTGGGTGTCAAATCACGTGTTTTTGCCCCCCCGCTGAGCCGGACTCCGACAACCGTGTCTCCAACGGCCTGACTAAGCTGCTCCCAGCACTCTGCCGTAGCGTTGGTCTGTCCTGTCGCACTCTGTTCAAAGACAGAAGAAAGAAAAAGCTAACCTCCACGTCAGAGACAATGGTAGAAGGCTTGTTCCTTGCAACCGAGGAGAGTGAGTGTTCTCGGCACGAGCATCATGGGCGATCTGGAGGGTATTTTTGAGGGGAAAAAACGGGATCAGGACAAACAGAGGCCACAGACCGGGAATCTGGGCCCCAAAACGGCCTTTTCCCGTCGCAAAACCGGTCTACATACACCCCTTCGGCCCGCCACAGGCCGGTGTGAAAAACCCTAAAGCTTGCTTCAAACCAGACGGACGCACAGCAAGACACATCATGAAGAGTCACCTGCAGTATATATAGATCTGGGGATCCCCAGTAGACTGACCAAGCATACAAAA。 >SEQ ID NO: 49 YAT1 promoter GCCGCGATTTCCGTAAGTGAAAGTGGTATGAGGAGCTCTGTCACTGAACCTGTAAGTACTGTACCCCGATAGTTCGTTGATCGTGCCCCAGACTGCTAAGGTTGCTGATCCCCGCTACCCCAGACTGTCCCGCAAGAAGCAAGTCAGTTTTTACCTCTCTTTTTCTTCCTCGTTATGTCATATGTCTCACAAGATAGGTCATGATACTGGCATGCACAAACCAAGCACAAAGTGCATCAGCCGCAATTTGAATCCCTTGTGCACCCGTTGTTTCATAGAACTACTTGTTCAACTATGTTTTGCCTCGGCACCGTTGTACCGTCCAGTTCAACACTATAGTACATACTAGCCCACCGGTCCCGTGCCCTACACAGGAATATATGACTATGGGAACCTCAAGAACCTACTTTACGAACCTAACGTCGTGGCTTTTACCCGAATATTGTACCAGCTGTAATAATGTTACCCCGGACAAGCGGAGACCAAGAGAAAGGCAAAGGAAAAGGCAAAGTAGGACAATACAGGACCAAATAGCGATAGCCATTGCAGTCAACCTAATTGGAGAGTGTAGGAGTGGGTGGATGGGCTGTGACTGCTTGCAGTGGAGGTGCTCAAAATCAATTTTCATGACACAGTATACTTGATTCTAACCACACTAGTCTTGTTTACTTGTTTCCATCCGGTTGTATCTGTTGTCACTTATTACAGCTCCCCTAGAAAGCGTTCCGGTGATCCGACTTTAGCTCGGCCAGCATATGCACCCCGGACTTCTGAACCTTTGACTTTGTTTGCAAAAGTTGGTTTGCAAACCCTGTCGCATCATCATGACTGCAACCTGAGCGAGATATAAGTGTCCACCAGTCCGTCGTCAAAACACACACCCCACCAGCAGTCTATCAAACTAACACTAGCGGTACCAGCGCACAGAAAACTGCCACTCGCACACTAACCTCACGCCCACACTACCCGTAACTACACAATCGCTTATTACACATCCACC。 >SEQ ID NO: 50 CTR1 promoterGCCGCGATTTCCGTAAGTGAAAGTGGTATGAGGAGCTCTGTCACTGAACCTGTAAGTACTGTACCCCGATAGTTCGTTGATCGTGCCCCAGACTGCTAAGGTTGCTGATCCCCGCTACCCCAGACTGTCCCGCAAGAAGCAAGTCAGTTTTTACCTCTCTTTTTCTTCCTCGTTATGTCATATGTCTCACAAGATAGGTCATGATACTGGCATGCACAAACCAAGCACAAAGTGCATCAGCCGCAATTTGAATCCCTTGTGCACCCGTTGTTTCATAGAACTACTTGTTCAACTATGTTTTGCCTCGGCACCGTTGTACCGTCCAGTTCAACACTATAGTACATACTAGCCCACCGGTCCCGTGCCCTACACAGGAATATATGACTATGGGAACCTCAAGAACCTACTTTACGAACCTAACGTCGTGGCTTTTACCCGAATATTGTACCAGCTGTAATAATGTTACCCCGGACAAGCGGAGACCAAGAGAAAGGCAAAGGAAAAGGCAAAGTAGGACAATACAGGACCAAATAGCGATAGCCATTGCAGTCAACCTAATTGGAGAGTGTAGGAGTGGGTGGATGGGCTGTGACTGCTTGCAGTGGAGGTGCTCAAAATCAATTTTCATGACACAGTATACTTGATTCTAACCACACTAGTCTTGTTTACTTGTTTCCATCCGGTTGTATCTGTTGTCACTTATTACAGCTCCCCTAGAAAGCGTTCCGGTGATCCGACTTTAGCTCGGCCAGCATATGCACCCCGGACTTCTGAACCTTTGACTTTGTTTGCAAAAGTTGGTTTGCAAACCCTGTCGCATCATCATGACTGCAACCTGAGCGAGATATAAGTGTCCACCAGTCCGTCGTCAAAACACACACCCCACCAGCAGTCTATCAAACTAACACTAGCGGTACCAGCGCACAGAAAACTGCCACTCGCACACTAACCTCACGCCCACACTACCCGTAACTACACAATCGCTTATTACACATCCACC。 >SEQ ID NO: 51 CTR2 promoter TTCAGCCGCCATTTTAGATGGAAGGGACGTCAATCTTTTGGCAGAAGGCCTTGCATGTGCTGAAGGAAGTCATACCCGGTAGTGTAATACCTGGCTAATTCCTCACATAATGGTGAGATGTCACCACATAGTGATGAGAGAAATCAGCTGGGTCGTAGACGACGAGATGTCCATCCTGTAGATAGTTTTATCCACTGATCTAGTCTTTTCTGGCGAATGAGCAAGCACACACCCTTCTGGTGTGTGCTACTATCGGAGTGGGTTGTAAGTTGTTAACTATATCACATCAATAATACTTATGATCTTCAATAGGATGTTGGACAATTGACATAAAGATGCTCGTGAAAATAAAAATAAATAAATAAAATAAAATAAAATAAAATAAATAATAATATATATATAAATAACCCTAGGGAAATACGTGATCTGCAGTCATCATCGGACCCTCAAGTGAATAGCCGCTGCACAATCCACAGCCTTACGAAGTTCGATTCCCTCCTGCACTTAACGGCCAAACCACACGGAATAGGGAGTAACAGCGGGGCGAGAATACCGAAAGAAAGGAAAAGAAAGACAAAAAAGGAAAGAAAGACCAAAAGGAACCCTTCCGCATTCATACAACTTGCCGATTCATTGAATCGAGCATGTACGTACTCCCCGATAAGCGATACAGCAGTTGACTCTAGATACACTTGCGGGGACATCATACTCTGTACAGACTTGACAAGTGATCAGGATCAGACTTGACAAGTGATCAGGACCTTATCCAGCAAACGACATGAACCGCAATTGCCACACAATATTCCGGTACAAGTGTTCGCACTCGAACTGTGCGCCTCAAGCTAGTTTTCCGGGTGGCGCCAGCCACATTTGCCCTCTATCTCCATGCAGGTTCGAGCATTTTTGAAGCGTGTGTGAGATGGCTATTTAATTAACCCACTCCCAACCCAGAGAACATCAGATAACACAACCTCACACGTCCACACGTCCACACAAAAAA。 >SEQ ID NO: 52 pYALI0B18194 promoter GATTTGTGTCTCCACTCCATCTCCAAGGGCCGAAAACCTGTTGCTGACACTCTCCCCATCACGAGAAACTATCTACTCCCGGAAATGCTGAACCGCATCTGGAAATCCCATGCATCGTGACGCATATCTGTTTTACAGATCGTAAATGCTCCCGTATACACTCTGCAGGGCTTTTTGTACACCTGCAGCCCGTATGCAGGCTATCTACTACTGCGGGAGCTTCCGCTCGTTGGGTGGAAGGACTTGTCCAAAATGTCCTGGCATCCTTGTGCTAATATCTCTACATAGATCAGCACTGAAAAGTCCACCCAACCACATAGATTAGCAGATTTCCAGAGGACACATGGAAATACCCACTGTTGGGCATCCAACCGTACCCTTGATTACCCCACCAACAACCCCTGTGGTTTCTTGTATCGTACTGTACTTGTACATGTACAAAGCCGGCATTGAGATCCCGTATTCCGCGTGCCGCGCATTTGCTCAATACACTGTTGCAAATTTGGTTTTCTCAACAATACGGCCCTGCACAATATCCAAACCAGAGCAAAGTGACTGTGAGATTGGCGAACATGGACAGCAACATCTTCAAGACTATCCGCGAATGCTATGCAGAGAGTTGGACAACTTGACGATCCAATGGACCCGGACCCAGCTTTGATAGATAATATACCATATCAAATGACACTCGATATATCTCTCGACCGCTTCAAAATGCTTGACAAGCGCGTCGCAGTTTGTCAATGTTCACGAAGACAAAGCCGCTGTACCTTACAGTACAGTAGGTAAACTGCAGCCGCTTCCGGCTTGCGAAATTTCCCATCAACCTTCGTACATTCCGGGAATGGACCAGTTGGGAAGGGCAACTTCTGCAAGGTGCCAGCTCGAGATGGCAGTCTATATAACCGCTCACATCTGCTTGTTTGGAGGTTAGAATCAAAAGCTTCCCCACCCACCACAGTCTCTCGTTATGAAGTTCTCAGTCCTCACTCTCGCCGCC。 >SEQ ID NO: 53 pYALI0C11165 promoter GCAACACGAACAAGACAGTTAAAACTGCCGCCAACACTTTTCCTACTCCCACAACTCCCCAAAACACATCCATTTGAAGACCTCAGTACAGTACTAACTGGATGCAAATATTGCAGGACCCTGCACCGTGACGCACCCCTTTGGAGATCGGTGTGTAAAGCCCCGTATACACGCTGCAGGGCGGTCCGTATGCCTGCAGCTCTCACGCAGGCCATTTGTACCCTGCGGGAGCTTCCGCTTCTCGTCTAGACATTTCTCGAAGGTCGAACGATTGTCGACACTTGTCTACATGGTTCGATGTCAATACCACGGCGGAACGACACTAACCGACAACGTCCCCAGGAAACACGGGAAAATGATGTTGTTGTCATCATCTGTTCCTGGTTCCGTCATGAATACCCCTGAGACCCCCCAAAGTCGACCCGCATGTACGCCGCCGAGATGTACGGCGTGCCGCGCATTTGCTCACAATGCTGAGCTGAATTTCCTTCTTTCAGGAGTACTGTACGTCTGCGGACCGTGAGAGATTGCGCGATATCTTGTATACAGTACCAAGATAACTTCCTTCGAACGTGAGCTGTTTGGTGAAAGAAGTCCGCCTAGTGGTTGGTACCTCGAGTCTATCATACCAAAATATGTTCAACCGAAACACGTCTGGATATATATCCTTGTTAAACACATACATTTGACATGACGGACAATACGAGCGCGCGCGCCGTTGTCAAACGCTCGTGAAGACAAAGATACAGCCTGGCACAGTACAGGAACGTACAGGGGTTGCCGGCTTCAGCCGCTTCCCGCTTCGGAAATCTTTTACCAACTTACTTGCGCGCACATTCCAAGAATGGAACTGGTGCACGTTGGAAATTCCCACAAGATAGCAGGGGTCTCGCTCCCGTCTATATAAACACATGCCATCCCTTCTCTGCAGTTCAGAATCAACATCCTCACCCCACCCTAGTCTTTCGTTATGAAGTTCTCAGTCCTCGCGCTCGCCACC。 >SEQ ID NO: 54 pYALI0C15004 promoter TACAGTACATACGGTACAGTACATACATTACAGGACAAGTACCGTCATGGATCGGAACGACGTCTGGTCGATAAACCTGTAGAGATAGAGCAATTACATCACCAGGGAGCGTCCCAGTACCTTCCACCAACCAGACAGTTGCTTTCCACATCTGCATCGTGACGCAACTGCCTTTTTGATTTTTTTTTTGAATTTTTTAGAGATCGTAAAATTTCCGTATATACCCTGCAGGGCTTTTCGTACACCTGCAGCAGGAATGCAGACTCAATCTCGTTGCGGAAGATTCCGTCCCTTACAAAAAATATTGTGCTAATTGAACGATAGTCGTCGTATTTCTTTATGGAATTGAAAGAATGATGCAGATCGAGCTCCTTAGGAGAATATCCTAGGATGTGACATACATGGAAGGTGGGGGCATCGTGATGGGGAAGGCATTGCACCCTCATTTTTCATCGTGAAGAACCACCATTATATTCACTGATAAGAAAACTGCAACTCATTCCCGGAGACCGCATGCCGTGCGTTTGCTCAGTATATCTGCAGAGTCATGGTTTTCTCAACAATACGTGCGGAACGCAGAGGTTGACAATGGCTGTTCGGGCCCAAACGCTTTCCCAACTTCGATACTGCTCTGCACCCGTAGTGAGCACGTTTGCTAGGGCCTTACCGGTGAGAAAAAAAAAAACGATGACAGAACTCGTGAAAAGTCTCAGTAGCGCCGATCTTAGACTCATTGCGACAACGGTTTTCAAGCGTCTTTGCAAACAAAGGCATCGTACCTTACAGTACAGGAGGTTAACTTCAGCCGCTTCGGTGTGAGTGAGGAACGTTTCCGCAACTGGCGCACATTCCAGGAGTGGAAAAGTTGCACATGAACGGGATATCTGCATGAAAATTGCTCCCGGTCTGCCCCTATCCAATCTATATAAACCCATGGAAGACCCTGACTACTTCTTTAGAATCAACAGCCTCAGCTTCTCTCTTACCAAAGTCTCTCATT。 >SEQ ID NO: 55 pYALI0E14256 promoter ACTTGTTTGACAAGAGCCAAACGATACACCAATGTGTATGAAACCTACGCACTCTTGGCATATCTACTGGTACTGTAGCGACAGATTCACTTGTTGAGAGCTGTGCTTCCGAGCATCGGATGTACCTCTTTCTCATATAATTATCGTCAATAATACCGCGCATAACCCAGGCACTCATCAGGGCTGTACACCCTCCTCTCCAATGGCAGGCGCTCGTAGCAGCAACTAAACCTTGGGGAGGGGGCGTGATCGAGGAAAGGGCTTCCAGTGCGTACCACACACGTATATCGACGTAATCGTGCCATGCAGACGGCGTGAGATAGTGTAGTTTGAGCTGTATTCTGAAGCCGGTCTGCCACCGTATGTATAGGATCCACGTCCAAGAAGCCGCCTCGCTGGAGCCACCGGATCATACCCCATGTTCCAATACCCCGCTGAAAGGACAAACAGAAGCCGGACCGTGCGGTGCGGCGAGATATTCGGATTTGGCTCCATTATCTTTGTGTATCCGGTGCAAGTCGGCTTTTGCGGCTCGGAAATGGCTACTTGTAGCTCTGGGTTTGTGTTTGAGGGGAGAGTTGGATATGGAAAAACGTGGATGGTGAAGCCTTCGGGGAATTGGTGTGGTTCCCAATCAACTACTAGGTCAATTGATGCCGTCTTTTGGAGATTTCTGGACGCCATTGAATTGCTGTCCATGAGACACCCCATATTCGCTTAAGCAGCTTCCTTACCTTAGCGAGGCACAGAACATTCCGCCTGTCAGCCCCAACCCAATCTCTGAGGGCCACAACTCTCCCCCAATAGCCAGCTGCCCCAGTTGCTCGATCAGCCACCGAAGCTTCAGACAAGGCAGTTACACACTGAGCCTCAAGGTTGTGCGGGCGGATGGGGTATAAGGGTTGAGGTGGTAACCGTGTGAGCTCAGAAGATATATAAAGGGGTGGCCATGTCCCCCTATCGCTCCTTACCAAACAACAAACAACAAACAACTACAAT >SEQ ID NO: 56 pYALI0F13937 promoter 。 >SEQ ID NO: 58: pXPR2 promoter TGTATAGTACTGTACCTTCAGTAGACTATTGTAGCTAACATGTCGTTGCGTGGCGTATGTACCAAGCCACAGAAATTATGTCAGAGATAAGGTCGCGACAGTTAGAGCAGCAACGCGTGGAGAGTTTGGGTTTTGGGTTACGTACGTAGAGCCGTTTGATAGATGGTACATCCACCGGCTAGCGGAACACAGTGTCAAGACAAGCCTGCAACACAGTCATAATATTTGCGATATTCAGGCGTATCAGGTACAATCTGAGGTGTCTCACAAGTGCCGTGCAGTCCCGCCCCCACTTGCTTCTCTTTGTGTGTAGTGTACGTACATTATCGAGACCGTTGTTCCCGCCCACCTCGATCCGGGGTCCTATGCATCCCTGAAACATTGATTGGAAATTAACATATGAGCTGCGTGCTTTTTGCATTCAAGGGCGCAGCTTATCTTGTATCCTTAATTACACATGACCTCTTGAGCGCCACGGTACATTCCTGGCGTCAGTTCGGTGGAGCGGACACTTTTCTCTCCTTTGTCTGACATGTTGGTTAAGTTGTAGTCCAGGGACACAAGGGGTTCCAACGGCAGTGGCAGCCTACCCCACGCTACCCACCACTGGCCCTGGTCTAACTTCGACGATCGGCATCAGGGTTCATGGATAGGCGGTGTGATTTACGATGTGATGGACAATGTTAGAGAGATCCCACTACTTGTAGTCAGGCCATCTTTTACGTACGCACTGTACCATGATGTCAATGGAGTATGATGAACCGACTTTGAGAGACTCACATCTGCACAACACCATGTTTCAGCGGAATCCGACTTCCAACCCAAACCCAAGCCCCTGTCAGATATCGTGAGAAGGCACGGCACCAACTAATGCACACACTCCACCTGTATTGCACCAAGATAATGAGGGCATCGTCTTGGCGCGTCTTGGCGAGAGCCGTGTTTCGTGACGCAATCAGAGCAGTTTCTGGATAGTATCTTGTCCAGAAACACGATATAA。 >SEQ ID NO: 60 - according to Figure 13 , consensus sequence between BpsA-X5 and BaspA-X5-UNC: ……kslq#TnliEdd..lrgdpcLld$LakraaeHPEaiAYadrdakhTYrElahrssanA#gLrhagddqddC!GL%fePSaD$nlGAWgILaanaAYLPLal#Ype#e#RLRYM!E#Sqaq!!Laq#rLkerLreiap#d!r!VTLrEl#a..Lpegqkananedarp#rLAYVIYTSGSTGKPKGVMIEHrSIVaQLrWLrrcggidreks! LQKTPMSFDAAQWEiLalAnGaTVVMGapG!YaDPEaiIeT!qrhGVTTLQCVPTLLQaLlDhelFp#CeSL#QIFSGGEALsRaLAq#lfre$PgraL!NlYGPTE CT!NaSs%aldqaeineaP#aISIGaPVA#TeYH!L.rEDgePagVnEIGELhIgGQlarGYhnRP#LTAErFienekaegaGherLYKTGDLaqwNaDGTVQFaGR AD.QVKLRGYRVELDEIrLAIENH#WVrnAAV!VK#DaRTGhQNLIACVELdEk#AALMDQGr.dagHHaSKkSKLQVKAQLaNPGLR#dA#LaaRpayrLPGaeasaEQRqaaFARKsYRFYEGgaVTraDiLaLLGgrpraapSaraaDiaga.ELGqILRnFGrhLS#ERLLPKYGYASPGALYATQLYFELeGmeGLQPGyYYYqPqrHc L!hIralaadcahcanIHFIGKRgaIEhVYKNN!qEVLEiEaGHilGllE#VLPalGL#!Rdda%ePAardrLDVaEEDhYLGTFalaP.HgGrRe#eaEl%VQaHG drVanLPeGqYRYa#GsLerlg##!VdrrqVIAINQrVY#aadFGISm!crae#edmHYVhLGraLqrlqmNgqgLGmrS…….GnPLPASrRiDdlLqaaGVeaaP.SYFFVGGr!SaEQaeHEGMrEDaVHmrGPAELir##LeqqLP#YMIPNrVlVl#rLPqsaNGK!DaaALaalDalnaed!#RemValrTEaEKaIa#!Wars$rrESVS aQDDFFESGGNSLhaVal!rrlNrRlGtrLPLQsVl#sPmladLaari#reaaqeaSR$VRLnAE.GkDrqVlaWPGLGGYPMNLRpLAaaiGleRpfhG!QAHGINeG EaPYad!raMAaAD!EAIrEiQPhGPYlLcGYSFGARVAFETAaQLEaaG#eVdaL%LiAPGaPttraEreKaYgrEArFaNra%laILFSVFagrIeGkdL#rCL#la rdE#dFagFIc#.lkei#e#LaKRIIrVVaqTYeFeYqFtELA#RkLaaP!TTlKArGDdYSFiENSng%SaePPk!I#L#aDHYqlLreadIqELkqhir..hdLeE…. in: !-Isoleucine or Valine β-Leucine or Methionine % - Phenylalanine or tyrosine # - Asparagine, aspartic acid, glutamine, or glutamic acid - Gap Example Example 1 - Production of natural blue pigment Yarrowia lipolytica Strains By integrating expression boxes into Yarrowia lipolytica A proof-of-concept strain was produced in the genome that overexpressed codon-optimized genes encoding BpsA and PPTase enzymes.

[0225] Figure 1 This demonstrates the process from engineering Yarrowia lipolytica Natural blue pigment obtained from the biomass of cells Yarrowia lipolytica yeast Microscopic analysis of natural blue pigment extract.

[0226] Example 2 - Effect of carbon source on engineered resolution of Yarrowia liposa native blue pigment biosynthesis in bacterial strains Overexpression generated in Example 1 BpsA and PPTase of Yarrowia lipolyticaThe strain was cultured in flasks in two liquid media—rich (YPD and YP4D) and basal media (YNB glucose and YNB glycerol). The tested media contained 20 g / L of carbon source (40 g / L in YP4D). The highest product titers were achieved in basal YNB medium, reaching 2.6 g / L and 2.5 g / L with glucose and glycerol as substrates, respectively. Figure 2 A). The amount of natural blue pigment produced was significantly lower in nutrient-rich media, reaching 1.5 g / L in YP4D and 1.1 g / L in YPD. Conversely, cells grew better in nutrient-rich media, such as… Figure 2 As shown in B. Tracking the pH change of the culture over time ( Figure 2 C). In nutrient-rich media, the pH increased slightly between 72 and 96 hours. However, in basal media, the pH dropped significantly from 7 to 3 at 48 hours, and then stabilized at pH 5.

[0227] When cells cultured in nutrient-rich media are compared with those cultured in basal media, cell morphology differs. Transformations cultured in YPD medium are oval-shaped, while those in YNB medium show a significant change in shape, exhibiting both oval and elongated hyphae (e.g., ...). Figure 2 (As shown in D). Additionally, when cultured in YNB medium, a blue ring of the natural blue pigment was observed on the flask wall (as shown in Figure D). Figure 2 E), which may be due to the rupture of elongated cells and the subsequent release of dye.

[0228] Next, we tested a range of carbon sources to determine the optimal feedstock for the production of natural blue pigment. The proof-of-concept strain was tested in an analytical grade (20 g / L) carbon solution. Figure 3 A) and renewable and waste carbon sources (at different concentrations, Figure 3 B Figure 3 C) The culture was carried out in YNB basal medium in deep-well plates for 120 hours. Therefore, the optimal substrate was glucose (5.2 g / L natural blue pigment), followed by monosaccharides and mannose, and then the polyol glycerol. Acetate, along with vegetable oil, resulted in natural blue pigment yields ranging from 0.34 g / L to 0.56 g / L. When citrate was used as the sole carbon source, the achieved titers were negligible (0.07 g / L).

[0229] Carbon sources can account for up to 30% of the total cost of bioprocesses, making the search for cost-effective raw materials valuable. Lignocellulose hydrolysates offer a cheap and sustainable source of raw materials for bioprocesses. A proof-of-concept strain was able to convert carbon present in the hydrolysates into a natural blue pigment, with the highest titer (2.9 g / L) obtained from urban trimming waste. Figure 3 B). Additionally, using waste glycerin from biodiesel plants and waste cooking oil (WCO) from local restaurants, 0.73 g / L to 0.79 g / L of natural blue pigment was produced. Figure 3 C).

[0230] Example 3 - Nitrogen quality and availability on engineered Yarrowia lipolytica biosynthesis of natural blue pigments in strains Influence Natural blue pigment molecules contain four nitrogen atoms (23% of the total mass), making this element a significant component of the pigment. To assess the quality of nitrogen (reduced or oxidized form)... Yarrowia lipolytica The influence of natural blue pigment biosynthesis on the study was investigated using several nitrogen sources ( Figure 4 A). This experiment determined that ammonium sulfate and urea are the most suitable nitrogen sources for the biosynthesis of natural blue pigment.

[0231] The C / N ratio (nitrogen availability) is another important parameter in microbial bioprocessing because it affects biosynthesis and growth. Therefore, the effects of the C / N ratio on the yield and growth of natural blue pigment were analyzed. Figure 4 B). The optimal C / N ratio for this process is in the range of 2 to 20.

[0232] Example 4 - Effect of aeration on engineered Yarrowia lipolytica natural blue pigment biosynthesis in a strain Oxygen availability is a crucial factor for biosynthesis and growth. To assess the effect of aeration on the yield of natural blue pigment, experiments were conducted in flasks with three different shaking speeds (150, 200, and 250 rpm) and in 250 mL flat-bottomed conical flasks. Yarrowia lipolytica Cultivation of the proof-of-concept strain. Experiments showed that a higher aeration rate (caused by a higher stirring speed) is beneficial for the production of natural blue pigment. Figure 5 ).

[0233] Example 5 – Sustainability of Bioprocesses Maintaining the optimal bioprocess temperature (30°C in this case) is a significant component of the total production cost, as both heating (in cold climates) and cooling (in warm climates) require substantial energy. The effects of three different temperatures (room temperature, 25°C, and 30°C) on the yield of natural blue pigment in YNB medium containing 20 g / L glucose were analyzed. Figure 6 A). The final titer decreases as the temperature decreases, but can reach up to 1.1 g / L even without a thermostat.

[0234] Water can also be a significant cost component, as the most commonly used distilled water (Milli-Q) requires energy to produce. We analyzed three different types of water in YNB medium containing 20 g / L glucose—Milli-Q, tap water (TW), and seawater (SW). Figure 6 B). A slight decrease in final titer was observed compared to Milli-Q water, indicating that both tap water and seawater can be used directly for this bioprocess. This is particularly important if future production plants have their own wells or are located near the coast.

[0235] In addition, we designed a superbasic culture medium SMm, which contains readily available and renewable raw materials (Table 1). Then, it will produce natural blue pigments. Yarrowia lipolytica The strains were cultured in deep-well plates containing these five SMm species. For example... Figure 6 As shown in C, natural blue pigment was produced in all the culture media, with the highest titers (1.8 g / L and 2.3 g / L, respectively) reaching SM4 and SM5 after 120 hours of culture.

[0236] Table 1. Composition of the superbasic culture medium.

[0237]

[0238] Example 6 – Bioreactor Cultivation under Optimized Conditions The results from the above experiments were used for bioreactor cultivation. A nitrogen source of 40 g / L glucose, urea, or ammonium sulfate was used, with a C / N ratio of 4, and sterile air was introduced via a shaking speed of 800 rpm and a flow rate of 1 lpm. Figure 7 The proof-of-concept strain was cultured in batch mode at a high aeration rate. The highest natural blue pigment titer was obtained in a medium containing ammonium sulfate as a nitrogen source, reaching a peak of 1.9 g / L at 72 hours.

[0239] Example 7 – The effect of precursor supply on the biosynthesis of natural blue pigment The direct precursors for the biosynthesis of natural blue pigment are the amino acids L-glutamic acid and L-glutamine. Figure 8 A). To identify bottlenecks in the pathway, natural blue pigments are produced. Yarrowia lipolytica The strain is supplemented with glutamine and glutamic acid ( Figure 8 (B) The results showed that the conversion of L-glutamate to L-glutamine was the rate-limiting step in this pathway. Compared with unsupplemented medium (YNB containing 20 g / L glucose), the yield levels were increased by 2.5-fold and 5.8-fold, respectively, with the addition of 100 mM glutamate and 100 mM glutamine.

[0240] Example 8 - Overexpression of GS and GDH in Yarrowia lipolytica native blue pigment biosynthesis in the strain Supplementing with direct precursors for biosynthesis is an effective strategy; however, the cost of these precursors makes the process economically unfeasible. Therefore, a genome-wide search was conducted on genes encoding glutamate dehydrogenase (GDH) and glutamine synthase (GS) to increase the supply of endogenous precursors. Through computer simulation Four target genes were identified (two for each enzymatic reaction) and cloned and expressed in proof-of-concept strains (Table 2).

[0241] Table 2. Overexpression of GDH-encoding genes and GS-encoding genes Yarrowia lipolytica strains.

[0242]

[0243] Prepare strains according to standard laboratory protocols, such as those described in the YaliCraft manual, see https: / / assets.researchsquare.com / files / rs-1570357 / v1 / 9174b2d69b59349a43c2c41e.pdf (researchsquare.com) .

[0244] The constructed strains were cultured in a medium with glucose as the sole carbon source. After 120 hours of culture, strain PHS40, which overexpressed the putative GS-encoding gene and the GDH-encoding gene, obtained the highest natural blue pigment titer (3.65 g / L). Figure 9 ).

[0245] Example 9 - Co-cultures Yarrowia lipolytica and Gerolactobacillus to produce blue bacterial cellulose fibers From engineering Yarrowia lipolytica The pigment produced by the strain can be directly applied in the textile industry. However, we decided to go a step further and use a co-culture strategy to create a sustainable blue biomaterial. This involved combining a proof-of-concept strain with a bacterial cellulose (BC)-producing strain. Gerolactobacillus The strain was cultured. Within 7 days, this preliminary experiment produced uniformly colored cellulose fibers ( Figure 10 This biomaterial could be directly used, for example, in shoemaking. Unfortunately, we can only obtain small and thin BC fragments, likely due to the antibacterial activity of the natural blue pigment. We hypothesize that engineered... Yarrowia lipolytica The natural blue pigment produced by the strain inhibits Gerolactobacillus The growth of BC leads to low BC production.

[0246] For the reasons mentioned above, we are optimizing the protocol to allow for more reliable BC production and coloring. In addition to co-cultivation, we are also exploring the decoupling of BC formation and coloring in a two-step process. First, we will... GerolactobacillusThe bacteria were cultured for 5 days under static conditions in a nutrient-rich medium (YPD). At the end of this process, a thick layer of cellulose, called a film, was produced at the liquid-air interface. Figure 11 A). Next, the membrane is separated, sterilized in ethanol, washed with sterile water, and then reacted with the product of the natural blue pigment. Yarrowia lipolytica The strains were inoculated together in basal YNB medium (20 g / L glucose). We then applied shaking conditions (e.g., incubator or rotary shake) to promote membrane colonization of the naturally occurring blue pigment yeast. We hypothesized that when in basal medium, Yarrowia lipolytica Enhanced filamentation may lead to better colonization patterns.

[0247] Example 10 - Use of colorants Yarrowia lipolytica Biomass as textile dyeing agent At the end of the natural blue pigment production process (e.g., bioreactor culture), the biomass is collected and left to dry overnight at room temperature. The dried cells are then mechanically ground, in our case, using a pestle and mortar. The final product is a fine, deep blue powder. A small amount (<1 mL) of DMSO is then added to produce a staining paste.

[0248] Different textiles, whether untreated or soap-washed, should be soaked in dye paste for 4 to 12 hours. Agitation conditions can be applied to promote dye incorporation into the fabric. The fabric is then sterilized by autoclaving and subsequently washed with soap and water to remove excess dye.

[0249] Continuous processing applications can be performed to adjust the color and hue of textiles, where repeated applications result in higher dye incorporation into the textiles. Figure 12 We are also testing in-situ dyeing methods, adding textiles to growing plants. Fat-reducing Saccharomyces cerevisiae In cultures, yeast filamentization in basal media may increase dye incorporation due to entanglement between fibers.

[0250] Example 11 - Cloning and expression of semi-synthetic BpsA protein in Yarrowia lipolytica E. coli Using internal methods Through computer simulation A semi-synthetic BpsA protein (BpsA_X5-UNC) was designed. The amino acid sequence is similar to that from... Streptomyces lavender (BpsA_X5; Figure 13The natural sequence of protein A is 50% identical. AlphaFold was used to predict both the natural and designed protein structures. (See, for example, https: / / alphafold.ebi.ac.uk / Jumper et al., 2021 Nature 596: 583-589; and Varadi et al., 2022 NAR D439-D444). The designed protein structures showed nearly identical structures to the natural protein. Figure 13 B).

[0251] The BpsA_X5-UNC encoding DNA sequence was synthesized by an external company and cloned into an expression vector. Several vector variants are currently being constructed, which will be used for transformation. Yarrowia lipolytica The constructed BpsA_X5-UNC expression strain will be tested for natural blue pigment production. This work will be completed approximately two months after the submission of this form.

[0252] Example 12 – Cloning and expression of target genes identified by FBA Our aim was to further increase the titer of natural blue pigment by overexpressing and knocking out target genes identified through flux balance analysis (FBA, Table 3). For gene overexpression, natural blue pigment titers were... Yarrowia lipolytica The gene is cloned into an expression vector, which will then be used to transform the product into a natural blue pigment. Yarrowia lipolytica Strains. For gene knockout, a disruption cassette will be constructed, cloned into a vector, and transformed into... Yarrowia lipolytica The study will then analyze the effect of the introduced modifications on the yield of natural blue pigment.

[0253] Table 3 – Overexpression Target Genes

[0254] equivalent The above embodiments, examples, and illustrations are applicable to any aspect of this disclosure and should be interpreted as such.

[0255] While this disclosure has been described in various aspects, embodiments, and examples, it should be understood that variations, modifications, and equivalents will occur to those skilled in the art. Such variations, modifications, and equivalents are contemplated in this disclosure and are all within the scope of what is disclosed and claimed herein.

Claims

1. A recombinant yeast cell capable of producing a natural blue pigment, wherein the cell expresses: a) a heterologous nonribosomal peptide synthase (NRPS) polypeptide capable of converting glutamine into natural blue pigment, derived from nucleic acid encoding said NRPS; and b) A heterologous phosphopantoylthioethylaminotransferase (PPTase) polypeptide derived from the nucleic acid encoding the PPTase.

2. The recombinant yeast cell according to claim 1, wherein the cell overexpresses: a glutamine dehydrogenase (GDH) polypeptide derived from a nucleic acid encoding the GDH polypeptide; and a glutamine synthase (GS) polypeptide derived from a nucleic acid encoding the GS polypeptide.

3. The recombinant yeast cell according to claim 1, wherein the cell overexpresses: glutamine dehydrogenase (GDH) polypeptide derived from nucleic acid encoding the GDH polypeptide; or glutamine synthase (GS) polypeptide derived from nucleic acid encoding the GS polypeptide.

4. The recombinant yeast cell according to any one of claims 1 to 3, wherein the heterologous NRPS polypeptide: a) is BpsA; arbitrarily: i) Streptomyces lavender BpsA; or ii) Synthesized BpsA; b) is IndC; optionally, it is... Brown Streptomyces IndC; c) is IndB; optionally, it is... Brown Streptomyces IndB; d) Contains or consists of the following: The amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; or the amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; and / or e) Encoded by: a nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 7; or a nucleotide sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5 or SEQ ID NO:

7.

5. The recombinant yeast cell according to any one of claims 1 to 4, wherein the heterologous PPTase polypeptide: a) for E. coli PPTase; b) is Bacillus subtilis PPTase; c) Containing an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 17, or SEQ ID NO: 19; or having an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 17, or SEQ ID NO: 18; and / or d) Encoded by a nucleotide sequence of SEQ ID NO: 2, SEQ ID NO: 16 or SEQ ID NO: 18; or encoded by a nucleic acid having sequence identity with SEQ ID NO: 2, SEQ ID NO: 16 or SEQ ID NO: 18 having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

6. The recombinant yeast cell according to any one of claims 1 to 5, wherein the GDH polypeptide and / or GS polypeptide are endogenous GDH and / or GS polypeptides.

7. The recombinant yeast cell according to any one of claims 1 to 5, wherein the GDH polypeptide and / or GS polypeptide is a heterologous GDH and / or GS polypeptide.

8. The recombinant yeast cell according to any one of claims 1 to 7, wherein: The GDH polypeptide: a) Encoded by an open reading frame defined by YALI0E09603g or YALI0F17820g; b) Containing an amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 21; or an amino acid sequence having 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 20 or SEQ ID NO: 21; and / or c) Encoded by a nucleotide sequence of SEQ ID NO: 11 or SEQ ID NO: 12; or encoded by a nucleic acid having sequence identity with SEQ ID NO: 11 or SEQ ID NO: 12 of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%; and / or The GS polypeptide: a) Encoded by an open reading frame defined by YALI0D13024g or YALI0F00506g; b) Containing an amino acid sequence of SEQ ID NO: 22 or SEQ ID NO: 23; or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 22 or SEQ ID NO: 23; and / or c) Encoded by a nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 10; or encoded by a nucleic acid having sequence identity with SEQ ID NO: 9 or SEQ ID NO: 10 having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the sequence.

9. The recombinant yeast cell according to any one of claims 1 to 8, wherein the nucleotide sequences encoding the heterologous NRPS polypeptide, the heterologous PPTase polypeptide, the GDH polypeptide, and / or the GS polypeptide are each operatively linked to a promoter selected from or the group consisting of: a) A constitutive promoter selected from the group consisting of or including the following: ATP1 promoter [SEQ ID NO: 24], ATP2 promoter [SEQ ID NO: 25], FBAin promoter [SEQ ID NO: 26], PGK1 promoter [SEQ ID NO: 27], GPM1 promoter [SEQ ID NO: 28], HHF1 promoter [SEQ ID NO: 29], CYC1 promoter [SEQ ID NO: 30], HHT1 promoter [SEQ ID NO: 31], HTB1 promoter [SEQ ID NO: 32], EXP1 promoter [SEQ ID NO: 33], TDH1 promoter [SEQ ID NO: 34], RPL25 promoter [SEQ ID NO: 35], TEF1 promoter [SEQ ID NO: 36], TEFin promoter [SEQ ID NO: 37], TEF2UAS promoter [SEQ ID NO: 38], CYC1 promoter [SEQ ID NO: 39], HHT ... CYC1 promoter [SEQ ID NO: 36], CYC1 promoter [SEQ ID NO: 37], CYC1 promoter [SEQ ID NO: 38], CYC1 promoter [SEQ ID NO: 39], CYC1 promoter [SEQ ID NO: 30], CY 38], TEF4UAS bootloader [SEQ ID NO: 39] and TEF8UAS bootloader [SEQ ID NO: 40], pTEF bootloader [SEQ ID NO: 13 and 14], GAP bootloader [SEQ ID NO: 57], or b) Inducible promoters selected from the group consisting of or including the following: pPOX2 promoter [SEQ ID NO: 41], pXPR2 promoter [SEQ ID NO: 42], pFBP1 promoter [SEQ ID NO: 43], pMDH1a promoter [SEQ ID NO: 44], ACL2 promoter [SEQ ID NO: 45], XPR2 promoter [SEQ ID NO: 58], POT1 promoter [SEQ ID NO: 46], LIP2 promoter [SEQ ID NO: 47], ICL promoter [SEQ ID NO: 48], YAT1 promoter [SEQ ID NO: 49], CTR1 promoter [SEQ ID NO: 50], CTR2 promoter [SEQ ID NO: 51], pYALI0B18194 promoter [SEQ ID NO: 52], pYALI0C11165 promoter [SEQ ID NO: 52], pYALI0C11165 promoter [SEQ ID NO: 52], pYALI0B18194 ...B18194 promoter [SEQ ID NO: 52], pYALI0C11165 promoter [SEQ ID NO: 52], pYALI0B18194 promoter [ [53], pYALI0C15004 promoter [SEQ ID NO: 54], pYALI0E14256 promoter [SEQ ID NO: 55] and pYALI0F13937 promoter [SEQ ID NO: 56].

10. The recombinant yeast cell according to any one of claims 1 to 9, wherein the nucleotide sequences encoding the heterologous NRPS polypeptide, the heterologous PPTase polypeptide, the GDH polypeptide and / or the GS polypeptide are each operatively linked to a promoter, the promoter being a pTEF promoter; optionally, wherein the pTEF promoter has a nucleotide sequence encoded by SEQ ID NO: 13 or SEQ ID NO:

14.

11. The recombinant yeast cell according to any one of claims 1 to 10, wherein the nucleic acid encoding the NRPS polypeptide, the PPTase polypeptide, the GDH polypeptide and / or the GS polypeptide is integrated into the genome of the cell.

12. The recombinant yeast cell according to any one of claims 1 to 10, wherein the following is omitted: Endogenous chromosomal GDH and / or GS genes; and / or At least one gene selected from open reading frames selected from or consisting of open reading frames selected from the group consisting of YALI0E09603g, YALI0F17820g, YALI0D13024g, YALI0F00506g, YALI0E18634g, YALI0B00572g and / or YALI0F01606g or any combination thereof.

13. The recombinant yeast cell according to any one of the preceding claims, wherein the yeast cell is an oil-producing yeast cell, but not... genus Rhodotorula Cells, optionally, wherein said cells are not Rhodotorula buergerianum cell.

14. The recombinant yeast cell according to any one of the preceding claims, wherein the yeast cell is... Yeast genus Cells, optionally selected from the group consisting of or including the following: Yogurtia spp. cell, Deformed Yersinia cell, Yarrowia lipolytica cell, Yeast rice cell, Yarrowia Yakushima cell, Parofonymycosis cell, Chicken Saccharomyces cerevisiae cell, Yeast Oslo cell, Yeast in food cell, Yarrowia hollandiensis Cells and Yarrowia pangaensis Cells; optionally, the recombinant cells Yersinia Cells are Yarrowia lipolytica cell.

15. A method for producing natural blue pigment, the method comprising culturing recombinant yeast cells according to any one of claims 1 to 14.

16. The method of claim 15, wherein the cells are cultured in a culture medium selected from: a) Enriched culture medium, optionally YPD or YP4D; b) Basic culture medium, optionally YNB containing glucose or YNB containing glycerol; or c) A superbasic culture medium; optionally, wherein the superbasic culture medium is SM1, SM2, SM3, SM4 or SM5.

17. The method according to any one of claims 15 or 16, wherein the carbon-to-nitrogen (C / N) ratio of the culture medium is: a) Between 2 and 160, 2 and 150, 2 and 140, 2 and 130, 2 and 120, 2 and 110, 2 and 100, 2 and 90, 2 and 80, 2 and 70, 2 and 60, 2 and 50, 2 and 40, 2 and 30, 2 and 20, 2 and 10, etc. 2 and John 9, John 2 and John 8, John 2 and John 7, John 2 and John 6, John 2 and John 5, John 2 and John 4, John 2 and John 3, John 4 and John 160, John 4 and John 150, John 4 and John 140, John 4 and John 130, John 4 and John 140, John 4 and John 110, John 4 and John 100, John 4 and John 90, John 4 and John 80, John 4 and John 70, John 4 and John 60, John 4 and John 50, John 4 and John 40, John 4 and John 30, John 4 and John 20, John 4 and John 10, John 4 and John 9, John 4 and John 8, John 4 and John 7, John 4 and John 6, John 4 and John 5, John 6 and John 160, John 6 and John 150, John 6 and John 140, John 6 and John 130, John 6 and John 160, John 6 and John 110, John 6 and John 100, John 6 and John 90, John 6 and John 80, John 6 and John 70, John 6 and John 60, John 6 and John 50, John 6 and John 40, John 6 and John 30, John 6 and John 20, John 6 and John 10, John 6 and John 9, John 6 and John 8, John 6 and John 7, John 8 and John 160, John 8 and John 150, John 8 and John 140, John 8 and John 130, John 8 and John 180, John 8 and John 110, John 8 and John 100, John 8 and John 90, John 8 and John 80, John 8 and John 70, John 8 and John 60, John 8 and John 50, John 8 and John 40, John 8 and John 30, John 8 and John 20, John 8 and John 10, John 8 and John 9, John 8 and John 8, John 8 and John 7, John 8 and John 6, John 8 and John 5, John 8 and John 4, John 8 and John 3, John 10 and John 160, John 10 With about 150, about 10 with about 140, about 10 with about 130, about 10 with about 1100, about 10 with about 110, about 10 with about 100, about 10 with about 90, about 10 with about 80, about 10 with about 70, about 10 with about 60, about 10 with about 50, about 10 with about 40, about 10 with about 30, about 10 with about 20, about 20 with about 160, Approximately 20 and approximately 150, approximately 20 and approximately 140, approximately 20 and approximately 130, approximately 20 and approximately 1200, approximately 20 and approximately 110, approximately 20 and approximately 100, approximately 20 and approximately 90, approximately 20 and approximately 80, approximately 20 and approximately 70, approximately 20 and approximately 60, approximately 20 and approximately 50, approximately 20 and approximately 40, approximately 20 and approximately 30, approximately 30 and approximately 160, approximately 30 and approximately 150, about 30 and about 140, about 30 and about 130, about 30 and about 1300, about 30 and about 110, about 30 and about 100, about 30 and about 90, about 30 and about 80, about 30 and about 70, about 30 and about 60, about 30 and about 50, about 30 and about 40, about 40 and about 160, about 40 and about 150, about 40 and about 140,Between approximately 40 and approximately 130, approximately 40 and approximately 1400, approximately 40 and approximately 110, approximately 40 and approximately 100, approximately 40 and approximately 90, approximately 40 and approximately 80, approximately 40 and approximately 70, approximately 40 and approximately 60, approximately 40 and approximately 50, approximately 50 and approximately 160, approximately 50 and approximately 150, approximately 50 and approximately 140, approximately 50 and approximately 130, approximately 50 and approximately 150, approximately 50 and approximately 110, approximately 50 and approximately 100, approximately 50 and approximately 90, approximately 50 and approximately 80, approximately 50 and approximately 70, or approximately 50 and approximately 60; b) Between 2 and 160, 2 and 150, 2 and 140, 2 and 130, 2 and 120, 2 and 110, 2 and 100, 2 and 90, 2 and 80, 2 and 70, 2 and 60, 2 and 50, 2 and 40, 2 and 30, 2 and 20, 2 and 10, 2 and 9, 2 and 8, 2 and 7, 2 and 6, 2 and 5, 2 and 4, 2 and 3, 4 and 160, 4 and 150, 4 and 140, 4 and 130, 4 and 140, 4 and 110, 4 and 100, 4 and 90, 4 and 80, 4 and 70, 4 and 60, 4 and 50, 4 and 40, 4 and 30, 4 and 20, 4 and 10, 4 and 9, 4 and 8, 4 and 7 4 and 6, 4 and 5, 6 and 160, 6 and 150, 6 and 140, 6 and 130, 6 and 160, 6 and 110, 6 and 100, 6 and 90, 6 and 80, 6 and 70, 6 and 60, 6 and 50, 6 and 40, 6 and 30, 6 and 20, 6 and 10, 6 and 9, 6 and 8, 6 and 7, 8 and 160, 8 and 150, 8 and 140, 8 and 130, 8 and 180, 8 and 110, 8 and 100, 8 and 90, 8 and 80, 8 and 70, 8 and 60, 8 and 50, 8 and 40, 8 and 30, 8 and 20, 8 and 10, 8 and 9, 8 and 8, 8 and 7, 8 and 6, 8 and 5, 8 and 4 8 and 3, 10 and 160, 10 and 150, 10 and 140, 10 and 130, 10 and 1100, 10 and 110, 10 and 100, 10 and 90, 10 and 80, 10 and 70, 10 and 60, 10 and 50, 10 and 40, 10 and 30, 10 and 20, 20 and 160, 20 and 150, 20 and 140, 20 and 130, 20 and 1200, 20 and 110, 20 and 100, 20 and 90, 20 and 80, 20 and 70, 20 and 60, 20 and 50, 20 and 40, 20 and 30, 30 and 160, 30 and 150, 30 and 140, 30 Between 130, 30 and 1300, 30 and 110, 30 and 100, 30 and 90, 30 and 80, 30 and 70, 30 and 60, 30 and 50, 30 and 40, 40 and 160, 40 and 150, 40 and 140, 40 and 130, 40 and 1400, 40 and 110, 40 and 100, 40 and 90, 40 and 80, 40 and 70, 40 and 60, 40 and 50, 50 and 160, 50 and 150, 50 and 140, 50 and 130, 50 and 150, 50 and 110, 50 and 100, 50 and 90, 50 and 80, 50 and 70 or 50 and 60; c) At least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150 or more; d) Less than approximately 160, less than approximately 150, less than approximately 140, less than approximately 130, less than approximately 120, less than approximately 110, less than approximately, less than approximately 100, less than approximately 90, less than approximately 80, less than approximately 70, less than approximately 60, less than approximately 50, less than approximately 40, less than approximately 30, less than approximately 20, less than approximately 10, less than approximately 9, less than approximately 8, less than approximately 7, less than approximately 6, less than approximately 5, less than approximately 4, less than approximately 3; and / or e) At least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150 or more; f) Less than 160, less than 150, less than 140, less than 130, less than 120, less than 110, less than approximately, less than 100, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3; g) Approximately 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, approximately 8, approximately 9, approximately 10, approximately 20, approximately 30, approximately 40, approximately 50, approximately 60, approximately 70, approximately 80, approximately 90, approximately 100, approximately 110, approximately 120, approximately 130, approximately 140, approximately 150; and / or h)2、3、4、5、6、7、8、9、10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160。 18. The method according to any one of claims 15 to 17, wherein: The culture medium has a carbon source selected from or consisting of the following: lignocellulose hydrolysate, glucose, fructose, mannose, citrate, acetate, rapeseed oil, sunflower oil, palm oil, waste cooking oil, rapeseed oil, and sunflower oil, glycerol and / or waste glycerol; optionally, the lignocellulose hydrolysate is selected from or consisting of the following: hydrolyzed flaxseed bran, pepper, vegetable mixtures, and urban trimmings, or any combination thereof; The culture medium contains water selected from or grouped with seawater, tap water, and distilled water; or any combination thereof; and The culture medium contains a nitrogen source selected from or grouped with the following: ammonium sulfate, urine, synthetic urine, urea, ammonium chloride, peptone, and any combination thereof.

19. The method according to claims 15 to 18, wherein the culture comprises stirring a liquid culture medium during cell growth; wherein the liquid culture medium is stirred at the following rotational speeds: a) Between approximately 150 and approximately 2000 RPM, approximately 150 and approximately 250 RPM, approximately 150 and approximately 200 RPM, approximately 200 and approximately 250 RPM, approximately 150 and approximately 800 RPM, approximately 200 and approximately 800 RPM, approximately 250 and approximately 800 RPM, approximately 800 and approximately 2000 RPM, approximately 1000 and approximately 1800 RPM, approximately 1200 and approximately 1400 RPM; b) Between 150 and 2000 RPM, 150 and 250 RPM, 150 and 200 RPM, 200 and 250 RPM, 150 and 800 RPM, 200 and 800 RPM, 250 and 800 RPM, 800 and 2000 RPM, 1000 and 1800 RPM, and 1200 and 1400 RPM; c) At least 150 RPM, at least 200 RPM, at least 250 RPM, at least 800 RPM, at least 1000 RPM, at least 1200 RPM, at least 1400 RPM, at least 1600 RPM, at least 1800 RPM; d) Less than 2000 RPM, less than 1800 RPM, less than 1600 RPM, less than 1400 RPM, less than 1200 RPM, less than 1000 RPM, less than 800 RPM, less than 250 RPM, or less than 200 RPM; c) Approximately 150 RPM, approximately 200 RPM, approximately 250 RPM, approximately 800 RPM, approximately 1000 RPM, approximately 1200 RPM, approximately 1400 RPM, approximately 1600 RPM, approximately 1800 RPM, or approximately 2000 RPM; or d) 150 RPM, 200 RPM, 250 RPM, 800 RPM, 1000 RPM, 1200 RPM, 1400 RPM, 1600 RPM, 1800 RPM or 2000 RPM.

20. The method according to any one of claims 15 to 19, wherein: a) The culture medium is supplemented with glutamine, and the concentration of glutamine is: i) Between approximately 1 mm and approximately 100 mm, approximately 2 mm and approximately 90 mm, approximately 3 mm and approximately 80 mm, approximately 4 mm and approximately 70 mm, approximately 5 mm and approximately 60 mm, approximately 6 mm and approximately 50 mm, approximately 7 mm and approximately 40 mm, approximately 8 mm and approximately 30 mm, approximately 9 mm and approximately 20 mm, approximately 10 mm and approximately 15 mm, approximately 1 mm and approximately 10 mm, approximately 2 mm and approximately 9 mm, approximately 3 mm and approximately 8 mm, approximately 4 mm and approximately 7 mm, approximately 5 mm and approximately 6 mm, approximately 10 mm and approximately 10 ... Between mM and approximately 100mM, approximately 20mM and approximately 90mM, approximately 30mM and approximately 80mM, approximately 40mM and approximately 70mM, approximately 50mM and approximately 60mM, approximately 50mM and approximately 150mM, approximately 60mM and approximately 140mM, approximately 70mM and approximately 130mM, approximately 80mM and approximately 120mM, or approximately 90mM and approximately 110mM glutamine; ii) Between 1mM and 100mM, 2mM and 90mM, 3mM and 80mM, 4mM and 70mM, 5mM and 60mM, 6mM and 50mM, 7mM and 40mM, 8mM and 30mM, 9mM and 20mM, 10mM and 15mM, 1mM and 10mM, 2mM and 9mM, 3mM and 8mM, 4mM and 7mM, 5mM and 6mM, 10 Between mM and 100mM, 20mM and 90mM, 30mM and 80mM, 40mM and 70mM, 50mM and 60mM, 50mM and 150mM, 60mM and 140mM, 70mM and 130mM, 80mM and 120mM, or 90mM and 110mM glutamine; iii) At least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, at least 10 mM, at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 110 mM, at least 120 mM, at least 130 mM, at least 140 mM, at least 150 mM or more glutamine; iv) Less than 150mM, less than 140mM, less than 130mM, less than 120mM, less than 120mM, less than 110mM, less than 100mM, less than 90mM, less than 80mM, less than 70mM, less than 60mM, less than 50mM, less than 40mM, less than 30mM, less than 20mM, less than 10mM, less than 9mM, less than 8mM, less than 7mM, less than 6mM, less than 5mM, less than 4mM, less than 3mM, less than 2mM or less glutamine; v) Approximately 1 mM, approximately 2 mM, approximately 3 mM, approximately 4 mM, approximately 5 mM, approximately 6 mM, approximately 7 mM, approximately 8 mM, approximately 9 mM, approximately 10 mM, approximately 20 mM, approximately 30 mM, approximately 40 mM, approximately 50 mM, approximately 60 mM, approximately 70 mM, approximately 80 mM, approximately 90 mM, approximately 100 mM, approximately 110 mM, approximately 120 mM, approximately 130 mM, approximately 140 mM, or approximately 150 mM glutamine; and / or vi) 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM or 150mM glutamine; And / or b) The culture medium is supplemented with glutamic acid, and the concentration of the glutamic acid is: i) Between approximately 1 mm and approximately 100 mm, approximately 2 mm and approximately 90 mm, approximately 3 mm and approximately 80 mm, approximately 4 mm and approximately 70 mm, approximately 5 mm and approximately 60 mm, approximately 6 mm and approximately 50 mm, approximately 7 mm and approximately 40 mm, approximately 8 mm and approximately 30 mm, approximately 9 mm and approximately 20 mm, approximately 10 mm and approximately 15 mm, approximately 1 mm and approximately 10 mm, approximately 2 mm and approximately 9 mm, approximately 3 mm and approximately 8 mm, approximately 4 mm and approximately 7 mm, approximately 5 mm and approximately 6 mm, approximately 10 mm and approximately 10 ... Between mM and approximately 100mM, approximately 20mM and approximately 90mM, approximately 30mM and approximately 80mM, approximately 40mM and approximately 70mM, approximately 50mM and approximately 60mM, approximately 50mM and approximately 150mM, approximately 60mM and approximately 140mM, approximately 70mM and approximately 130mM, approximately 80mM and approximately 120mM, or approximately 90mM and approximately 110mM glutamate; ii) Glutamate between 1mM and 100mM, 2mM and 90mM, 3mM and 80mM, 4mM and 70mM, 5mM and 60mM, 6mM and 50mM, 7mM and 40mM, 8mM and 30mM, 9mM and 20mM, 10mM and 15mM, 1mM and 10mM, 2mM and 9mM, 3mM and 8mM, 4mM and 7mM, 5mM and 6mM, 10mM and 100mM, 20mM and 90mM, 30mM and 80mM, 40mM and 70mM, 50mM and 60mM, 50mM and 150mM, 60mM and 140mM, 70mM and 130mM, 80mM and 120mM, or 90mM and 110mM; iii) At least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, at least 10 mM, at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 110 mM, at least 120 mM, at least 130 mM, at least 140 mM, at least 150 mM or more glutamate; iv) Less than 150mM, less than 140mM, less than 130mM, less than 120mM, less than 120mM, less than 110mM, less than 100mM, less than 90mM, less than 80mM, less than 70mM, less than 60mM, less than 50mM, less than 40mM, less than 30mM, less than 20mM, less than 10mM, less than 9mM, less than 8mM, less than 7mM, less than 6mM, less than 5mM, less than 4mM, less than 3mM, less than 2mM or less glutamate; v) Approximately 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, or 150 mM glutamate; and / or vi) 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM or 150mM glutamate.

21. The method according to any one of claims 15 to 20, wherein the method further comprises extracting and / or purifying the natural blue pigment from the cells; optionally The extraction and / or purification of the natural blue pigment from said cells includes: a) Lysing the cells; optionally, wherein the cells are lysed by homogenization in DMSO; b) Collect the yeast cells, dry the yeast cells, and lyse the yeast by grinding the yeast cells.

22. A method for producing bacterial cellulose or cellulose film stained with natural blue pigment, the method comprising co-culturing recombinant yeast cells according to any one of claims 1 to 14 with cellulose-producing bacterial cells, wherein the cells are co-cultured under conditions that allow the cellulose-producing bacterial cells to produce cellulose or cellulose film.

23. A method for producing bacterial cellulose or cellulose films dyed with natural blue pigment, the method comprising: a) Culturing cellulose-producing bacterial cells to form bacterial cellulose or a cellulose film; And subsequently b) Contact the cellulose or cellulose film produced by the cellulose-producing second cell in (a) with the recombinant yeast cell according to any one of claims 1 to 14.

24. A bacterial cellulose or cellulose film stained with natural blue pigment obtained or available from the method according to any one of claims 22 or 23.

25. A natural blue pigment obtained or available from recombinant yeast cells according to any one of claims 1 to 14 or by the method according to any one of claims 15 to 23.

26. A method for producing dyed textiles or garments, the method comprising contacting the textiles or garments with a natural blue pigment according to claim 25.

27. A kit comprising any one or more of the following: a) Recombinant yeast cells according to any one of claims 1 to 14; b) Bacterial cellulose; c) Cellulose film; d) The natural blue pigment according to claim 25; e) textiles, clothing or thread; and / or f) Cellulose-producing bacterial cells.

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