WO2022003130A2 - Système d'expression de levure - Google Patents

Système d'expression de levure Download PDF

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WO2022003130A2
WO2022003130A2 PCT/EP2021/068252 EP2021068252W WO2022003130A2 WO 2022003130 A2 WO2022003130 A2 WO 2022003130A2 EP 2021068252 W EP2021068252 W EP 2021068252W WO 2022003130 A2 WO2022003130 A2 WO 2022003130A2
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amino acid
host cell
seq
modified
saccharomyces
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PCT/EP2021/068252
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WO2022003130A3 (fr
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Valmik Kanubhai VYAS
Peter Louis HOUSTON
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Dsm Ip Assets B.V.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P23/00—Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01084—Alcohol O-acetyltransferase (2.3.1.84)

Definitions

  • the present invention is related to expression of heterologous enzymes under control of constitutive promoters, particularly expression of acetyl transferase enzymes in a host selected from Saccharomyces.
  • Fungal host cells such as particularly Saccharomyces cerevisiae, are widely used as host organisms for production of vitamins or carotenoids.
  • retinoids as well as carotenoids are not stable as such.
  • the production of acetylated forms, as it is known for carotenoids, such as e.g. astaxanthin has been reported to enable industrial production of said compounds (WO2014096992).
  • acetylation of retinol has been reported in Yarrowia lipolytica by action of fungal acetyl transferases (ATFs) [EC 2.3.1.84] (W02019058001).
  • fungal ATFs have been reported in fungal hosts such as e.g. Yarrowia lipolytica (W02019058001)
  • expression of fungal ATFs in yeast such as e.g. Saccharomyces turned out to be more challenging as expected with very low transformation rates.
  • acetylating enzymes such as fungal ATFs
  • a constitutive promoter in a host cell selected from yeast such as Saccharomyces, wherein the host is transformed with fungal ATFs comprising one or more modification(s), such as amino acid substitution(s), leading to enhanced growth of the host cell compared to transformation with the respective wild-type enzymes.
  • the present invention is directed to a host cell, such as Saccharomyces, preferably Saccharomyces cerevisiae, and a process for production of acetylated compounds in said host cell, wherein the host cell is transformed with (and is expressing) heterologous fungal enzymes, particularly fungal ATFs, comprising one or more amino acid substitution(s) in a sequence with at least about 20%, such as e.g.
  • the transformation rate of such host cell expressing the modified ATF as defined herein is increased by at least about 2-fold compared to a host cell transformed with the respective non-modified ATF.
  • the present invention is concerned with an increase in the transformation rate by at least about 2-fold of fungal ATFs to be heterologous expressed in a host cell selected from Saccharomyces, wherein the ATF expressed under the control of a constitutive promoter is modified, i.e.
  • amino acid substitution(s) in a sequence with at least about 20%, such as 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, 98, 99% or up to 100% identity to SEQ ID NO:1, wherein the one or more amino acid substitution(s) are located at position(s) corresponding to amino acid residue(s) selected from the group consisting of position 68, 69, 72, 73, 171, 174, 176, 178, 291, 292, 294, 301, 307, 308, 311, 312, 320, 322, 334, 362, 405, 407, 409, 480, 483, 484, 490, 492, 520, 521, 522, 524, 525, 526 and combinations thereof, in the polypeptide according to SEQ ID N 0:1, preferably comprising one or more amino acids selected from the group consisting of residue 68 being threonine, residue 69 being asparagine, serine or
  • the process for increasing the transformation rate as described above comprises:
  • ATF fungal acetylating enzyme
  • modified "acetyl transferase”, "retinol acetylating enzyme”, “enzyme having retinol acetylating activity”, “ATF” or “ATF1” are used interchangeably herein and refer to enzymes of EC class [EC 2.3.1.84] which are capable of catalyzing the conversion of retinol into retinyl acetate, with particularly about up to 50 to 90% in the acetylated form based on total retinoids.
  • the modified enzymes are based on the respective non-modified or wild-type enzymes according to sequences with at least about 20% identity to SEQ ID NO:1 as defined herein, including e.g. SEQ ID NO:3, 5, 6, 7, 8.
  • a suitable ATF to be mutated and expressed in a host cell as defined herein might be selected from any fungal enzyme [EC 2.3.1.84] capable of catalyzing the conversion of retinol into retinyl acetate, with particularly at least about 10%, such as e.g. at least about 15, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95 or event up to 100% in the acetylated form based on total retinoids.
  • fungal enzyme EC 2.3.1.84
  • Particular useful ATFs are obtained from fungi including yeast, such as e.g.
  • Particularly suitable fungal ATFs to be used in a process according to the present invention are comprising a highly conserved partial amino acid sequence of at least 7 amino acid residues selected from [NDEHCS]- H-x(3)-D-[GA] (motifs are in Prosite syntax, as defined in https://prosite.expasy.org/scanprosite/scanprosite_doc.html), wherein "x” denotes an arbitrary amino acid and with the central histidine being part of the enzyme's binding pocket, preferably wherein the 7 amino acid motif is selected from [NDE]-H-x(3)-D-[GA], more preferably selected from [ND]-H-x(3)-D-[GA], most preferably selected from N-H-x(3)-D-[GA] corresponding to position N218 to
  • yeast such as Lachancea, Saccharomyces or Wickerhamomyces, particularly from L. mirantina, L. fermentati, S. bayanus, or W. anomalus, such as e.g. LmATFI according to SEQ ID NO:1, SbATFI according to SEQ ID NO:3, LffATFI according to SEQ ID NO:5, LfATFI according to SEQ ID NO:6, Wa1ATF1 according to SEQ ID NO:7 or Wa3ATF1 according to SEQ ID NO:8.
  • the ATF1 to be used for the present invention is obtainable from yeast, such as Lachancea mirantina (LmATFI), in particular selected from polypeptides with at least about 60%, 65, 70, 75, 80, 85, 90, 92, 95, 97, 98, 99% or up to 100% identity to SEQ ID NO:1.
  • yeast such as Lachancea mirantina (LmATFI)
  • ATF1 obtainable from yeast, such as Saccharomyces bayanus (SbATFI), in particular selected from polypeptides with at least about 60%, 65, 70, 75, 80, 85, 90, 92, 95, 97, 98, 99% or up to 100% identity to SEQ ID NO:3.
  • ATF1 obtainable from yeast such as Lachancea fermentati, in particular selected from polypeptides with at least about 60%, 65, 70, 75, 80, 85, 90, 92, 95, 97, 98, 99% or up to 100% identity to SEQ ID NO:5 or 6.
  • yeast such as Wickerhamomyces anomalus, in particular selected from polypeptides with at least about 60%, 65, 70, 75, 80, 85, 90, 92, 95, 97, 98, 99% or up to 100% identity to SEQ ID NO:7 or 8.
  • the term "fungal host cell” particularly includes yeast cells, i.e. retinol-producing yeast cells, such as e.g. Saccharomyces.
  • yeast cells i.e. retinol-producing yeast cells, such as e.g. Saccharomyces.
  • the invention is directed to a retinol-producing host cell, particularly S. cerevisiae, comprising retinol mix being converted by modified fungal ATFs, particularly modified Atfl, as defined herein wherein the transformation rate with regards to expression ofATF is at least about 2-fold higher compared to transformation rate for expression of the respective non- modified ATF under similar conditions, such as e.g. under control of a constitutive promoter, particularly strong constitutive promoter, such as e.g. HSP26.
  • the modified Atfl enzyme as defined herein comprises an amino acid substitution at a position corresponding to residue 69 in the polypeptide according to SEQ ID NO:1 leading to asparagine, serine or alanine at said residue, such as e.g. via substitution of histidine by asparagine (H69N), serine (H69S) or alanine (H69A), with preference for H69A.
  • Said modified enzyme might be originated from yeast, such as e.g. L. mirantina, L. fermentati, W. anomalus or S. bayanus, preferably from L. mirantina.
  • Such modified enzyme comprising said mutation together with the 7 amino acid motif according to N-H-x(3)-D-[GA] as defined herein, in a fermentation process using a host cell selected from Saccharomyces and a suitable carbon source such as e.g. glucose particularly might lead to transformation rate that is increased by 2- fold compared to using a non-modified ATF with histidine at the corresponding position.
  • the mutation might furthermore be combined with 1, 2, 3, 4 or more mutation(s) as defined herein, such as particularly with one or more amino acid substitution(s) at position(s) corresponding to residue(s) 334 and/or 407 and/or 409 and/or 480 and/or 484 in the polypeptide accordingto SEQ ID NO:1.
  • the modified Atfl enzyme as defined herein comprises an amino acid substitution at a position corresponding to residue 334 in the polypeptide accordingto SEQ ID NO:1 leading to leucine at said residue, such as e.g. via substitution of valine by leucine (V334L).
  • Said modified enzyme might be originated from yeast, such as e.g. L. mirantina, L. fermentati, W. anomalus or S. bayanus, preferably from L. mirantina.
  • yeast such as e.g. L. mirantina, L. fermentati, W. anomalus or S. bayanus, preferably from L. mirantina.
  • the mutation might furthermore be combined with 1, 2, 3, 4 or more mutation(s) as defined herein, such as particularly with one or more amino acid substitution(s) at position(s) corresponding to residue(s) 69 and/or 407 and/or 409 and/or 480 and/or 484 in the polypeptide accordingto SEQ ID NO:1.
  • the modified Atfl enzyme as defined herein comprises an amino acid substitution at a position corresponding to residue 407 in the polypeptide according to SEQ ID NO:1 leading to isoleucine at said residue, such as e.g. via substitution of valine by isoleucine (V407I).
  • Said modified enzyme might be originated from yeast, such as e.g. L. mirantina, L. fermentati, W. anomalus or S. bayanus, preferably from L. mirantina.
  • Such modified enzyme comprising said mutation together with the 7 amino acid motif accordingto N-H-x(3)-D-[GA] as defined herein, in a fermentation process using a host cell selected from Saccharomyces and a suitable carbon source such as e.g. glucose particularly might lead to transformation rate that is increased by 2- fold compared to using a non-modified ATF with valine at the corresponding position.
  • the mutation might furthermore be combined with 1, 2, 3, 4 or more mutation(s) as defined herein, such as particularly with one or more amino acid substitution(s) at position(s) corresponding to residue(s) 69 and/or 334 and/or 409 and/or 480 and/or 484 in the polypeptide accordingto SEQ ID NO:1.
  • the modified Atfl enzyme as defined herein comprises an amino acid substitution at a position corresponding to residue 409 in the polypeptide accordingto SEQ ID NO:1 leading to alanine at said residue, such as e.g. via substitution of glycine by alanine (G409A).
  • Said modified enzyme might be originated from yeast, such as e.g. L. mirantina, L. fermentati, W. anomalus or S. bayanus, preferably from L. mirantina or W. anomalus.
  • Such modified enzyme comprising said mutation together with the 7 amino acid motif accordingto N-H-x(3)-D-[GA] as defined herein, in a fermentation process using a host cell selected from Saccharomyces and a suitable carbon source such as e.g. glucose particularly might lead to transformation rate that is increased by 2-fold compared to using a non-modified ATF with valine at the corresponding position.
  • the mutation might furthermore be combined with 1, 2, 3, 4 or more mutation(s) as defined herein, such as particularly with one or more amino acid substitution(s) at position(s) corresponding to residue(s) 69 and/or 334 and/or 407 and/or 480 and/or 484 in the polypeptide accordingto SEQ ID NO:1.
  • the modified Atfl enzyme as defined herein comprises an amino acid substitution at a position corresponding to residue 480 in the polypeptide according to SEQ ID NO:1 leadingto glutamic acid, lysine, methionine, phenylalanine or glutamine at said residue, such as e.g. via substitution of serine by glutamic acid (S480E), lysine (S480L), methionine (S480M), phenylalanine (S480F) or glutamine (S480Q).
  • Said modified enzyme might be originated from yeast, such as e.g. L. mirantina, L. fermentati, W. anomalus or S. bayanus, preferably from L. mirantina or W. anomalus.
  • modified enzyme comprising said mutation together with the 7 amino acid motif according to N-H-x(3)-D-[GA] as defined herein
  • a host cell selected from Saccharomyces and a suitable carbon source such as e.g. glucose
  • a suitable carbon source such as e.g. glucose
  • the mutation might furthermore be combined with 1, 2, 3, 4 or more mutation(s) as defined herein, such as particularly with one or more amino acid substitution(s) at position(s) correspondingto residue(s) 69 and/or 334 and/or 407 and/or 409 and/or 484 in the polypeptide according to SEQ ID NO:1.
  • the modified Atfl enzyme as defined herein comprises an amino acid substitution at a position corresponding to residue 484 in the polypeptide according to SEQ ID NO:1 leadingto leucine at said residue, such as e.g. via substitution of isoleucine by leucine (I484L).
  • Said modified enzyme might be originated from yeast, such as e.g. L. mirantina, L. fermentati,
  • W. anomalus or S. bayanus preferably from L. mirantina.
  • modified enzyme comprising said mutation together with the 7 amino acid motif according to N-H-x(3)-D-[GA] as defined herein, in a fermentation process using a host cell selected from Saccharomyces and a suitable carbon source such as e.g. glucose particularly might lead to transformation rate that is increased by 2- fold compared to using a non-modified ATF with valine at the corresponding position.
  • the mutation might furthermore be combined with 1, 2, 3, 4 or more mutation(s) as defined herein, such as particularly with one or more amino acid substitution(s) at position(s) corresponding to residue(s) 69 and/or 334 and/or 407 and/or 409 and/or 484 in the polypeptide accordingto SEQ ID NO:1.
  • Atfl enzymes as defined herein comprising a combination of at least 2 mutations, i.e. combination of at least 2 amino acid substitutions at positions correspondingto residues S480L or S480Q with V334L, S480L or S480Q with G409A in the polypeptide according to SEQ ID NO:1, together with the 7 amino acid motif according to N-H-x(3)-D-[GA] as defined herein, to be used in a fermentation process using a host cell selected from Saccharomyces and a suitable carbon source such as e.g. glucose particularly leading to transformation rates that are increased by at least about 4 to 50-fold compared to using the respective non- modified ATFs.
  • a host cell selected from Saccharomyces and a suitable carbon source such as e.g. glucose particularly leading to transformation rates that are increased by at least about 4 to 50-fold compared to using the respective non- modified ATFs.
  • modified enzymes comprising combinations of at leasts mutations, i.e. combinations of at least s amino acid substitutions at positions corresponding to residues
  • a host cell selected from Saccharomyces and a suitable carbon source such as e.g. glucose particularly leading to transformation rates that are increased by at least about 2-fold compared to using the respective non- modified ATFs.
  • mutagenesis may be performed in different ways, such as for instance by random or side- directed mutagenesis, physical damage caused by agents such as for instance radiation, chemical treatment, or insertion of a genetic element.
  • agents such as for instance radiation, chemical treatment, or insertion of a genetic element.
  • the skilled person knows how to introduce mutations.
  • the present invention is directed to a retinol-producing host cell, particularly selected from Saccharomyces, as described herein comprising an expression vector or a polynucleotide encoding modified ATFs, particularly modified Atfl enzymes, as described herein which has been integrated in the chromosomal DNA of the host cell under the conditions as defined herein.
  • a retinol-producing host cell such as S. cerevisiae, comprising a heterologous polynucleotide on an expression vector under the control of a constitutive promoter encoding modified ATFs, particularly modified Atfl enzymes, as described herein is called a recombinant host cell.
  • the present invention is directed to a process for production of retinoids including retinal, retinol and retinyl acetate, via enzymatic activity of modified Atfl enzyme as defined herein under the conditions as defined herein, comprising contacting retinol, such as e.g. a retinol mix comprising trans- and cis-retinol and preferably with a percentage of at least about 65 to 90% in the form of trans-retinol, with said modified Atfl enzyme, and optionally isolating and/or purifying the formed retinyl acetate from the host cell.
  • retinol such as e.g. a retinol mix comprising trans- and cis-retinol and preferably with a percentage of at least about 65 to 90% in the form of trans-retinol
  • the invention is directed to a process for production of vitamin A, said process comprising (a) introducing a nucleic acid molecule encoding one of the modified Atfl enzymes as defined herein under the conditions as defined herein into a suitable retinol-producing host cell, particularly fungal host cell, as defined herein, (b) enzymatic conversion, i.e. acetylation, of retinol, such as e.g. a retinol-mix comprising at least about 65-90% retinol as trans-retinol, via action of said expressed modified Atfl into particularly at least about 10%, such as e.g.
  • retinyl acetate based on total retinoids, and optionally furthermore (c) conversion of said retinyl acetate into vitamin A under suitable conditions known to the skilled person.
  • sequence identity in order to determine the percentage of sequence identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes. In order to optimize the alignment between the two sequences gaps may be introduced in any of the two sequences that are compared. Such alignment can be carried out over the full length of the sequences being compared. Alternatively, the alignment may be carried out over a shorter length, for example over about 20, about 50, about 100 or more nucleic acids/bases or amino acids.
  • sequence identity is the percentage of identical matches between the two sequences over the reported aligned region.
  • the percent sequence identity between two amino acid sequences or between two nucleotide sequences may be determined usingthe Needleman and Wunsch algorithm for the alignment of two sequences (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be aligned by the algorithm.
  • the Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE.
  • the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite
  • the percentage of sequence identity between a query sequence and a sequence of the invention is calculated as follows: number of corresponding positions in the alignment showing an identical amino acid or identical nucleotide in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment.
  • the identity as defined herein can be obtained from NEEDLE by usingthe NOBRIEF option and is labeled in the output of the program as "longest identity”. If both amino acid sequences which are compared do not differ in any of their amino acids, they are identical or have 100% identity.
  • modified ATFs particularly modified Atfl enzymes, as defined herein also encompass enzymes carrying (further) amino acid substitution(s) which do not alter enzyme activity, i.e. which show the same properties with respect to the enzymes defined herein and catalyze the conversion of retinol to retinyl acetate with conversion ratios of particularly at least about 10%, such as e.g. at least about 15, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95 or event up to 100% retinyl acetate based on the total amount of retinoids.
  • Such mutations are also called "silent mutations", which do not alter the (enzymatic) activity of the enzymes according to the present invention.
  • the present invention is directed to a retinol-producing host cell, particularly S. cerevisiae, comprising polynucleotides encoding modified fungal ATFs, in particular modified Atfl enzymes, expressed under control of a constitutive promoter as defined herein which are optimized for expression in the respective host cell.
  • organisms such as e.g. microorganisms, fungi, algae or plants also include synonyms or basonyms of such species having the same physiological properties, as defined by the International Code of Nomenclature of Prokaryotes or the International Code of Nomenclature for algae, fungi, and plants (Melbourne Code).
  • strain Lachancea mirantina is a synonym of strain Zygosaccharomyces sp. IFO 11066, originated from Japan.
  • the present invention is directed to a process for production of retinyl acetate, wherein the retinyl acetate is generated via acetylation of retinol (particularly at least 65% as trans-retinol) as disclosed herein by the action of modified ATF, particularly modified Atfl enzymes, as described herein, wherein the acetylating enzymes are heterologous expressed in a suitable host cell under suitable conditions as described herein including the use of constitutive promoters.
  • the produced retinyl acetate might be isolated and optionally further purified from the medium and/or host cell.
  • Said acetylated retinoids defined herein can be used as building blocks in a multi-step process leading to vitamin A.
  • Vitamin A might be isolated and optionally further purified from the medium and/or host cell as known in the art.
  • the host cell such as e.g. S. cerevisiae, capable of producing retinol
  • beta-carotene which might be enzymatically converted into retinal, optionally further converted into retinol.
  • the skilled person knows which genes to be used/expressed for either biosynthesis of beta-carotene and/or bio-conversion of beta-carotene into retinol.
  • Such retinol and optionally carotenoid-producing host cell further being capable of expressing the modified ATF genes, particularly modified ATF1 genes, as defined herein, and/or further genes required for biosynthesis of vitamin A is cultured in an aqueous medium comprising suitable carbon sources, such as e.g. glucose, galactose, xylose or the like, optionally supplemented with appropriate nutrients under aerobic or anaerobic conditions and as known by the skilled person to enable production of retinyl acetate.
  • suitable carbon sources such as e.g. glucose, galactose, xylose or the like
  • suitable nutrients such as e.g. glucose, galactose, xylose or the like
  • the cultivation/growth of the host cell may be conducted in batch, fed-batch, semi- continuous or continuous mode, particularly in fed-batch mode under suitable culture conditions. Fermentation products including retinyl acetate may be harvested from the cultivation at a suitable moment, e.g. when one or more of the nutrients are exhausted.
  • retinoids such as e.g. vitamin A, precursors and/or derivatives thereof such as retinal, retinol, retinyl acetate, particularly retinyl acetate, can vary, as it is known to the skilled person.
  • the retinoids including but not limited to retinol, retinyl acetate, vitamin A might be used as ingredients/formulations in the food, feed, pharma or cosmetic industry. Cultivation and isolation of beta-carotene and retinoid-producing host cells selected from Saccharomyces is described in e.g. W02008042338.
  • the term "specific activity" or "activity" with regards to enzymes means its catalytic activity, i.e. its ability to catalyze formation of a product from a given substrate. The specific activity defines the amount of substrate consumed and/or product produced in a given time period and per defined amount of protein at a defined temperature.
  • An enzyme is active, if it performs its catalytic activity in vivo, i.e. within the host cell as defined herein or within a suitable (cell-free) system in the presence of a suitable substrate. The skilled person knows how to measure enzyme activity. Analytical methods to evaluate the capability of a suitable ATF (wild-type or modified), particularly Atfl, as defined herein for retinyl acetate production, i.e.
  • acetylation of retinol are known in the art, such as e.g. described in Example 4 of WO2014096992.
  • titers of products such as retinyl acetate, retinol, trans-retinal, cis-retinal, beta-carotene and the like can be measured by HPLC.
  • Suitable host cells comprising specific enzymes involved in biosynthesis of beta-carotene and that are expressed and active in vivo leading to production of carotenoids, e.g. beta-carotene
  • both genes and methods to generate carotenoid-producing host cells are known in the art, see e.g. W02006102342.
  • different genes might be involved.
  • a "retinol-producing host cell” is a host cell, wherein the respective polypeptides are expressed and active in vivo, leading to production of retinoids, e.g. vitamin A and its precursors including retinol, via enzymatic conversion of beta-carotene via retinal into retinol.
  • retinoids e.g. vitamin A and its precursors including retinol
  • These polypeptides include the modified ATFs as defined herein.
  • the genes of the vitamin A pathway and methods to generate retinoid-producing host cells are known in the art.
  • retinoid includes retinol, which is used as a substrate for the modified acetylating enzymes as defined herein leading to production of retinyl acetate and thus to a retinyl acetate-producing host cell.
  • Retinoids as used herein include beta-carotene cleavage products also known as apocarotenoids, including but not limited to retinal, retinolic acid, retinol, retinoic methoxide, retinyl acetate, retinyl esters, 4-keto-retinoids, 3 hydroxy- retinoids or combinations thereof. Biosynthesis of retinoids is described in e.g. W02008042338. "Retinal” as used herein is known under lUPAC name (2E,4E,6E,8E)-3,7-Dimethyl- 9-(2,6,6-trimethylcyclohexen-1-yl)nona-2,4,6,8-tetraenal.
  • retinaldehyde It is herein interchangeably referred to as retinaldehyde or vitamin A aldehyde and includes both cis- and trans-isoforms, such as e.g. 11-cis retinal, 13-cis retinal, trans- retinal and all-trans retinal.
  • carotenoids as used herein is well known in the art. It includes long, 40 carbon conjugated isoprenoid polyenes that are formed in nature by the ligation of two 20 carbon geranylgeranyl pyrophosphate molecules. These include but are not limited to phytoene, lycopene, and carotene, such as e.g. beta-carotene, which can be oxidized on the 4-keto position or 3-hydroxy position to yield canthaxanthin, zeaxanthin, or astaxanthin. Biosynthesis of carotenoids is described in e.g. W02006102342.
  • Vitamin A as used herein may be any chemical form of vitamin A found in aqueous solutions, in solids and formulations, and includes retinol, retinyl acetate and retinyl esters. It also includes retinoic acid, such as for instance undissociated, in its free acid form or dissociated as an anion.
  • Example 1 General methods and plasmids All basic molecular biology and DNA manipulation procedures described herein are generally performed according to Sambrook et al. (eds.), Molecular Cloning:
  • UPLC reverse phase retinol method For rapid screening this method does not separate cis-isomers, only major functional groups.
  • a Waters Acquity UPLC with PDA detection (or similar) with auto sampler was used to inject samples.
  • An Acquity UPLC HSS T31.8um P/N 186003539 was used to resolve retinoids.
  • the mobile phase consisted of either, 1000 mL hexane, 30 mL isopropanol, and 0.1 mL acetic acid for retinoid related compounds.
  • the flow rate for each was 0.6 mL per minute. Column temperature was 20 C. The injection volume was 5 pL. The detector was a photodiode array detector collecting from 210 to 600 nm. Analytes were detected according to Table 1. Table 1a: list of analytes using reverse phase retinol method. The addition of all added intermediates gives the total amount retinoids. Beta-carotene can be detected in 325nm and will interfere with retinyl ester quantification, therefore care must be taken to observe the carotene peak and not include them in the retinoid quantification. "N/A" means "not available”. For more details, see text.
  • Table 1B UPLC Method Gradient with solvent A: water; solvent B: acetonitrile; solvent C: methanol; solvent D: tert-butyl methyl ether.
  • Method Calibration Method is calibrated on retinyl acetate, retinols and retinals are quantitated against retinyl-acetate using the indicated response factor.
  • Retinyl Acetate is dissolved in THF at ⁇ 200pg/ml for stock solution using a volumetric flask. Using volumetric flasks, x20, x50 and x100 dilutions of stock solution in 50/50 methanol/MTBE were made. UV absorbance of retinyl acetate becomes nonlinear fairly quickly, so care must be taken to stay within the linear range. Consequently, lower concentrations might be better.
  • Retinyl palmitate can also be used as retinyl ester calibration. Peaks for retinyl acetate at about 3 minutes and peaks for retinyl esters (long-chain retinyl esters) at around 3.5 minutes.
  • Sample preparation Samples were prepared by various methods depending on the conditions. For whole broth or washed broth samples the broth was placed in a Precellys ® tube, weighted, and mobile phase was added. Briefly in a 2ml Precellys ® tube, add 25pl of well mixed broth and 975mI of THF. The samples were then processed in a Precellys ® homogenizer (Bertin Corp, Rockville, MD, USA) on the highest setting 3X according to the manufacturer's directions, typically 3x15x7500tpms.
  • the samples were spun in a 1.7 ml tube in a microfuge at lOOOOrpm for 1 minute, the broth decanted, 1ml water added, mixed, pelleted and decanted, and brought up to the original volume.
  • the mixture was pelleted again and brought up in appropriate amount of mobile phase and processed by Precellys ® bead beating.
  • the sample was spun at 4000RPM for 10 minutes and the oil was decanted off the top by positive displacement pipet (Eppendorf, Hauppauge, NY, USA) and diluted into mobile phase mixed by vortexing and measured for retinoid concentration by UPLC analysis.
  • the cells were then pelleted and resuspended in liquid YPD media, and permitted to grow for 3 hours at 30°C or overnight at 22°C to allow for expression of the HygR antibiotic resistance gene before plating on the selective media, containing 100pg/ml hygromycin.
  • Most of the DNA sequences used herein are codon-optimized for expression in the respective host system and as indicated in the sequence listing.
  • first strain CAR-0002 was constructed as follows: Three carotenoid gene expression cassettes, crtE, crtYB and crtl from Xanthophyllomyces dendrorhous codon optimized for expression in Saccharomyces cerevisiae were transformed into strain CEN.PK113-7D (van Dijken et al., Enzyme Microb Technol. 26(9-10), p. 706- 714, 2000). The expression cassettes were integrated in the INT1 integration site usingthe CRISPR approach as described in Example 9 of W02016110512.
  • Expression cassettes containing strong constitutive promoters (see SEQ ID NO:139, SEQ ID NO:142 and SEQ ID NO:148 of W02016110512) and donor DNA flank sequences (see SEQ ID NO:149 and SEQ ID NO:152 of W02016110512) were transformed into CEN.PK113-7D.
  • the sequence of the INT1 genomic target (protospacer) is set out in SEQ ID NO:176 of W02016110512.
  • the INT1 integration site is located at the non-coding region between NTR1 (YOR071c) and GYP1 (YOR070c) located on chromosome XV.
  • Strain CEN.PK113-7D was transformed usingthe LiAc/salmon sperm (SS) carrier DNA/PEG method (Gietz and Woods, Methods in Enzymology, Vol. 350, p. 87-86, 2002) using DNA concentrations as described by Verwaal et al. (Yeast, 35, p. 201-211, 2018).
  • SS LiAc/salmon sperm
  • the retinol producing strain MY4834 was created by serial transformation of CAR-0002 with Sfil-linearized plasmid MB8433 (SEQ ID N0:11), containing a constitutive expression cassette for DmBCO (Saccharomyces cerevisiae codon optimized SEQ ID NO:9; TDH3 promoter, PGK1 terminator) with selection on hygromycin, and with a PCR amplicon using primers MO12301 (SEQ ID NO:13) and MO12302 (SEQ ID N0:14) from plasmid MB8431 (SEQ ID NO:12), containing an expression cassette for HsRDH12 (Saccharomyces cerevisiae codon optimized SEQ ID NO:10; TDH3 promoter, PGK1 terminator), targeting the PCR amplicon to a URA3 locus with selection on fluoroorotic acid (FOA).
  • DmBCO Sacharomyces cerevisiae codon optimized SEQ ID NO
  • the NatR marker in plasmid MB7621 and derivatives was removed by transient expression of Cre recombinase that resulted in antibiotic-sensitive excisants due to the flanking lox sites. Plasmids were made using standard molecular genetics techniques or are available by synthetic biology methods (GenScript, Piscataway, NJ).
  • Plasmids comprising the respective wild-type LmATFI (codon- optimized, originated from L. mirantina) and SbATFI (codon-optimized, originated from S. bayanus) enzymes that were used are listed in Table 2 and the sequence listing.
  • Table 2 list of plasmids used for construction of the strains carrying the heterologous ATF1-genes from Lachancea mirantina LmATFI or Saccharomyces bayanus SbATFI.
  • the codon- optimized sequences were used. Fermentation conditions. Fermentations were identical to the previously described conditions using preferably a silicone oil overlay and stirred tank that was preferably glucose in a bench top reactor with 0.5L to 5L total volume (see WO2016172282). Generally, the same results were observed with a fed batch stirred tank reactor with an increased productivity demonstrating the utility of the system for the production of retinoids.
  • fermentations were batched with 5% glucose and 20% silicone oil was added after dissolved oxygen dropped below about 20% and feed was resumed to achieve 20% dissolved oxygen throughout the feeding program.
  • Example 2 Expression of ATF in Saccharomyces cerevisiae
  • heterologous LmATFI For expression of heterologous LmATFI, SbATFI and the respective mutants (wt based on Saccharomyces cerevisiae codon optimized SEQ ID NO:2 and 4) in Saccharomyces cerevisiae as a host, the retinol producing strain MY4834 (see Ex. 1) was transformed with purified Sfil-digested gene fragments containing Hygromycin resistance marker (HygR) for selection on rich media (YPD) containing 100pg/ml hygromycin and HSP26 as strong constitutive promoter. Prior to plating the cultures are outgrown in YPD for 3 hours to synthesize the antibiotic resistance gene.
  • Hygromycin resistance marker Hygromycin resistance marker
  • Transformations were done in parallel for the backbone expression plasmid lacking an ATF (MB7621), but with a HygR marker.
  • Strains transformed with expression plasmids for non-modified SbATF or LmATF produced no transformants, mutant ATF derivatives produced few transformants, while those with an empty plasmid produced the most transformants (Table 3).
  • Table 3 Transformant count of MY4834 with 2.5-3pg of linearized plasmid. All constructs were expressed under control of strong constitutive promoter HSP26. For more details, see text.
  • Transformants from Example 2 expressing the mutant ATFs were plated on glucose and screened for production of retinyl acetate in culture tubes grown as described above (Ex. 1). After growth, oil overlays were sampled from centrifuged cultures, and subjected to UPLC analysis (see Example 1). Control reactions revealed a range of about 1.6 to 2% retinyl acetate based on total retinoids present in the cell with the mutant LmATFI. No retinyl acetate was detected with the empty plasmid MB7621.

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Abstract

La présente invention concerne l'expression d'enzymes hétérologues sous le contrôle de promoteurs constitutifs, en particulier l'expression d'enzymes de type acétyltransférase chez un hôte sélectionné à partir de Saccharomyces.
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WO2006102342A2 (fr) 2005-03-18 2006-09-28 Microbia, Inc. Production de caroténoïdes dans de la levure ou des champignons oléagineux
WO2008042338A2 (fr) 2006-09-28 2008-04-10 Microbia, Inc. Production de caroténoïdes dans des levures et des champignons oléagineux
WO2014096992A1 (fr) 2012-12-20 2014-06-26 Dsm Ip Assets B.V. Acétyltransférases et leur utilisation pour la production de caroténoïdes
WO2016110512A1 (fr) 2015-01-06 2016-07-14 Dsm Ip Assets B.V. Système crispr-cas pour une cellule hôte de levure
WO2016172282A1 (fr) 2015-04-21 2016-10-27 Dsm Ip Assets B.V. Production microbienne de terpénoïdes
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WO2008042338A2 (fr) 2006-09-28 2008-04-10 Microbia, Inc. Production de caroténoïdes dans des levures et des champignons oléagineux
WO2014096992A1 (fr) 2012-12-20 2014-06-26 Dsm Ip Assets B.V. Acétyltransférases et leur utilisation pour la production de caroténoïdes
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WO2024160658A1 (fr) * 2023-01-30 2024-08-08 Dsm Ip Assets B.V. Nouveaux mutants d'acétyl-transférase sb-atf

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