Methods for producing modified betalains in yeast
AssigneeDanmarks Tekniske Universitet
InventorsIrina Borodina, Jane Dannow Dyekjær, Christiane Ursula Glitz, Mahsa Babaei, Philip Tinggaard Thomsen
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Abstract
The present invention relates to microbial cell factories, in particular yeast cell factories, for production of modified betalains, such as acylated and di-glycosylated betalains.
Claims17
A yeast cell producing a modified betalain in the presence of betanin, isobetanin and/or glycosylated cyclo-DOPA, wherein the modified betalain is selected from the group consisting of acylated betalains and di-glycosylated betalains, and wherein the yeast cell expresses:
- a. a heterologous BAHD acyltransferase capable of acylating a betalain and/or glycosylated cyclo-DOPA, thereby producing an acylated betalain;
- and/or
- b. a heterologous glycosyltransferase capable of glycosylating a betalain and/or glycosylated cyclo-DOPA, thereby producing a di-glycosylated betalain.
The yeast cell according to any one of the preceding claims, wherein:
- a. the acylated betalain is an acylated betacyanin, such as an acylated betanin and/or isobetanin, such as a betanin and/or isobetanin acylated at position 4 of the glycosyl moiety of betanin and/or isobetanin, such as phyllocactin II and/or isophyllocactin II;
- and/or
- b. the acylated betalain is an acylated betacyanin, such as an acylated betanin and/or isobetanin, such as a betanin and/or isobetanin acylated at position 6 of the glycosyl moiety of betanin and/or isobetanin, such as phyllocactin and/or isophyllocactin;
- and/or
- c. the di-glycosylated betalain is a di-glycosylated betacyanin, such as a glycosylated betanin and/or isobetanin, such as a betanin and/or isobetanin glycosylated at position 2 of the glycosyl moiety of betanin and/or isobetanin, such as a betanin and/or isobetanin glucosylated at position 2 of the glycosyl moiety of betanin and/or isobetanin, such as bougainvillein-r-I and/or isobougainvilleinen-r-I;
- and/or
- d. the yeast cell is supplemented with and/or capable of producing UDP-glucuronic acid and the di-glycosylated betalains is a di-glycosylated betacyanin, such as a glycosylated betanin and/or isobetanin, such as a betanin and/or isobetanin glycosylated at position 2 of the glycosyl moiety of betanin and/or isobetanin, such as a betanin and/or isobetanin glucoronidated at position 2 of the glycosyl moiety of betanin and/or isobetanin, such as amaranthin and/or isoamaranthin.
The yeast cell according to any one of the preceding claims, wherein:
- a. the BAHD acyltransferase is native to a plant, such as a plant of the genus Hylocereus, such as Hylocereus polyrhizus, optionally wherein the BAHD acyltransferase is HpBAHD3 as set forth in SEQ ID NO:
- 29 or a functional variant thereof having at least 70% sequence identity thereto;
- and/or
- b. the glycosyltransferase is native to a plant, such as a plant of the genus Amaranthus, Chenopodium and/or Celosia, such as Amaranthus hypochondriacus, Chenopodium quinoa or Celosia cristata, optionally the wherein the glycosyltransferase is AhAmaSy1 as set forth in SEQ ID NO:
- 38, CqAmaSy1 as set forth in SEQ ID NO:
- 41 and/or CcAmaSy1 as set forth in SEQ ID NO:
- 44 or a functional variant thereof having at least 70% sequence identity to SEQ ID NO:
- 38, SEQ ID NO:
- 41 or SEQ ID NO 44.
The yeast cell according to any one of the preceding claims, wherein the genus of said yeast cell is selected from the group consisting of Saccharomyces, Pichia, Yarrowia, Kluyveromyces, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon or Lipomyces, such as wherein the yeast is of a species selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces boulardi, Candida tropicalis, Pichia pastoris, Kluyveromyces marxianus, Cryptococcus albidus, Lipomyces lipofera, Lipomyces starkeyi, Rhodosporidium toruloides, Rhodotorula glutinis, Trichosporon pullulan or Yarrowia lipolytica, preferably wherein the yeast is Saccharomyces cerevisiae or Yarrowia lipolytica.
The yeast cell according to any one of the preceding claims, wherein said yeast cell further expresses:
- a. a first heterologous enzyme which is a tyrosine hydroxylase (TYH) capable of hydroxylating L-tyrosine and oxidizing L-DOPA;
- b. a second heterologous enzyme which is a 4,5-DOPA extradiol dioxygenase (DOD);
- and
- c. a third heterologous enzyme having glycosyltransferase activity;
- whereby the yeast cell produces betanin and/or isobetanin;
- optionally wherein:
- a. the TYH is native to a plant, such as of the genus Abronia, Acleisanthes, Basella, Beta, Cleretum, Ercilla, Mirabilis, Optunia or Phytolacca, such as Abronia nealleyi, Acleisanthes obtusa, Basella alba, Beta vulgaris, Cleretum bellidiforme, Ercilla volubilis, Mirabilis multiflora, Optunia ficus-indic, or Phytolacca dioica, or a functional variant thereof having at least 80% identity thereto;
- and/or
- b. the DOD is native to a plant, such as of the genus Amaranthus, Beta, Bougainvillea, Mirabilis Phytolacca, Portulaca, Spinacia or Suaeda, such as Amaranthus hypochondriacus, Amaranthus tricolour, Beta vulgaris, Bougainvillea glabra, Mirabilis jalapa, Phytolacca americana, Portulaca grandiflora, Spinacia oleracea or Suaeda salsa, or a functional variant thereof having at least 80% identity thereto;
- and/or
- c. the enzyme having glycosyltransferase activity is native to a plant, such as of the genus Abronia, Beta, Bougainvillea, Ercilla or Portacula, such as Abronia nealleyi, Beta vulgaris, Bougainvillea glabra, Ercilla volubilis or Portacula grandiflora, or a functional variant thereof having at least 80% identity thereto;
- optionally wherein:
- a. the TYH is selected from:
- i. Abronia nealleyi TYH, such as AnTYH as set forth in SEQ ID NO:
- 49 or a functional variant thereof having at least 70% sequence identity thereto;
- ii. Ercilla volubilis TYH, such as EvTYH as set forth in SEQ ID NO:
- 6 or a functional variant thereof having at least 70% sequence identity thereto;
- iii. Beta vulgaris TYH, such as BvCYP76AD1W13L as set forth in SEQ ID NO:
- 4 or a functional variant thereof having at least 70% sequence identity thereto;
- and/or
- b. the DOD is selected from:
- i. Mirabilis jalapa DOD, such as MjDOD as set forth in SEQ ID NO:
- 1 or a functional variant thereof having at least 70% sequence identity thereto;
- ii. Portulaca grandiflora DOD, such as PgDOD as set forth in SEQ ID NO:
- 55 or a functional variant thereof having at least 70% sequence identity thereto;
- iii. Bougainvillea glabra DOD, such as BgDOD2 as set forth in SEQ ID NO:
- 52 or a functional variant thereof having at least 70% sequence identity thereto;
- and
- c. the enzyme having glycosyltransferase activity is selected from:
- i. Chenopodium quinoa glycosyltransferase CqSGT2 set forth in SEQ ID NO:
- 57, or a functional variant thereof having at least 70% sequence identity thereto;
- ii. Beta vulgaris glycosyltransferase BvSGT2 set forth in SEQ ID NO:
- 8, or a functional variant thereof having at least 70% sequence identity thereto;
- iii. Bougainvillea glabra glycosyltransferase BgGT2 set forth in SEQ ID NO:
- 11, or a functional variant thereof having at least 70% sequence identity thereto;
- and
- iv. Beta vulgaris glycosyltransferase BvSGT4 set forth in SEQ ID NO:
- 59, or a functional variant thereof having at least 70% sequence identity thereto.
The yeast cell according to any one of the preceding claims, wherein said yeast cell comprises a mutation resulting in reduced activity in one or more genes encoding one or more native β-glucosidases,
- wherein the activity is reduced as compared the activity in a yeast cell in which the one or more native β-glucosidases have not been mutated, but otherwise identical, when cultivated in the same conditions,
- optionally wherein said one or more genes are selected from the group consisting of:
- i) YALI1_B23300g (SEQ ID NO:
- 61);
- ii) YALI1_F21504g (SEQ ID NO:
- 62);
- iii) YALI1_B05024g (SEQ ID NO:
- 63);
- iv) YALI1_B18845g (SEQ ID NO:
- 64);
- v) YALI1_B18887g (SEQ ID NO:
- 65);
- vi) YALI1_D22997g (SEQ ID NO:
- 66);
- vii) YALI1_E40502g (SEQ ID NO:
- 67);
- viii) YALI1_F02592g (SEQ ID NO:
- 68);
- ix) YALI1_F08075g (SEQ ID NO:
- 69);
- x) YALI1_F17788g (SEQ ID NO:
- 70);
- xi) YALI1_E23994g (SEQ ID NO:
- 79);
- xii) YALI1_E39796g (SEQ ID NO:
- 80);
- xiii) YALI1_F03075g (SEQ ID NO:
- 81);
- xiv) YALI1_E25163g (SEQ ID NO:
- 82);
- xv) YALI0F16027g (SEQ ID NO:
- 217);
- xvi) YALI0B14289g (SEQ ID NO:
- 218);
- and
- xvii) YALI0F05390g (SEQ ID NO:
- 219);
- preferably wherein said one or more genes are selected from the group consisting of YALI1_D22997g (SEQ ID NO:
- 66), YALI1_B23300g (SEQ ID NO:
- 61), YALI1_F21504g (SEQ ID NO:
- 62), YALI1_F08075g (SEQ ID NO:
- 69), YALI1_B18845g (SEQ ID NO:
- 64), YALI1_E23994g (SEQ ID NO:
- 82), YALI0F16027g (SEQ ID NO:
- 217), YALI0B14289g (SEQ ID NO:
- 218) and YALI0F05390g (SEQ ID NO:
- 219).
The yeast cell according to any one of the preceding claims, wherein said yeast cell comprises:
- a. a mutation resulting in reduced activity of 4-hydroxyphenylpyruvate dioxygenase (4-HPPD), wherein the activity is reduced as compared the activity in a yeast cell in which 4-HPPD has not been mutated, but otherwise identical, when cultivated in the same conditions;
- b. a mutation resulting in increased production of L-tyrosine, wherein the production is increased as compared the production in a yeast cell not carrying said mutation resulting in increased production of L-tyrosine, but otherwise identical, when cultivated in the same conditions;
- and/or
- c. a mutation resulting in less sensitivity to feedback inhibition by aromatic amino acids, wherein the sensitivity is reduced as compared the sensitivity in a yeast cell not carrying said mutation resulting in less sensitivity to feedback inhibition by aromatic amino acids, but otherwise identical, when cultivated in the same conditions.
The yeast cell according to any one of the preceding claims, wherein said yeast cell comprises a mutation in:
- a. 3-deoxy-7-phosphoheptulonate synthase, such as in Yarrowia lipolytica Aro4 or Saccharomyces cerevisiae Aro4, such as wherein amino acid no. 221 of Yarrowia lipolytica Aro4 is substituted with a leucine or wherein amino acid no. 229 of Saccharomyces cerevisiae Aro4 is substituted with leucine;
- and/or
- b. chorismate mutase, such as in Yarrowia lipolytica Aro7 or Saccharomyces cerevisiae Aro7, such as wherein amino acid no. 139 of Yarrowia lipolytica Aro7 is substituted with a serine or wherein amino acid no. 141 of Saccharomyces cerevisiae Aro7 is substituted with a serine.
The yeast cell according to any one of the preceding claims, wherein said yeast cell comprises as a partial or total deletion of the gene encoding 4-HPPD.
A method of producing a modified betalain in the presence of betanin, isobetanin and/or glycosylated cyclo-DOPA in a yeast cell, wherein the modified betalain is selected from the group consisting of acylated betalains and di-glycosylated betalains, said method comprising the steps of:
- a. incubating a yeast cell in a medium;
- b. expressing in said yeast cell:
- i. a heterologous BAHD acyltransferase capable of acylating a betalain and/or glycosylated cyclo-DOPA, thereby obtaining an acylated betalain;
- and/or
- ii. a heterologous glycosyltransferase capable of glycosylating a betalain and/or glycosylated cyclo-DOPA, thereby obtaining a di-glycosylated betalain.
Use of a glycosyltransferase:
- a. in a method of producing a di-glycosylated betalain, such as a glycosylated betanin and/or isobetanin;
- b. to catalyse the glycosylation of glycosylated cyclo-DOPA and/or a betalain, such as betanin and/or isobetanin, thereby obtaining a di-glycosylated betalain;
- and/or
- c. for glucuronidation of position 2 of the glycosyl moiety of betanin and/or isobetanin.
A method of increasing the titer and/or purity of a betalain produced by a yeast cell, wherein the betalain is selected from the group consisting of glycosylated betalains, acylated betalains and di-glycosylated betalains, said method comprising the steps of:
- a. providing a yeast cell comprising a mutation resulting in reduced activity of one or more native β-glucosidases, said yeast cell producing a glycosylated betalain;
- b. incubating said yeast cell in a medium;
- c. expressing in said yeast cell:
- i. a first heterologous enzyme which is a tyrosine hydroxylase (TYH) capable of hydroxylating L-tyrosine and oxidizing L-DOPA;
- ii. a second heterologous enzyme which is a 4,5-DOPA extradiol dioxygenase (DOD);
- and
- iii. a third heterologous enzyme having glycosyltransferase activity;
- d. optionally, further expressing in said yeast cell:
- i. a heterologous BAHD acyltransferase capable of acylating a betalain and/or glycosylated cyclo-DOPA;
- and/or
- ii. a heterologous glycosyltransferase capable of glycosylating a betalain and/or glycosylated cyclo-DOPA;
- e. optionally recovering the modified betalain;
- thereby obtaining a betalain with an improved purity and/or titer as compared to the purity and/or titer of a betalain produced by a yeast cell expressing said TYH, DOD, enzyme having glycosyltransferase activity and optionally said BAHD acyltransferase and/or glycosyltransferase, but not comprising a mutation resulting in reduced activity of one or more native β-glucosidases, and cultivated under the same conditions.
Dependencies are inferred from the claim text.