Glycosyltransferase glycosylating flavokermesic acid and/or kermesic acid
10724012 · 2020-07-28
Assignee
Inventors
- Rubini Maya Kannangara (Frederiksberg, DK)
- Mads Bennedsen (Græsted, DK)
- Bjørn Madsen (Helsingør, DK)
- Kim Binderup (Charlottelund, DK)
- Ulf Thrane (Helsinge, DK)
- Rasmus John Normand Frandsen (Allerød, DK)
- Uffe Hasbro Mortensen (Copenhagen N, DK)
- Birger Lindberg Møller (Brønshøj, DK)
- Finn Thyge Okkels (Roskilde, DK)
Cpc classification
C12N15/63
CHEMISTRY; METALLURGY
International classification
C12N15/63
CHEMISTRY; METALLURGY
Abstract
An isolated glycosyltransferase (GT) polypeptide capable of: (I): conjugating glucose to flavokermesic acid (FK); and/or (II): conjugating glucose to kermesic acid (KA) and use of this GT to e.g. make Carminic acid.
Claims
1. A method for producing flavokermesic acid glycoside or kermesic acid glycoside comprising the acts of: providing an aglycon wherein said aglycon comprises flavokermesic acid or kermesic acid; providing a sugar, wherein said sugar comprises a nucleotide activated glucose; providing a recombinant cell wherein said recombinant cell comprises an expression cassette comprising an isolated polynucleotide comprising a nucleotide sequence which encodes an isolated glycosyltransferase polypeptide capable of: (I): conjugating nucleotide activated glucose to flavokermesic acid (FK); and/or (II): conjugating nucleotide activated glucose to kermesic acid (KA); and wherein the glycosyltransferase polypeptide is at least one polypeptide selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence which has at least 95% identity with amino acids 1 to 515 of SEQ ID NO:2; and (b) a polypeptide comprising an amino acid sequence which has at least 95% identity with amino acids 20 to 468 of SEQ ID NO:2; and wherein said polynucleotide is operably linked to one or more control sequences that direct the production of the polypeptide in an expression host; and contacting said glycosyltransferase polypeptide with said aglycon and said nucleotide activated glucose to produce flavorkermesic acid glycoside or kermesic acid glycoside.
2. The method of claim 1, wherein said cell is Saccharomyces spp. or Pichia spp.
3. The method of claim 2, wherein said cell is Saccharomyces cerevisiae.
4. The method of claim 1, wherein said nucleotide activated glucose comprises UDP-glucose.
5. The method of claim 1, wherein said expression cassette comprises a plasmid.
6. The method of claim 1, further comprising the act of purifying said produced flavokermesic acid glycoside or kermesic acid glycoside.
7. A method for producing flavokermesic acid glycoside or kermesic acid glycoside in vitro comprising the acts of: providing an aglycon wherein said aglycon comprises flavokermesic acid or kermesic acid; providing a sugar, wherein said sugar comprises a nucleotide activated glucose; providing an isolated recombinant glycosyltransferase produced by recombinant cell wherein said recombinant cell comprises an expression cassette comprising an isolated polynucleotide comprising a nucleotide sequence which encodes an isolated glycosyltransferase polypeptide capable of: (I): conjugating nucleotide activated glucose to flavokermesic acid (FK); and/or (II): conjugating nucleotide activated glucose to kermesic acid (KA); and wherein the glycosyltransferase polypeptide is at least one polypeptide selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence which has at least 95% identity with amino acids 1 to 515 of SEQ ID NO:2; and (b) a polypeptide comprising an amino acid sequence which has at least 95% identity with amino acids 20 to 468 of SEQ ID NO:2; and wherein said polynucleotide is operably linked to one or more control sequences that direct the production of the polypeptide in an expression host; and contacting said glycosyltransferase polypeptide with said aglycon and said nucleotide activated glucose to produce flavokermesic acid glycoside or kermesic acid glycoside.
8. The method of claim 7, wherein said cell is Saccharomyces spp. or Pichia spp.
9. The method of claim 8, wherein said cell is Saccharomyces cerevisiae.
10. The method of claim 7, wherein said nucleotide activated glucose comprises UDP-glucose.
11. The method of claim 7, wherein said isolated recombinant glycosyltransferase is isolated by the act of extraction from said recombinant cell comprising a expression cassette which encodes for a glycosyltransferase.
Description
DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
(10) The present application includes a Sequence Listing which has been submitted in ASCII computer readable format (CFR) and in paper format, both via EFS-Web, and are hereby incorporated by reference in their entirety.
(11) A Novel Isolated Glycosyltransferase Polypeptide as Described Herein
(12) When there herein is referred to an isolated glycosyltransferase polypeptide as described herein there is referred to an isolated glycosyltransferase polypeptide of the first aspect of the invention and/or herein relevant embodiments thereof.
(13) As discussed abovethe term isolated polypeptide essentially relates to that the polypeptide is isolated from its natural environment. The herein described novel glycosyltransferase polypeptide as shown in SEQ ID NO: 2 was isolated from the insect Dactylopius coccus. Accordingly, as understood by the skilled person in the present contextthe term isolated polypeptide does not cover the glycosyltransferase polypeptide of SEQ ID NO: 2 when it is naturally present in the genome of Dactylopius coccus.
(14) Preferably, the isolated glycosyltransferase polypeptide as described herein denotes a polypeptide preparation which contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% by weight of other polypeptide material with which it is natively associated.
(15) As understood by the skilled person, the term other polypeptide material with which it is natively associated may in relation to the novel glycosyltransferase polypeptide as shown in SEQ ID NO: 2 be seen as relation to other polypeptide material with which it is natively associated in Dactylopius coccus.
(16) In some caseit may be preferred that the isolated glycosyltransferase polypeptide as described herein refers to a polypeptide which is at least 20% pure, preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, most preferably at least 90% pure, and even most preferably at least 95% pure, as determined by SDS-PAGE.
(17) Based on e.g. the sequence information disclosed hereinit is routine work for the skilled person to obtain an isolated glycosyltransferase polypeptide as described herein.
(18) This may e.g. be done by recombinant expression in a suitable recombinant host cell according to procedures known in the art.
(19) Accordingly, it is not believed necessary to describe such standard known recombinant expression procedures in many details herein.
(20) Preferably, the isolated glycosyltransferase polypeptide as described herein is capable of: (I): conjugating glucose to flavokermesic acid (FK); and (II): conjugating glucose to kermesic acid (KA).
(21) A preferred embodiment relates to wherein the glycosyltransferase polypeptide of the first aspect is: (a) a polypeptide comprising an amino acid sequence which has at least 80% identity with amino acids 1 to 515 of SEQ ID NO:2; more preferably (a) a polypeptide comprising an amino acid sequence which has at least 90% identity with amino acids 1 to 515 of SEQ ID NO:2; even more preferably (a) a polypeptide comprising an amino acid sequence which has at least 95% identity with amino acids 1 to 515 of SEQ ID NO:2; and most preferably (a) a polypeptide comprising an amino acid sequence which has at least 98% identity with amino acids 1 to 515 of SEQ ID NO:2.
(22) It may be preferred that the glycosyltransferase polypeptide of the first aspect is a polypeptide comprising an amino acid sequence with amino acids 1 to 515 of SEQ ID NO:2.
(23) A preferred embodiment relates to wherein the glycosyltransferase polypeptide of the first aspect is: (b) a polypeptide comprising an amino acid sequence which has at least 80% identity with amino acids 20 to 468 of SEQ ID NO:2; more preferably (b) a polypeptide comprising an amino acid sequence which has at least 90% identity with amino acids 20 to 468 of SEQ ID NO:2; even more preferably (b) a polypeptide comprising an amino acid sequence which has at least 95% identity with amino acids 20 to 468 of SEQ ID NO:2; and most preferably (b) a polypeptide comprising an amino acid sequence which has at least 98% identity with amino acids 20 to 468 of SEQ ID NO:2.
(24) It may be preferred that the glycosyltransferase polypeptide of the first aspect is a polypeptide comprising an amino acid sequence with amino acids 20 to 468 of SEQ ID NO:2.
(25) A preferred embodiment relates to wherein the glycosyltransferase polypeptide of the first aspect is: (c) a polypeptide which is encoded by a polynucleotide which hybridizes under at least medium-high stringency conditions with (i) nucleotides 1 to 1548 of SEQ ID NO:1 or (ii) a complementary strand of (i); more preferably (c) a polypeptide which is encoded by a polynucleotide which hybridizes under at least high stringency conditions with (i) nucleotides 1 to 1548 of SEQ ID NO:1 or (ii) a complementary strand of (i); and even more preferably (c) a polypeptide which is encoded by a polynucleotide which hybridizes under at least very stringency conditions with (i) nucleotides 1 to 1548 of SEQ ID NO:1 or (ii) a complementary strand of (i).
(26) It is routine work for the skilled person to make a variant of an isolated glycosyltransferase polypeptide as described hereini.e. a variant, wherein e.g. one or more amino acids of e.g. SEQ ID NO:2 have been modified/altered.
(27) Furtheras known to the skilled person if such variant changes are not too drastic it will be plausible that the enzyme would maintain its relevant GT activity.
(28) A preferred embodiment relates to wherein the glycosyltransferase polypeptide of the first aspect is:
(29) (a) a polypeptide comprising an amino acid sequence with amino acids 1 to 515 of SEQ ID NO:2 or a variant thereof, wherein the variant comprises an alteration at one or more (several) positions of SEQ ID NO:2 and wherein the variant comprises less than 50 alterations, more preferably less than 40 alterations, even more preferably less than 20 alterations and most preferably less than 10 alterations.
(30) In a preferred embodiment the term an alteration at one or more (several) positions of SEQ ID NO:2 refers to 1 to 10 alterations in SEQ ID NO:2.
(31) According to the artthe term variant means herein a peptide having the relevant GT activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (several) positions. A substitution means a replacement of an amino acid occupying a position with a different amino acid; a deletion means removal of an amino acid occupying a position; and an insertion means adding 1-3 amino acids adjacent to an amino acid occupying a position.
(32) The amino acid may be natural or unnatural amino acidsfor instance, substitution with e.g. a particularly D-isomers (or D-forms) of e.g. D-alanine could theoretically be possible.
(33) In a preferred embodiment the glycosyltransferase polypeptide of the first aspect is a GT which is membrane bound or insoluble in water.
(34) Isolated Polynucleotide Comprising a Nucleotide Sequence which Encodes the Glycosytransferase Polypeptide as Described Herein
(35) As discussed abovea second aspect of the present invention relates to an isolated polynucleotide comprising a nucleotide sequence which encodes the polypeptide of the first aspect and/or herein relevant embodiments thereof.
(36) The term isolated polynucleotide may herein alternatively be termed cloned polynucleotide.
(37) As discussed abovethe term isolated polynucleotide essentially relates to that the polynucleotide is isolated from its natural environmentsaid in other words that the polynucleotide preparation is essentially free of other polynucleotide material with which it is natively associated. The polynucleotide sequence encoding the described isolated/cloned novel glycosyltransferase is shown in SEQ ID NO: 1 and it was isolated from the insect Dactylopius coccus. Accordingly, as understood by the skilled personthe term isolated polynucleotide does not cover the polynucleotide of SEQ ID NO: 1 when it is naturally present in the genome of Dactylopius coccus.
(38) The term isolated polynucleotide essentially relates to that the isolated polynucleotide is in a form suitable for use within genetically engineered protein production systems.
(39) Thus, an isolated polynucleotide contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% by weight of other polynucleotide material with which it is natively associated.
(40) Based on e.g. the sequence information disclosed hereinit is routine work for the skilled person to obtain an isolated polynucleotide as described herein.
(41) This may e.g. be done by recombinant expression in a suitable recombinant host cell according to procedures known in the art.
(42) Accordingly, it is not believed necessary to describe such standard known recombinant expression procedures in many details herein.
(43) A Nucleic Acid Construct Comprising the Isolated Polynucleotide as Described Herein
(44) As discussed abovea third aspect of the present invention relates to a nucleic acid construct comprising the isolated polynucleotide of the second aspect and/or herein relevant embodiments thereof operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.
(45) According to the artthe term nucleic acid construct as used herein refers to a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature.
(46) The term nucleic acid construct is synonymous with the term expression cassette when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present invention. As known in the art control sequences include all components, which are necessary or advantageous for the expression of a polynucleotide encoding a polypeptide of the present invention.
(47) Based on e.g. the sequence information disclosed hereinit is routine work for the skilled person to make a relevant nucleic acid constructfor instance, based on the prior art the skilled person knows numerous different suitable control sequences for the expression of a polynucleotide encoding a polypeptide of the present invention. Accordingly, it is not believed necessary to describe such standard known technical elements in many details herein.
(48) A Recombinant Expression Vector Comprising the Nucleic Acid Construct as Described Herein
(49) As discussed abovea fourth aspect of the present invention relates to a recombinant expression vector comprising the nucleic acid construct of the third aspect and/or herein relevant embodiments thereof.
(50) According to the artthe term recombinant expression vector relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleic acids and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleotide sequence encoding the polypeptide at such sites.
(51) Based on e.g. the sequence information disclosed hereinit is routine work for the skilled person to make a relevant recombinant expression vectorfor instance, based on the prior art the skilled person knows numerous different suitable promoter, and transcriptional and translational stop signals.
(52) Accordingly, it is not believed necessary to describe such standard known technical elements in many details herein.
(53) A Recombinant Host Cell Comprising the Nucleic Acid Construct as Described Herein
(54) As discussed abovea fifth aspect of the present invention relates to a recombinant host cell comprising the nucleic acid construct of the third aspect and/or herein relevant embodiments thereof.
(55) The term recombinant host cell should herein be understood according to the art. As known in the art, recombinant polynucleotide (e.g. DNA) molecules are polynucleotide (e.g. DNA) molecules formed by laboratory methods of genetic recombination (such as molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms. As understood by the skilled persona recombinant host cell comprises recombinant polynucleotide (e.g. DNA) molecules and a recombinant host cell will therefore not be understood as covering a natural wildtype cell, such as e.g. a natural wildtype Dactylopius coccus cell.
(56) Based on e.g. the sequence information disclosed hereinit is routine work for the skilled person to make a relevant recombinant host cellfor instance, based on the prior art the skilled person knows numerous different suitable recombinant host cells that for years have been used as recombinant host cells for e.g. expression of different polypeptides of interest.
(57) The recombinant host cell may be any suitable cell such as any eukaryotic cell [e.g. mammalian cells (such as e.g. Chinese hamster ovary (CHO) cells) or a plant cell] or any prokaryotic cell.
(58) Particularly preferred is wherein the recombinant host cell is a plant cell producing flavokermesic acid/kermesic acid or other related compound such as e.g. rhubarb plant cell.
(59) Preferably the recombinant host cell is a cell selected from the group consisting of a filamentous fungal cell and a microorganism cell.
(60) Filamentous fungi include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are characterized by a vegetative mycelium composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
(61) It may be preferred that the filamentous fungal cell is a cell of a species of, but not limited to, Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, and Trichoderma or a teleomorph or synonym thereof.
(62) A preferred Aspergillus cell is Aspergillus niger or Aspergillus oryzae.
(63) A preferred microorganism cell herein is a microorganism cell selected from the group consisting of a yeast cell and prokaryotic cell.
(64) A preferred yeast cell is a yeast cell selected from the group consisting of Ascomycetes, Basidiomycetes and fungi imperfecti. Preferably a yeast cell selected from the group consisting of Ascomycetes.
(65) Preferred Ascomycetes yeast cell selected from the group consisting of Ashbya, Botryoascus, Debaryomyces, Hansenula, Kluveromyces, Lipomyces, Saccharomyces spp e. g. Saccharomyces cerevisiae, Pichia spp., Schizosaccharomyces spp.
(66) A preferred yeast cell is a yeast cell selected from the group consisting of Saccharomyces spp, e. g. Saccharomyces cerevisiae, and Pichia spp.
(67) A preferred prokaryotic cell is selected from the group consisting of Bacillus, Streptomyces, Corynebacterium, Pseudomonas, lactic acid bacteria and an E. coli cell.
(68) A preferred Bacillus cell is B. subtilis, B. amyloliquefaciens or B. licheniformis.
(69) A preferred Streptomyces cell is S. setonii or S. coelicolor.
(70) A preferred Corynebacterium cell is C. glutamicum.
(71) A preferred Pseudomonas cell is P. putida or P. fluorescens.
(72) A Method for Producing Flavokermesic Acid (FK) Glycoside and/or Kemesic Acid (KA)
(73) As discussed abovea sixth aspect of the present invention relates to a method for producing flavokermesic acid (FK) glycoside and/or kermesic acid (KA) glycoside, wherein the method comprises following steps:
(74) (A): contacting in vitro or in vivo in a recombinant host cell comprising a glycosyltransferase gene encoding a glycosyltransferase:
(75) (a1): flavokermesic acid (FK) with a glycosyltransferase capable of glycosylating the flavokermesic acid under suitable conditions wherein there is produced the flavokermesic acid glycoside; and/or (a2): kermesic acid (KA) with a glycosyltransferase capable of glycosylating the kermesic acid under suitable conditions wherein there is produced the kermesic acid glycoside.
(76) It may be preferred that the recombinant host cell in step (A) is a recombinant host cell comprising a recombinant glycosyltransferase gene encoding a glycosyltransferase.
(77) Preferably, the glycosyltransferase in step (a2) is a glucosyltransferase and there thereby in step (a2) is produced kermesic acid glucoside, preferably wherein the produced kermesic acid glucoside is Carminic acid (
(78) It may be preferred that the glycosyltransferase in step (a1) is a glucosyltransferase and there thereby in step (a1) is produced flavokermesic acid glucoside, preferably wherein the produced flavokermesic acid glucoside is the compound DcII (
(79) When the produced compound in step (a1) is DcII it may be preferred to use this DcII as an intermediate to make Carminic acid.
(80) This may be done by chemical synthesis and the skilled person knows based on his common general knowledge how to do this.
(81) Alternatively, it may be done enzymatically by e.g. using a suitable oxygenase. An example of a suitable oxygenase is cytochrome P450 superfamily of monooxygenases (officially abbreviated as CYP) enzyme. Other examples are flavine monooxygenases or different types of dioxygenases, this list not to be considered excluding the involvement of other classes of enzymes
(82) As known in the artthe most common reaction catalyzed by cytochromes P450 is a monooxygenase reaction, e.g., insertion of one atom of oxygen into a substrate.
(83) As understood by the skilled person in the present contextthe terms flavokermesic acid (FK) and/or kermesic acid (KA) aglycons of step (a) of the method of the sixth aspect as discussed herein should be understood as the FK and/or KA specific compounds shown in
(84) As understood in by the skilled personif FK methyl ester is used as aglycon in step (a) of the method of the sixth aspect then there will via the glycosylation step be generated a FK methyl ester glycoside, which by routine removal of the methyl group will generate DcIIaccordingly FK methyl ester aglycon may be seen as equivalent to FK aglycon in relation to the method of the sixth aspect as discussed herein.
(85) In step (a) of the method of the sixth aspect is specified that there is used a glycosyltransferase capable of glycosylating FK and/or KAaccordingly it is understood that the GT must be capable of doing this.
(86) It may be preferred to purify the glycoside produced in step (A)i.e. in step (a1) and/or in step (a2).
(87) Accordingly it may be preferred that the method of the sixth aspect comprises a further step (B) with following steps: (B): purifying the produced glycoside in step (a1) and/or in step (a2) whereby one gets a composition, wherein at least 5% w/w (preferably at least 10% w/w, more preferably at least 50% w/w and most preferably at least 80% w/w) of the compounds in the composition is the produced flavokermesic acid glycoside and/or kermesic acid glycoside.
(88) The skilled person knows how to purify such glycoside compounds and it may be done according to the art.
(89) The purifying step (B) may be particularly preferred when: the produced glycoside in step (a2) is Carminic acid; the produced glycoside in step (a1) is compound DcII; and/or the produced glycoside in step (a1) is compound DcII and it is used as an intermediate to make Carminic acid.
(90) As discussed hereinin working Examples there was made a contacting in vitro of flavokermesic acid (FK) and/or kermesic acid (KA) with the glycosyltransferase of SEQ ID NO:2. It may be seen as routine work for the skilled person to perform such an in vitro contacting step.
(91) The glycosyltransferase of SEQ ID NO:2 was recombinantly expressed in a yeast cell (see working Example herein)accordingly, in a working Example herein there was made a recombinant yeast host cell comprising a recombinant glycosyltransferase gene encoding a glycosyltransferase of SEQ ID NO:2.
(92) It is believed that if flavokermesic acid (FK) and/or kermesic acid (KA) would be added under suitable condition to a fermentation medium the FK and/or KA compound(s) would enter into e.g. yeast cells fermented in the mediumaccordingly, if e.g. the yeast cells are recombinant yeast host cells comprising a recombinant glycosyltransferase gene encoding a glycosyltransferase then there would be made a contacting in vivo in a recombinant host cell of FK and/or KA with a glycosyltransferase.
(93) In a preferred embodiment the contacting in step (A) is in vivo and the recombinant host cell is a yeast cell, preferably wherein the yeast cell is selected from the group consisting of Saccharomyces spp (e.g. Saccharomyces cerevisiae) and Pichia spp.
(94) Above is described preferred recombinant host cellsthese preferred recombinant host cells may also be preferred recombinant host cells in relation to the method of the sixth aspect of the present invention.
(95) In the present contextit may be said that it is within the skilled person's common knowledge to identify a suitable recombinant host cell to perform the in vivo contacting step (A) of the method of the sixth aspect and it is not believed that it is necessary to describe this in many details herein.
(96) Above is discussed that preferred recombinant host cells may e.g. be a microorganism cell or a filamentous fungal cellthese cells may be preferred recombinant host cells in relation to the method of the sixth aspect.
(97) It may be possible to make a recombinant host cell (e.g. a recombinant host microorganism cell) which comprises a gene encoding a product involved in the biosynthesis pathway leading to flavokermesic acid (FK) and/or kermesic acid (KA) and such a recombinant host cell could be preferred herein.
(98) Accordingly, it may be preferred that the contacting in step (A) is contacting in vivo in a recombinant host cell comprising a recombinant glycosyltransferase gene encoding a glycosyltransferase and a gene encoding a product involved in the biosynthesis pathway leading to flavokermesic acid (FK) and/or kermesic acid (KA).
(99) As discussed in working Example hereinthe GT of SEQ ID NO:2 is membrane bound or hydrophobic/insoluble in vivo and in water. When production cells or fractions of cells containing the membrane bound GT are separated from the product (e.g. carminic acid), the GT can essentially not be present in the fraction where the more soluble product/hydrophilic product is present. This is an advantage for obtaining a final product (e.g. carminic acid product/composition) which is essentially totally free of the recombinant GT.
(100) Because the substrates glycosylated by the GT may be hydrophobic aglycons, the aglycons would be expected to partly accumulate in membranes and other hydrophobic parts of the production cells. By the use of a membrane bound GT a more efficient glycosylation of hydrophobic compounds present in e.g. membranes is obtained
(101) Accordingly, in a preferred embodiment the glycosyltransferase used in the method of the sixth aspect is a GT which is membrane bound or insoluble in water.
(102) In a preferred embodimentthe glycosyltransferase in step (A) of the method of the sixth aspect is a glycosyltransferase of the first aspect and/or herein relevant embodiments thereof.
(103) As discussed hereinthe identified data/results of working Examples 4 show that herein relevant GT enzymes can be identified in e.g. Sorghum and rice plants.
(104) The Sorghum polypeptide sequence (Genbank ID number: AAF17077.1) is shown as SEQ ID NO: 4 herein.
(105) The rice polypeptide sequence (Genbank ID number: CAQ77160.1) is shown as SEQ ID NO: 5 herein.
(106) It may be relevant that the glycosyltransferase in step (A) of the method of the sixth aspect is a glycosyltransferase comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90% and even more preferably at least 98%) identity with amino acids 1 to 492 of SEQ ID NO:4.
(107) It may be relevant that the glycosyltransferase in step (A) of the method of the sixth aspect is a glycosyltransferase comprising an amino acid sequence which has at least 70% (preferably at least 80%, more preferably at least 90% and even more preferably at least 98%) identity with amino acids 1 to 471 of SEQ ID NO:5.
EXAMPLES
Example 1Cloning of D. Coccus GT and Test of its FK and KA Activity
(108) Materials and Methods
(109) Purification of DNA and mRNA
(110) Fresh frozen Dactylopius coccus (were obtained from Lanzarote). Fresh frozen Porphyrophora polonica were obtained from Poland. The frozen insects were ground into powder under liquid nitrogen and DNA/RNA was purified: DNA was purified using DNeasy Blood & Tissue kit (Qiagen), according to the protocol of the supplier. RNA was purified using RNeasy mini kit (Qiagen) according to the protocol of the supplier. Eucaryote mRNA was made into cDNA using RT.sup.2 Easy First Strand Kit (Qiagen) according to the protocol of the supplier using poly-dT priming of the revers transcriptase reaction.
(111) Sequencing of DNA and RNA:
(112) DNA and cDNA were sent for sequencing at BGI (Shenzen, China) for sequencing using 100 bp paired-end Illumina technology according to the protocol of Illumina at a coverage of approximately 60-100 and the output in fastq data format.
(113) Analysis of DNA and RNA/cDNA Sequences:
(114) The obtained fastq-sequences of DNA and RNA/cDNA were imported into Genomic Workbench version 5.4 (CLC-bio, rhus, Denmark) and assembled using the de novo assembling algorithm into contigs. Output files were exported as FASTA format. RNA/cDNA FASTA files were then imported into IOGMA v. 10 (Genostar, Grenoble, France) and putitative genes were identified using the hidden Markov-Matrix-based prokaryote gene-finder.
(115) Putative genes were annotated using BLAST (basic local alignment sequence tool) against genbank (NCBI) using as well the nucleotide sequence as the translated protein sequence. The putative genes were also annotated by similarity comparison to PFAM databases of protein families.
(116) Preparation of Protein Fractions from D. Coccus
(117) Three grams of fresh D. coccus insects were homogenized in 120 mL of isolation buffer [350 mM sucrose, 20 mM Tricine (pH 7.9), 10 mM NaCl, 5 mM DTT, 1 mM PMSF) containing 0.3 g polyvinylpolypyrrolidone. The homogenate was filtered through a nylon cloth (22 m mesh) and centrifuged for (10 min, 10,000g at 4 C.). The supernatant was centrifuged (1 h, 105,000g, at 4 C.), yielding a soluble and a membrane bound protein fraction. The soluble protein fraction was concentrated to 1 mL and buffer-exchanged with 20 mM Tricine (pH 7.9), 5 mM DTT by using Amicon ultrafugation-3K devices (Millipore). The membrane bound protein pellet was washed 3 times by resuspending the pellet in 60 mL of 20 mM Tricine (pH 7.9), 5 mM DTT using a marten paintbrush followed by re-centrifugation. The membrane bound protein pellet was finally resuspended in 1 mL 20 mM Tricine (pH 7.9), 5 mM DTT. The soluble protein fraction and the membrane bound protein fraction were analyzed for glycosylation activity.
(118) Purification of a Flavokermesic Acid/Kermesic Acid-Specific GT Activity from D. Coccus Membrane Proteins
(119) A membrane bound protein fraction isolated from 3 g fresh D. coccus insects was solubilized by adding 1% (v/v) Triton x-100 (reduced form) and gently stirring for 1.5 h in the cold. The Triton x-100 treated solution was centrifuged (1 h, 105,000g, at 4 C.) and the supernatant was isolated and applied to a column packed with 2 mL Q-sepharose Fast flow (Pharmacia). The column was washed in 4 mL of buffer A [20 mM Tricine (pH 7.9), 0.1% (v/v) Triton x-100 (reduced form), 50 mM NaCl] and proteins were eluted with 20 mM Tricine (pH 7.9), 0.1% (v/v) Triton x-100 (reduced form)] using a discontinuous NaCl gradient from 100 mM-500 mM, (with 50 mM increments). 0.5-ml-fractions were collected, desalted, analyzed by SDS-PAGE and monitored for glucosylation activity using the described radiolabeled glucosylation enzyme assay. A fraction containing enriched flavokermesic acid/kermesic acid-specific GT activity was subjected to peptide mass fingerprinting analysis.
(120) Enzyme Assays and Glucoside Product Detection
(121) Glucosylation of flavokermesic acid and kermesic acid was performed in assay mixtures of 60 L containing 20 mM Tricine (pH 7.9), 3.3 m UDP[14C]glucose and 20 uL protein extract (membrane bound or soluble protein). Reactions were incubated for 0.5 h at 30 C. and terminated by adding 180 L of methanol. Samples were centrifuged at 16,000g for 5 min at 4 C. and supernatant was spotted on TLC plates (silica gel 60 F254 plates; Merck). Assay products were resolved in dichloromethane:methanol:formic acid (7:2:2, by volume). Radiolabeled products were visualized using a STORM 840 PhosphorImager (Molecular Dynamics, http://www.moleculardynamics.com).
(122) Expression of Codon Optimized DcUGT2, DcUGT4 and DcUGT5 in S. cerevisiae
(123) A synthetic codon optimized version of DcUGT2 and two other putative GT sequences from the D. coccus transcriptome termed DcUGT4 and DcUGT5 for yeast expression was purchased from GenScript with flanking gateway recombination attL sites. The synthetic fragments were used as PCR templates with specific primers to generate the corresponding C-terminal StrepII-tagged versions. The six gene constructs (tagged and non-tagged fragments) were cloned into the gateway destination plasmid pYES-DEST52 (Invitrogen) using LR clonasell enzyme mix. The six pYES-DEST52 plasmid constructs were transformed separately into the Invsc1 yeast strain (Invitrogen) and positive transformants were verified by PCR. Heterologous protein production was performed according to the instructions of the pYES-DEST52 gateway vector (Invitrogen). Production of heterologous StrepII-tagged protein was verified by western blotting using anti-Strep antibody. A membrane bound protein fraction was prepared from verified yeast transformants as described in (D. Pompon, B. Louerat, A. Bronine, P. Urban, Yeast expression of animal and plant P450s in optimized redox environments, Methods Enzymol. 272 (1996) 51-64) and screened for glucosylation activity towards flavokermesic acid/kermesic acid. The yeast optimized sequence is shown in SEQ ID NO: 3 herein.
(124) LC-MS-MS
(125) LC/MS was performed on an Agilent Q-TOF with the following HPLC system:
(126) Column Kinetix 2.6 XB-C18 100A (1004.60 mm, Phenomenex). Solvent A (900 ml deionized water, 100 ml methanol and 50 ml formic acid). Solvent B (700 ml methanol, 300 ml deionized water and 50 ml formic acid).
(127) Flow 0.8 ml/min. 35 C.
(128) Gradient elution. 0-1 min 100% A. Linear gradient to 83% A 3 min. linear gradient to 63% A 6 min, linear gradient to 45% A 9 min, linear gradient to 27% A 12 min, linear gradient to 10% A 15 min, linear gradient to 3% A 17 min, linear gradient to 2% A 19 min, linear gradient to 0% A 20 min, 0% A 22 min, linear gradient to 100% A 25 min. Retention times were 7.6 min for carminic acid, 7.8 min for DC II, 13.7 min for flavokermesic acid and 13.9 min for kermesic acid.
(129) Results:
(130) The ability to glycosylate flavokermesic acid/kermesic acid using C14-UDP-glucose as a substrate was detected in homogenized D. coccus insects. The activity was shown to be membrane bound and the activity was purified and the purified proteins were submitted to proteomics analysis. It was shown that the enzymatic activity was to come from a polypeptide with a sequence corresponding to our candidate gene DcUGT2.
(131) As discussed abovethe herein relevant glycosyltransferase enzyme of SEQ ID NO: 2 may herein be termed DcUGT2.
(132) The amino acid sequence of DcUGT2 shows less than 45% homology to any known glycosyl transferase.
(133) Knowing that cloning the wildtype sequence into yeast had given no relevant enzyme activity, we redesigned the nucleotide sequence of DcUGT2 to a sequence coding for the same polypeptide but using nucleotide codons optimized for S. cerevisiae, a process called codon optimization (the S. cerevisiae optimized sequence is shown as SEQ ID No. 3 herein). Subsequently the codon optimized sequence of DcUGT2 was cloned and expressed in yeast. The heterologous yeast strain contains a membrane bound enzyme activity capable of glucosylating kermesic acid and flavokermesic acid.
(134) After obtaining peptide mass fingerprinting data from a D. coccus protein fraction enriched with GT activity towards flavokermesic acid/kermesic acid, we matched the peptide masses to the transcriptomic dataset and identified three putative UGTs (DcUGT2, DcUGT4 and DcUGT5).
(135) Heterologous expression of the three candidates in yeast revealed that only one of these UGTs, namely DcUGT2 was responsible for the observed glucosylation activity towards flavokermesic acid/kermesic acid in the D. coccus protein fraction.
(136) A viscozyme treatment of the generated C-14 radiolabelled glucoside, showed that it was resistant towards hydrolysis, further suggesting that the DcUGT2 is a C-GT, responsible for producing DCII and carminic acid.
(137) A LC-MS-MS showed formation of products with the same retention time, spectrum, molecular mass and molecular degradation pattern as DcII and carminic acid respectively.
(138) Conclusion
(139) The result of this example 1 demonstrated that it was not an easy task to isolate/clone the herein relevant glycosyltransferase enzyme of SEQ ID NO: 2, which may herein be termed DcUGT2.
(140) For instance, the identified gene sequences of the genome and transcriptome of D. coccus insects were analyzed for similarity to herein relevant public known C-glycosyltransferase sequences and the result was negative in the sense that none of the identified gene sequences of the genome/transcriptome showed herein significant similarity to publicly known herein relevant C-glycosyltransferase sequences.
(141) However, even though the bioinformatic sequence similarity analysis could be said to indicate that the genome of Dactylopius coccus would not comprise a gene encoding a herein relevant glycosyltransferasethe present inventors continued to investigate the matter and the present inventors identified a Dactylopius coccus extract (including extracts of the endosymbionts present in D. coccus) with herein relevant GT activity and by a combination of herein relevant purification and testing steps the inventors were finally able to get a relatively pure fraction/composition wherefrom it was possible to obtain several partial amino acid sequences of possible GT enzyme candidates.
(142) The present inventors tested the activity of the herein described isolated/cloned novel glycosyltransferase of SEQ ID NO: 2 (DcUGT2) and found that it was able to conjugate glucose to the aglycons flavokermesic acid (FK) and kermesic acid (KA)see
Example 2 Testing KA GT Activity of Prior Art Known UrdGT2
(143) As discussed abovethe UrdGT2 is described in the article Baig et al (Angew Chem Int Ed Engl. 2006 Nov. 27; 45(46):7842-6).
(144) As discussed abovethis article describes that UrdGT2 is capable of glycosylating different aglycon molecules that may be considered structurally similar to the herein relevant Kermesic acid (KA) and Flavokermesic acid (FK) aglycons.
(145) A codon optimized synthetic version of UrdGT2 for E. coli expression was cloned and recombinantly expressed in E. coli. A crude soluble protein extract containing the the recombinant UrdGT2 was obtainedi.e. an extract comprising the UrdGT2
(146) The UrdGT2 GT activity was analyzed in vitro using either UDP-glucose or TDP-glucose as a sugar donor and FA/KA as aglycone substrates. No activity was detected towards these aglyconsi.e. no herein relevant GT activity was identified in relation to these aglycons.
(147) However, it was confirmed that the recombinant UrdGT2 was active, as demonstrated by the in vitro formation of a C14-radiolabelled glucoside derived from the glucosylation of an unidentified compound in the crude E. coli extract.
Example 3 GT Activity in Aloe Plant and Haworthia Plant
(148) Isolation and Test of GT Activity from Aloe 1) The plant was washed from soil particles and separated into: A) Root, B) Green leaf tissue and C) the gel material from the leaf 2) 5 g of tissue was frozen immediately in liquid nitrogen and ground in a cold mortar with a pestle to a fine powder. 3) 20 mL of cold extraction buffer [20 mM Tricine-HCl, 10 mM NaCl, 5 mM DTT, 1 mM PMSF, pH 7.9] containing a Complete protease inhibitor without EDTA (Roche), 0.1% (w/v) proteamine sulfate and 0.5 g of PVPP were added to the powder and vortexed. 4) The homogenate was gently stirred at 4 C. for 10 min and then centrifuged at 12,000g at 4 C. for 5 min. 5) Supernatant was isolated and 1 mL of 2% (w/v) proteamine sulfate in 20 mM Tricine-HCl, pH 7.9 was added dropwise over 2 min at 4 C. under constant stirring. 6) The supernatant was filtered through 2 pieces of nylon mesh. The filtered supernatant was then centrifuged at 12,000 xg at 4 C. for 5 min. 7) The supernatant was isolated and ultracentrifuged at 110,000g at 4 C. for 1 h. 8) The soluble protein fraction (supernatant) was isolated and buffer-exchanged 5 times with 20 mM Tricine-HCl, pH 7.9 containing 5 mM DTT using a Amicon Ultra centrifugal filter device-3K (Millipore) 9) 20 L soluble protein extract was incubated in a total reaction volume of 60 L containing UDP-glucose (1.25 mM final conc.) and either FK (50 M final conc.), KA (50 M final conc) or MeO-FK/EtO-FK (50 M/50 M final conc) for 2 h at 30 C., shaking at 650 rpm. 10) Enzyme reactions were terminated with 180 L cold methanol and filtered through a 0.45 micron filter and subjected to HPLC-MS analysis.
(149) TABLE-US-00001 TABLE 1 Glucosides formed in in vitro glucosylation assays using enzyme extracts from Aloe. m/z [M H].sup. values 475 m/z Aloe [M H].sup. 491 m/z 489 m/z [M H].sup. Soluble FK- [M H].sup. MeOFK- 503 m/z [M H].sup. protein monoglc KA-monoglc monoglc EtOFK-monoglc Leaf 3.73 3.71 5.81 6.63 Gel Root 3.71
(150) Crude soluble enzyme extracts of three Aloe tissues, green leaf material (Leaf), gel material from the leaf (Gel) and Root were tested for glucosylation activity towards flavokermesic acid (FK), kermesic acid (KA), methyl ester of flavokermesic acid (MeOFK) and ethyl ester of flavokermesic acid (EtOFK). Numbers correspond to retention times (min) after HPLC-MS separation of the novel glucosides formed in vitro (Table 1).
(151) The m/z values 475 and 491 are the same m/z values as are obtained for DcII and CA, respectively, solubilized in similar solutions. Both m/z values are 162 (m/z value of glucose in a glucoside) higher than the m/z values of the FK and KA indicating that the glucose moiety from UDP-glucose in the reaction buffer has been transferred to the aglycone by a GT in the extract. The m/z [M-H] values 489 and 503 are also 162 higher than the m/z values obtained with MeOFK and EtOFK, respectively, indicating that a glucose unit has been added to both MeOFK and EtOFK by a GT present in the extract.
(152) Isolation and Test of GT Activity from Haworthia limifolia
(153) The procedure was as described for Aloe but plant tissue analyzed were following: A) Green leaf tissue, B) Gel material from the leaf, C) Base tissue (pink part between root and stem) and D) Root tissue.
(154) Crude soluble enzyme extracts of four Haworthia limifolia tissues, green leaf material (Leaf), gel material from the leaf (Gel), pink tissue between root and stem (Base) and Root were tested for glucosylation activity towards flavokermesic acid (FK), kermesic acid (KA), methyl ester of flavokermesic acid (MeOFK) and ethyl ester of flavokermesic acid (EtOFK). Numbers correspond to retention times (min) after HPLC-MS separation of the novel glucosides formed in vitro (Table 2).
(155) TABLE-US-00002 TABLE 2 Glucosides formed in in vitro glucosylation assays using enzyme extracts from Haworthia limifolia. m/z [M H].sup. values 489 m/z Haworthia 475 m/z 491 m/z [M H].sup. Soluble [M H].sup. [M H].sup. MeOFK- 503 m/z [M H].sup. protein FK-monoglc KA-monoglc monoglc EtOFK-monoglc Leaf 3.73 3.71 5.81 6.63 Gel Base 3.73 3.71 5.81 6.63 Root 3.73 3.71 5.81 6.63
(156) The m/z values 475 and 491 are the same m/z values as are obtained for DcII and CA, respectively, solubilized in similar solutions. Both m/z values are 162 (m/z value of glucose in a glucoside) higher than the m/z values of the FK and KA indicating that the glucose moiety from UDP-glucose in the reaction buffer has been transferred to the aglycone by a GT in the extract. The m/z [M-H] values 489 and 503 are also 162 higher than the m/z values obtained with MeOFK and EtOFK, respectively, indicating that a glucose unit has been added to both MeOFK and EtOFK by a GT present in the extract.
(157) Conclusion
(158) The results of this example demonstrate that herein relevant glycosyltransferase (GT) enzymes can be identified in Aloe plants and Haworthia plants.
(159) Said in other words, Aloe plants and Haworthia plants comprise a glycosyltransferase which is capable of glycosylating flavokermesic acid in order to produce flavokermesic acid glycoside; and/or capable of glycosylating kermesic acid in order to produce kermesic acid glycoside.
Example 4 GT Activity in Sorghum and Rice Plant
(160) As known the artSorghum and rice plants comprise glycosyltransferases.
(161) As known in the artsome of the Sorghum and rice glycosyltransferases may glycosylate low molecular weight aglycone compounds.
(162) The in the art described glycosyltransferases from Sorghum and rice plants have significant less than 70% identity with amino acids 1 to 515 of SEQ ID NO:2 as disclosed herein.
(163) It is not known in the art if glycosyltransferases of Sorghum and/or rice plants would be a herein relevant glycosyltransferasei.e. a glycosyltransferase which is capable of glycosylating flavokermesic acid in order to produce flavokermesic acid glycosides; and/or capable of glycosylating kermesic acid in order to produce kermesic acid glycosides.
(164) The known glycosyltransferases from Sorghum (Sorghum bicolor), SbUGT85B1, with Genbank ID number AF199453.1 (nucleotide seq.)/AAF17077.1 (polypeptide seq) and rice (Oryza sativa), OsCGT, with Genbank ID number FM179712.1 (nucleotide seq.)/CAQ77160.1 (polypeptide seq) were expressed in E. coli strain Xjb and crude E. coli proteins extracts were prepared and tested for glucosylation activity on the substrates kermesic acid and flavokermisic acid as described by Kannangara et al. (2011) and Augustin et al. (2012).
(165)
(166) There were identified KA glycosides (491 m/z [M-H]the m/z[M-H] value of CA) for both glycosyltransferases and FK glycosides (475 m/z [M-H] the m/z[M-H] value of DcII) for OsCGT.
(167) Conclusion
(168) The result of this example demonstrated that herein relevant glycosyltransferase (GT) enzymes can be identified in Sorghum and/or rice plants.
(169) Said in other words, Sorghum and/or rice plants comprise a glycosyltransferase which is capable of glycosylating flavokermesic acid in order to produce flavokermesic acid glycoside; and/or capable of glycosylating kermesic acid in order to produce kermesic acid glycoside.
Example 5 Use of Endogenous GT Gene or GT Activity
(170) As known in the art glycosyltransferases able to glycosylate low molecular weight are present in a lot of different organisms. A method to contact the glycosyltransferase of the cells of an organism with a low molecular weight compound is to introduce one or more genes directing the biosynthesis of the low molecular weight compound and thus enabling the cells to glycosylate the low molecular weight compound. The low molecular weight compound may be e.g. flavokermesic acid or kermersic acid or decorated versions of these molecules.
(171) One or more genes directing the biosynthesis of flavokermesic acid or kermesic acid or decorated version of these molecules are introduced into a glycosyltransferase containing organism, e.g. the tobacco plant, Nicotiana benthamiana.
(172) When the gene/genes is/are transiently expressed according to the methods described in D'Aoust et al. (2008) in e.g. plant tissue the low molecular weight compound or compounds is/are produced. Cells stably expressing the gene/genes are produced and selected according to the methods described in Gelvin (2003).
(173) In cells containing either stably expressed and/or transiently expressed gene/genes the low molecular weight compounds come into contact with the endogenous glycosyltransferases, resulting in the formation of one or more glycosides of flavokermesic acid, kermesic acid or decorated versions of these molecules.
(174) The presence of the glycosides is demonstrated by the extraction and the analytical methods described in example 3.
(175) Samples are prepared for LC/MS by the method for extraction described by Rauwald and Sigler (1994).
(176) Conclusion
(177) The results of this example demonstrate that endogenous glycosyltransferases present in the cells of a recombinant organism can be used to convert flavokermesic acid, kermesic acid or decorated versions of these molecules into glycosides when a gene/genes directing the biosynthesis of the aglycons are introduced into the organism.
(178) Said in other words introduction of a gene or genes directing the biosynthesis of flavokermesic acid, kermesic acid, decorated versions of these molecules, or related low molecular weight compounds is a method to bring the low molecular weight compound in contact with glycosyltransferases and thus a method to produced glycosides of flavokermesic acid, kermesic acid or decorated version of these compounds.
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