UDP-glucosyltransferases
AssigneeE. I. Du Pont de Nemours and Company
InventorsPaul V. Viitanen, Knut Meyer, Drew E. Van Dyk
Life of this patent
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Abstract
This invention pertains to nucleic acid fragments encoding plant glucosyltransferases, heretofore undescribed, that exhibit catalytic activity with p-hydroxybenzoic acid (pHBA) as a substrate and only attach glucose to the aromatic carboxyl group of pHBA, to form the pHBA glucose ester. These enzymes have potential applications both in vitro and in vivo, and their primary amino acid sequences can be used to identify other proteins that have similar kinetic properties.
Claims21
An isolated nucleic acid molecule encoding a UDP-glucosyltransferase enzyme selected from the group consisting of:
- (a) an isolated nucleic acid molecule encoding the amino acid sequence set forth in SEQ ID NO:
- 18 or SEQ ID NO:
- 22;
- (b) an isolated nucleic acid molecule that hybridizes with (a) under the following stringent hybridization conditions:
- 0.1×SSC, 0.1% SDS at 65° C., and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS;
- and
- (c) an isolated nucleic acid molecule that is complementary to (a) or (b).
An isolated nucleic acid molecule encoding a UDP-glucosyltransferase enzyme selected from the group consisting of:
- (a) an isolated nucleic acid molecule encoding the amino acid sequence set forth in SEQ ID NO:
- 31;
- (b) an isolated nucleic acid molecule that hybridizes with (a) under the following stringent hybridization conditions:
- 0.1×SSC, 0.1% SDS at 65° C., and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS;
- and
- (c) an isolated nucleic acid molecule that is complementary to (a) or (b).
An isolated nucleic acid molecule encoding a UDP-glucosyltransferase enzyme having:
- a) at least 75% identity to the amino acid sequence set forth in SEQ ID NO:
- 18 or at least 72% identity to the amino acid sequence set forth in SEQ ID NO:
- 22;
- b) activity to catalyze the production of pHBA ester glucoside from pHBA;
- c) at least a 4.88-fold substrate preference for pHBA over sinapic acid at a 10 mM substrate concentration;
- and
- d) a turnover number of at least 1.77 sec−1 for the conversion of pHBA to pHBA ester glucoside.
An isolated nucleic acid molecule comprising
- (a) a nucleotide sequence encoding an UDP-glucosyltransferase enzyme having at least 82% identity over the length of 478 amino acids based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence set forth in SEQ ID NO:
- 18, or
- (b) a nucleotide sequence comprising the complement of the nucleotide sequence of (a).
An isolated nucleic acid molecule comprising
- (a) a nucleotide sequence encoding an UDP-glucosyltransferase enzyme having at least 82% identity over the length of 511 amino acids based on the Smith-Waterman method of alignment when compared to a polypeptide having the sequence set forth in SEQ ID NO:
- 22, or
- (b) a nucleotide-sequence comprising the complement of the nucleotide sequence of (a).
A method for increasing UDP-glucosyltransferase enzyme activity in a microorganism or green plant cell comprising,
- (a) transforming a host microorganism or green plant cell with an UDP-glucosyltransferase gene comprising the nucleotide sequence set forth in SEQ ID NO:
- 17, SEQ ID NO:
- 21, or SEQ ID NO:
- 30, the nucleic acid sequence operably linked to suitable regulatory sequences for protein expression;
- (b) growing the transformed host microorganism or green plant cell of step a) under appropriate conditions for expression of the UDP-glucosyltransferase gene.
A method for increasing the ratio of the pHBA ester glucoside to total pHBA glucose conjugates in pHBA-producing microorganisms and green plant cells, the method comprising:
- (a) tranforming a pHBA-producing microorganism or green plant cell with a nucleic acid fragment encoding a polypeptide for UDP-glucosyltransferase enzyme activity operably linked to suitable regulatory sequences, the polypeptide having
- 1) at least 75% identity to an amino acid sequence as set forth in SEQ ID NO:
- 18 or at least 72% identity to an amino acid sequence as set forth in SEQ ID NO:
- 22;
- 2) at least a 4.88-fold substrate preference for pHBA over sinapic acid at,a 10 mM substrate concentration;
- and
- 3) a turnover number of at least 1.77 sec−1 for conversion of pHBA to pHBA ester glucoside,
- (b) growing the transformed microorganism or green plant cell of step a) under suitable conditions for expressing UDP-glucosyltransferase activity and producing pHBA ester glucoside;
- and
- (c) recovering pHBA ester glucoside, the ratio of pHBA ester glucose to total pHBA glucose conjugates at least 10% greater than the ratio of pHBA ester glucose to total pHBA glucose conjugates of an untransformed green plant cell.
A method for the in vitro production of pHBA ester glucoside comprising
- i) contacting in vitro pHBA with UDP-glucose in the presence of an effective amount of a UDP-glucosyltransferase enzyme having
- a) at least 75% identity to the amino acid sequence set forth in SEQ ID NO:
- 18, or at least 72% identity to the amino acid sequence set forth in SEQ ID NO:
- 22;
- b) at least a 4.88-fold substrate preference for pHBA over sinapic acid at a 10 mM substrate concentration;
- and
- c) a turnover number of at least. 1.77 sec−1 for conversion of pHBA to the pHBA ester glucoside;
- and
- ii) isolating the pHBA ester glucoside.
Dependencies are inferred from the claim text.