Variant sucrose transporter polypeptides
AssigneeE. I. Du Pont de Nemours and Company
InventorsDANA MICHELLE WALTERS POLLAK, TINA K. VAN DYK
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Abstract
Variant sucrose transporter polypeptides that enable bacterial growth over a wide range of gene expression levels and sucrose concentrations are described. Additionally, recombinant bacteria comprising these variant sucrose transporter polypeptides, and methods of utilizing the bacteria to produce products such as glycerol and glycerol-derived products are provided
Claims12
A variant sucrose transporter polypeptide having an amino acid sequence that has at least 95% identity to an amino acid sequence as set forth in SEQ ID NO:26 based on a Clustal W method of alignment and having an amino acid change from arginine to alanine or arginine to leucine at position 300, and comprising:
- (a) at least one additional amino acid change selected from the group consisting of:
- (i) glutamine to histidine at position 353;
- (ii) leucine to proline at position 61;
- (iii) phenylalanine to leucine at position 159;
- (iv) glycine to cysteine at position 162;
- (v) proline to histidine at position 169;
- (vi) leucine to tryptophan at position 61;
- (vii) leucine to histidine at position 61;
- (viii) leucine to phenylalanine at position 61;
- and
- (ix) leucine to tyrosine at position 61;
- or
- (b) a length of 402 to 407 amino acids from the N-terminus;
- or
- (c) a length of 402 to 407 amino acids from the N-terminus, and having at least one of the amino acid changes of (a).
A variant sucrose transporter polypeptide having an amino acid sequence that has at least 95% identity based on a Clustal W method of alignment to an amino acid sequence selected from the group consisting of SEQ ID NOs:
- 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, and 98, and comprising an amino acid at an equivalent position when compared with a reference amino acid sequence of SEQ ID NO:26 selected from the group consisting of:
- (a) alanine at a position equivalent to position 300;
- and
- (b) leucine at a position equivalent to position 300.
A recombinant bacterium comprising in its genome or on at least one recombinant construct:
- (a) a nucleotide sequence encoding a variant sucrose transporter polypeptide having an amino acid sequence that has at least 95% identity based on a Clustal W method of alignment to an amino acid sequence selected from the group consisting of SEQ ID NOs:26, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, and 98, and an amino acid at an equivalent position when compared with a reference amino acid sequence of SEQ ID NO:26 selected from the group consisting of:
- (i) alanine at a position equivalent to position 300;
- and
- (ii) leucine at a position equivalent to position 300;
- and
- (b) a nucleotide sequence encoding a polypeptide having sucrose hydrolase activity;
- wherein (a) and (b) are each operably linked to the same or a different promoter, further wherein said recombinant bacterium is capable of metabolizing sucrose.
Dependencies are inferred from the claim text.