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US 7,615,365 B2Granted
α(1,6)-linked glucose oligosaccharide hydrolyzing enzyme polynucleotides and polypeptides and methods of use thereof
AssigneeE.I. du Pont de Nemours and Company
InventorsPerry G. Caimi, Mario W. Chen, Vasantha Nagarajan, Jean-Francois Tomb, Siqun Wang, Yuying Zhang
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Abstract
This invention relates to a method for utilizing less purified starch in fermentation processes. One example is a recombinant E. coli containing a exogenous extracellular isoamylase activity that is capable of utilizing small oligomers containing (1,6) linkages (including but not limited to isomaltose and panose) in fermentations to produce useful products. The invention is useful in large-scale industrial biofermentations by reducing the cost of the substrate carbohydrate.
Claims25
An isolated nucleic acid molecule encoding an α(1,6)-linked glucose oligosaccharide hydrolyzing polypeptide selected from the group consisting of:
- (a) an isolated nucleic acid molecule encoding the amino acid sequence SEQ ID NO:2;
- (b) a nucleic acid molecule that hybridizes with (a) under the following stringent hybridization conditions:
- 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS;
- and
- (c) a nucleic acid molecule that is fully complementary to (a) or (b).
2 more elements
An isolated nucleic acid having the SEQ ID NO:
- 1 encoding an α(1,6)-linked glucose oligosaccharide hydrolyzing polypeptide consisting of:
- (a) an isolated nucleic acid molecule encoding a chimeric protein comprised of a signal peptide operably linked to an α(1,6)-linked glucose oligosaccharide hydrolyzing polypeptide;
- (b) a nucleic acid molecule that hybridizes with (a) under the following stringent hybridization conditions:
- 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS;
- and
- (c) a nucleic acid molecule that is fully complementary to (a) or (b).
3 more elements
A method for the production of a target molecule in a recombinant host cell comprising:
- (a) contacting a transformed host cell comprising:
- (i) an isolated nucleic acid molecule encoding a chimeric protein comprised of a signal peptide operably linked to an α(1,6)-linked glucose oligosaccharide hydrolyzing polypeptide;
- (ii) a nucleic acid molecule that hybridizes with (i) under the following stringent hybridization conditions:
- 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS;
- or
- (iii) a nucleic acid molecule that is fully complementary to (i) or (ii);
- and
- (iv) at least one chimeric gene for converting mononsaccharides to the target molecule,
- in the presence of limit dextrin under suitable conditions whereby the target molecule is produced;
- and
- (b) optionally recovering the target molecule produced in (a).
8 more elements
A method for the production of glycerol in a recombinant host cell comprising:
- (a) contacting a transformed host cell comprising:
- (i) an isolated nucleic acid molecule encoding a chimeric protein comprised of a signal peptide operably linked to an α(1,6)-linked glucose oligosaccharide hydrolyzing polypeptide;
- (ii) a nucleic acid molecule that hybridizes with (i) under the following stringent hybridization conditions:
- 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS;
- or
- (iii) a nucleic acid molecule that is fully complementary to (i) or (ii);
- and
- (iv) at least one chimeric gene for converting mononsaccharides to glycerol,
- in the presence of limit dextrin under suitable conditions whereby glycerol is produced;
- and
- (b) optionally recovering the glycerol produced in (a).
8 more elements
A method for the production of 1,3-propanediol in a recombinant host cell comprising:
- (a) contacting a transformed host cell comprising:
- (i) an isolated nucleic acid molecule encoding a chimeric protein comprised of a signal peptide operably linked to an α(1,6)-linked glucose oligosaccharide hydrolyzing polypeptide;
- (ii) a nucleic acid molecule that hybridizes with (i) under the following stringent hybridization conditions:
- 0.1×SSC, 0.1% SES, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS;
- or
- (iii) a nucleic acid molecule that is fully complementary to (i) or (ii),
- (iv) at least one chimeric gene for converting mononsaccharides to 1,3-propanediol,
- in the presence of limit dextrin under suitable conditions whereby 1,3-propanediol is produced;
- and
- (b) optionally recovering the 1,3-propanediol produced in (a).
7 more elements
A method for the production of cell mass in a recombinant host cell comprising:
- (a) contacting a transformed host cell comprising:
- (i) an isolated nucleic acid molecule encoding a chimeric protein comprised of a signal peptide linked to an α(1,6)-linked glucose oligosaccharide hydrolyzing polypeptide;
- (ii) a nucleic acid molecule that hybridizes with (i) under the following stringent hybridization conditions:
- 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS;
- and
- (iii) a nucleic acid molecule that is fully complementary to (i) or (ii),
- under suitable conditions in the presence of limit dextrin;
- (b) optionally recovering the cell mass produced in (a).
5 more elements
A method for the production of a target molecule in a recombinant host cell comprising:
- (a) contacting a transformed host cell comprising:
- (i) an isolated nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:2;
- (ii) a nucleic acid molecule that hybridizes with (i) under the following stringent hybridization conditions:
- 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS;
- or
- (iii) a nucleic acid molecule that is fully complementary to (i) or (ii);
- and
- (iv) at least one chimeric gene for converting mononsaccharides to the target molecule,
- in the presence of limit dextrin under suitable conditions whereby the target molecule is produced;
- and
- (b) optionally recovering the target molecule produced in (a).
8 more elements
A method for the production of 1,3-propanediol in a recombinant host cell comprising:
- (a) contacting a transformed host cell comprising:
- (i) an isolated nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:2;
- (ii) a nucleic acid molecule that hybridizes with (i) under the following stringent hybridization conditions:
- 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS;
- or
- (iii) a nucleic acid molecule that is fully complementary to (i) or (ii);
- and
- (iv) at least one chimeric gene for converting mononsaccharides to 1,3-propanediol;
- in the presence of limit dextrin under suitable conditions whereby 1,3-propanediol is produced;
- and
- (b) optionally recovering the 1,3-propanediol produced in (a).
8 more elements
A method for the production of glycerol in a recombinant host cell comprising:
- (a) contacting a transformed host cell comprising:
- (i) an isolated nucleic acid molecule encoding the amino acid sequence of SEQ NO:2;
- (ii) a nucleic acid molecule that hybridizes with (i) under the following stringent hybridization conditions:
- 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS;
- or
- (iii) a nucleic acid molecule that is fully complementary to (i) or (ii);
- and
- (iv) at least one chimeric gene for converting mononsaccharides to glycerol;
- in the presence of limit dextrin under suitable conditions whereby glycerol is produced;
- and
- (b) optionally recovering the glycerol produced in (a).
8 more elements
A method for the production of cell mass in a recombinant host cell comprising:
- (a) contacting a transformed host cell comprising:
- (i) an isolated nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:2;
- (ii) a nucleic acid molecule that hybridizes with (i) under the following stringent hybridization conditions:
- 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS;
- or
- (iii) a nucleic acid molecule that is fully complementary to (i) or (ii),
- in the presence of limit dextrin under suitable conditions whereby cell mass is produced;
- and
- (b) optionally recovering the cell mass produced in (a).
6 more elements
A method for degrading limit dextrin comprising:
- (a) contacting a transformed host cell comprising:
- (i) a nucleic acid molecule encoding the enzyme having amino acid sequence of SEQ ID NO:2;
- (ii) a nucleic acid molecule that hybridizes with (i) under the following stringent hybridization conditions:
- 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS;
- or
- (iii) a nucleic acid molecule that is fully complementary to (i) or (ii),
- with an effective amount of limit dextrin substrate under suitable growth conditions;
- and
- (b) optionally recovering the product of step (a).
6 more elements
Dependencies are inferred from the claim text.