Utilization of starch for biological production by fermentation
AssigneeE.I. du Pont de Nemours and Company
InventorsPerry G. Caimi, Mario W. Chen, Vasantha Nigarajan, 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.
Claims34
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 NOs:2, 4, or 6;
- (b) a nucleic acid molecule that hybridizes with (a) under the following 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 complementary to (a) or (b).
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 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 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 complementary to (a) or (b).
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 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 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).
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 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 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).
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 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 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).
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 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 complementary to (i) or (ii).
- under suitable conditions in the presence of limit dextrin;
- (b) optionally recovering the cell mass produced in (a).
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 selected from the group consisting of SEQ ID NOs:2, 6, 17 and 31;
- (ii) a nucleic acid molecule that hybridizes with (i) under the following 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 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).
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 selected from the group consisting of SEQ ID NOs:2, 6, 17 and 31;
- (ii) a nucleic acid molecule that hybridizes with (i) under the following 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 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).
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 selected from the group consisting of SEQ NOs:2, 6, 17 and 31;
- (ii) a nucleic acid molecule that hybridizes with (i) under the following 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 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).
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 selected from the group consisting of SEQ ID NOs:2, 6, 17 and 31;
- (ii) a nucleic acid molecule that hybridizes with (i) under the following 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 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).
A method for degrading limit dextrin comprising:
- (a) contacting a transformed host cell comprising:
- (i) a nucleic acid molecule encoding the enzymes selected from the group consisting of SEQ ID NOs:2, 6, 17 and 31;
- (ii) a nucleic acid molecule that hybridizes with (i) under the following 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 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).
A polypeptide having an amino acid sequence that has at least 69% identity based on the BLASTP method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:17, the polypeptide having α(1,6)-linked glucose oligosaccharide hydrolyzing activity.
Dependencies are inferred from the claim text.