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US 2022/0098630 A1Published application
Cell-free production of allulose
AssigneeGreenLight Biosciences, Inc.
InventorsDaniel MacEachran, Drew S. Cunningham, William Jeremy Blake, Matthew Eduardo Moura
Life of this patent
Estimating the term…
Abstract
Provided herein, in some embodiments, are cell-free systems, methods, kits, and compositions (e.g., cells and cell lysates) for converting a polysaccharide to allulose via the use of enzymes, such as thermostable enzymes.
Claims84
A cell-free method of producing glucose 1-phosphate from a polysaccharide, the method comprising:
- converting a polysaccharide to glucose 1-phosphate using an α-glucan phosphorylase selected from the group consisting of AaGlgp (derived from Aquifex aeolicus), TzAgp (derived from Thermococcus zilligii), PtAgp (derived from Pseudothermotoga thermarum), Tm08495 (derived from Thermotoga maritima), TcGlgP (derived from Thermus caldophilus) and PfAgp (derived from Pyrococcus furiosus).
A cell-free method of producing fructose 6-phosphate from glucose 6-phosphate, the method comprising:
- converting glucose 6-phosphate to fructose 6-phosphate using a phosphoglucoisomerase selected from the group consisting of CtPgi (derived from Clostridium thermocellum), TtPgi (derived from Thermus thermophilus), MjPgi (derived from Methoanococcus jannaschii), PfPgi (derived from Pyrococcus furiosus), Ap0768 (derived from Aeropyrum pernix), and Cl1150 (derived from Caldisphaera lagunensis).
A cell-free method for producing allulose from a polysaccharide, the method comprising:
- converting a polysaccharide to glucose 1-phosphate using an α-glucan phosphorylase;
- converting glucose 1-phosphate to glucose 6-phosphate using phosphoglucomutase;
- converting glucose 6-phosphate to fructose 6-phosphate using a phosphoglucoisomerase;
- converting fructose 6-phosphate to allulose 6-phosphate using an allulose-6 phosphate epimerase;
- and
- converting allulose 6-phosphate to allulose using an allulose-6 phosphate phosphatase.
3 more elements
A cell lysate comprising:
- an α-glucan phosphorylase selected from the group consisting of TzAgp (derived from Thermococcus zilligii), AaGlgP (derived from Aquifex aeolicus), PtAgp (derived from Pseudothermotoga thermarum), and PfAgp (derived from Pyrococcus furiosus);
- a phosphoglucomutase;
- a phosphoglucoisomerase;
- an allulose 6-phosphate epimerase;
- and
- an allulose 6-phosphate phosphatase;
- and
- optionally a debranching enzyme.
5 more elements
A cell lysate comprising:
- a phosphoglucoisomerase selected from the group consisting of PfPgi (derived from Pyrococcus furiosus), CtPgi (derived from Clostridium thermocellum), TtPgi (derived from Thermus thermophilus)Ap0768 (derived from Aeropyrum pernix), and Cl1150 (derived from Caldisphaera lagunensis);
- an α-glucan phosphorylase;
- a phosphoglucomutase;
- an allulose 6-phosphate epimerase;
- an allulose 6-phosphate phosphatase;
- and
- optionally a debranching enzyme.
4 more elements
An engineered cell comprising:
- an α-glucan phosphorylase selected from the group consisting of TzAgp (derived from Thermococcus zilligii), AaGlgP (derived from Aquifex aeolicus), PtAgp (derived from Pseudothermotoga thermarum), and PfAgp (derived from Pyrococcus furiosus);
- a phosphoglucomutase;
- a phosphoglucoisomerase;
- an allulose 6-phosphate epimerase;
- and
- an allulose 6-phosphate phosphatase;
- and
- optionally a debranching enzyme.
5 more elements
An engineered cell comprising:
- a phosphoglucoisomerase selected from the group consisting of PfPgi (derived from Pyrococcus furiosus), CtPgi (derived from Clostridium thermocellum), TtPgi (derived from Thermus thermophilus) Ap0768 (derived from Aeropyrum pernix), and Cl1150 (derived from Caldisphaera lagunensis);
- an α-glucan phosphorylase;
- a phosphoglucomutase;
- an allulose 6-phosphate epimerase;
- an allulose 6-phosphate phosphatase;
- and
- optionally a debranching enzyme.
4 more elements
A mixture of cell lysates obtained from at least two cell populations, wherein the cells of each cell population express at least one or more enzymes selected from the group consisting of:
- α-glucan phosphorylases selected from the group consisting of TzAgp (derived from Thermococcus zilligii), AaGlgP (derived from Aquifex aeolicus), PtAgp (derived from Pseudothermotoga thermarum), and PfAgp (derived from Pyrococcus furiosus);
- phosphoglucomutases;
- phosphoglucoisomerases;
- allulose 6-phosphate epimerases;
- allulose 6-phosphate phosphatases;
- and
- debranching enzymes.
4 more elements
A mixture of cell lysates obtained from at least two cell populations, wherein the cells of each cell population express at least one or more enzymes selected from the group consisting of:
- phosphoglucoisomerases selected from the group consisting of PfPgi (derived from Pyrococcus furiosus), CtPgi (derived from Clostridium thermocellum), TtPgi (derived from Thermus thermophilus) Ap0768 (derived from Aeropyrum pernix), and Cl1150 (derived from Caldisphaera lagunensis);
- α-glucan phosphorylases;
- phosphoglucomutases;
- allulose 6-phosphate epimerases;
- allulose 6-phosphate phosphatases;
- and
- debranching enzymes.
4 more elements
A reaction mixture of cell lysates comprising:
- an α-glucan phosphorylase selected from the group consisting of TzAgp (derived from Thermococcus zilligii), AaGlgP (derived from Aquifex aeolicus), PtAgp (derived from Pseudothermotoga thermarum), and PfAgp (derived from Pyrococcus furiosus);
- a phosphoglucomutase;
- a phosphoglucoisomerase;
- an allulose 6-phosphate epimerase;
- an allulose 6-phosphate phosphatase;
- optionally a debranching enzyme;
- a polysaccharide;
- glucose 1-phosphate;
- glucose 6-phosphate;
- fructose 6-phosphate;
- allulose 6-phosphate;
- and
- allulose.
10 more elements
A reaction mixture of cell lysates comprising:
- an α-glucan phosphorylase;
- a phosphoglucomutase;
- a phosphoglucoisomerase selected from the group consisting of PfPgi (derived from Pyrococcus furiosus), CtPgi (derived from Clostridium thermocellum), TtPgi (derived from Thermus thermophilus) Ap0768 (derived from Aeropyrum pernix), and Cl1150 (derived from Caldisphaera lagunensis);
- an allulose 6-phosphate epimerase;
- an allulose 6-phosphate phosphatase;
- optionally a debranching enzyme;
- a polysaccharide;
- glucose 1-phosphate;
- glucose 6-phosphate;
- fructose 6-phosphate;
- allulose 6-phosphate;
- and
- allulose.
10 more elements
A cell lysate mixture comprising:
- an α-glucan phosphorylase selected from the group consisting of TzAgp (derived from Thermococcus zilligii), AaGlgP (derived from Aquifex aeolicus), PtAgp (derived from Pseudothermotoga thermarum), and PfAgp (derived from Pyrococcus furiosus);
- a phosphoglucomutase;
- a phosphoglucoisomerase;
- an allulose 6-phosphate epimerase;
- an allulose 6-phosphate phosphatase;
- optionally a debranching enzyme;
- a polysaccharide;
- glucose 1-phosphate;
- glucose 6-phosphate;
- fructose 6-phosphate;
- allulose 6-phosphate;
- and
- allulose for the synthesis of allulose.
10 more elements
A cell lysate mixture comprising:
- an α-glucan phosphorylase;
- a phosphoglucomutase;
- a phosphoglucoisomerase selected from the group consisting of PfPgi (derived from Pyrococcus furiosus), CtPgi (derived from Clostridium thermocellum), TtPgi (derived from Thermus thermophilus) Ap0768 (derived from Aeropyrum pernix), and Cl1150 (derived from Caldisphaera lagunensis);
- an allulose 6-phosphate epimerase;
- an allulose 6-phosphate phosphatase;
- optionally a debranching enzyme;
- a polymeric glucose;
- glucose 1-phosphate;
- glucose 6-phosphate;
- fructose 6-phosphate;
- allulose 6-phosphate;
- and
- allulose for the synthesis of allulose.
10 more elements
A cell-free method for producing allulose, the method comprising:
- (a) culturing cells engineered to express a thermostable α-glucan phosphorylase selected from the group consisting of TzAgp (derived from Thermococcus zilligii), AaGlgP (derived from Aquifex aeolicus), PtAgp (derived from Pseudothermotoga thermarum), and PfAgp (derived from Pyrococcus furiosus),
- a thermostable phosphoglucomutase, a thermostable phosphoglucoisomerase, a thermostable allulose 6-phosphate epimerase, and a thermostable allulose 6-phosphate phosphatase to produce cultured cells that express the thermostable enzymes;
- (b) lysing the cultured cells to produce a cell lysate;
- (c) heating the cell lysate to a temperature that inactivates native enzymatic activity but does not inactivate the thermostable enzymes of step (a) to produce a heat-inactivated lysate;
- and
- (d) incubating the heat-inactivated lysate in the presence of a starch, glycogen, or any partially hydrolyzed derivative thereof and inorganic phosphate to produce allulose.
3 more elements
A cell-free method for producing allulose, the method comprising:
- (a) culturing cells engineered to express a thermostable α-glucan phosphorylase, a thermostable phosphoglucomutase, a thermostable allulose 6-phosphate epimerase, a thermostable allulose 6-phosphate phosphatase, and
- a thermostable phosphoglucoisomerase selected from the group consisting of PfPgi (derived from Pyrococcus furiosus), CtPgi (derived from Clostridium thermocellum), TtPgi (derived from Thermus thermophilus) Ap0768 (derived from Aeropyrum pernix), and Cl1150 (derived from Caldisphaera lagunensis),
- to produce cultured cells that express the thermostable enzymes;
- (b) lysing the cultured cells to produce a cell lysate;
- (c) heating the cell lysate to a temperature that inactivates native enzymatic activity but does not inactivate the thermostable enzymes of step (a) to produce a heat-inactivated lysate;
- and
- (d) incubating the heat-inactivated lysate in the presence of a starch, glycogen, or any partially hydrolyzed derivative thereof and inorganic phosphate to produce allulose.
4 more elements
A cell-free method for producing allulose, the method comprising:
- (a) culturing at least two cell populations, wherein cells of each population are engineered to express at least one enzyme selected from the group consisting of:
- thermostable α-glucan phosphorylases selected from the group consisting of TzAgp (derived from Thermococcus zilligii), AaGlgP (derived from Aquifex aeolicus), PtAgp (derived from Pseudothermotoga thermarum), and PfAgp (derived from Pyrococcus furiosus),
- thermostable phosphoglucomutases, thermostable phosphoglucoisomerases, thermostable allulose 6-phosphate epimerases, and thermostable allulose 6-phosphate phosphatases, and thermostable debranching enzymes to produce at least two cultured populations of cells expressing different enzymes;
- (b) lysing cells of the at least two cultured populations to produce at least two cell lysates;
- (c) combining the at least two cell lysates to produce a cell lysate mixture that comprises a thermostable α-glucan phosphorylase, a thermostable phosphoglucomutase, a thermostable phosphoglucoisomerase, a thermostable allulose 6-phosphate epimerase, and a thermostable allulose 6-phosphate phosphatase, and optionally a thermostable debranching enzyme;
- (d) heating the cell lysate mixture to a temperature that inactivates native enzymatic activity but does not inactivate the thermostable enzymes of step (c) to produce a heat-inactivated lysate;
- and
- (e) incubating the reaction mixture in the presence of a starch, glycogen, or any partially hydrolyzed derivative thereof and inorganic phosphate to produce allulose.
5 more elements
A cell-free method for producing allulose, the method comprising:
- (a) culturing at least two cell populations, wherein cells of each population are engineered to express at least one enzyme selected from the group consisting of:
- thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable allulose 6-phosphate epimerases, thermostable allulose 6-phosphate phosphatases, phosphoglucoisomerases selected from the group consisting of PfPgi (derived from Pyrococcus furiosus), CtPgi (derived from Clostridium thermocellum), TtPgi (derived from Thermus thermophilus) Ap0768 (derived from Aeropyrum pernix), and Cl1150 (derived from Caldisphaera lagunensis),
- and optionally debranching enzymes to produce at least two cultured populations of cells expressing different enzymes;
- (b) lysing cells of the at least two cultured populations to produce at least two cell lysates;
- (c) combining the at least two cell lysates to produce a cell lysate mixture that comprises a thermostable α-glucan phosphorylase, a thermostable phosphoglucomutase, a thermostable phosphoglucoisomerase, a thermostable allulose 6-phosphate epimerase, and a thermostable allulose 6-phosphate phosphatase, and optionally a debranching enzyme;
- (d) heating the cell lysate mixture to a temperature that inactivates native enzymatic activity but does not inactivate the enzymes of step (c) to produce a heat-inactivated lysate;
- and
- (e) incubating the reaction mixture in the presence of a starch, glycogen, or any partially hydrolyzed derivative thereof and inorganic phosphate to produce allulose.
5 more elements
A cell-free method for producing allulose, the method comprising:
- (a) culturing at least two cell populations, wherein cells of each population are engineered to express at least one enzyme selected from the group consisting of:
- α-glucan phosphorylases selected from the group consisting of TzAgp (derived from Thermococcus zilligii), AaGlgP (derived from Aquifex aeolicus), PtAgp (derived from Pseudothermotoga thermarum), and PfAgp (derived from Pyrococcus furiosus),
- phosphoglucomutases, phosphoglucoisomerases, allulose 6-phosphate epimerases, and allulose 6-phosphate phosphatases, and debranching enzymes to produce at least two cultured populations of cells expressing different enzymes wherein at least one of the foregoing enzymes is thermostable;
- (b) lysing cells of the at least two cultured populations to produce at least two cell lysates;
- (c) optionally heating one or more of the cell lysates of step (b) to a temperature that inactivates native enzymatic activity but does not inactivate the enzymes of step (a) to produce a heat-inactivated lysate;
- (d) combining the cell lysates of step (b) and (c) to produce a cell lysate mixture that comprises an α-glucan phosphorylase, a phosphoglucomutase, a phosphoglucoisomerase, an allulose 6-phosphate epimerase, and an allulose 6-phosphate phosphatase, and optionally a debranching enzyme, wherein at least one of the foregoing enzymes is thermostable;
- and
- (e) incubating the cell lysate mixture in the presence of a starch, glycogen, or any partially hydrolyzed derivative thereof and inorganic phosphate to produce allulose.
5 more elements
A cell-free method for producing allulose, the method comprising:
- (a) culturing at least two cell populations, wherein cells of each population are engineered to express at least one enzyme selected from the group consisting of:
- α-glucan phosphorylases, phosphoglucomutases, allulose 6-phosphate epimerases, allulose 6-phosphate phosphatases, a phosphoglucoisomerase selected from the group consisting of PfPgi (derived from Pyrococcus furiosus), CtPgi (derived from Clostridium thermocellum), TtPgi (derived from Thermus thermophilus) Ap0768 (derived from Aeropyrum pernix), and Cl1150 (derived from Caldisphaera lagunensis),
- and optionally debranching enzymes to produce at least two cultured populations of cells expressing different enzymes wherein at least one of the foregoing enzymes is thermostable;
- (b) lysing cells of the at least two cultured populations to produce at least two cell lysates;
- (c) optionally heating one or more of the cell lysates of step (b) to a temperature that inactivates native enzymatic activity but does not inactivate the enzymes of step (a) to produce a heat-inactivated lysate;
- (d) combining the cell lysates of step (b) and (c) to produce a cell lysate mixture that comprises an α-glucan phosphorylase, a phosphoglucomutase, a phosphoglucoisomerase, an allulose 6-phosphate epimerase, and an allulose 6-phosphate phosphatase, and optionally a debranching enzyme, wherein at least one of the foregoing enzymes is thermostable;
- and
- (e) incubating the cell lysate mixture in the presence of a starch, glycogen, or any partially hydrolyzed derivative thereof and inorganic phosphate to produce allulose.
5 more elements
Dependencies are inferred from the claim text.