Cell-free production of sugars

Abstract

Provided herein, in some embodiments, are systems, methods, and compositions (e.g., cells and cell lysates) for enzymatically converting a polymeric glucose carbohydrate (e.g., starch) to sugar.

1 . A method for producing an allulose compound comprising the steps of:
 converting a polymeric glucose carbohydrate to glucose 1-phosphate (G1P) catalyzed by an α-glucan or a cellodextrin phosphorylase; converting said glucose 1-phosphate (G1P) to produce glucose 6-phosphate (G6P) catalyzed by a phosphoglucomutase; converting said glucose 6-phosphate (G6P) to fructose 6-phoshpate (F6P) catalyzed by a phosphoglucoisomerase; converting said fructose 6-phosphate (F6P) to produce allulose 6-phosphate (A6P) catalyzed by an allulose 6-phosphate epimerase (A6PE); and converting said allulose 6-phosphate epimerase (A6P) to allulose catalyzed by an allulose 6-phosphate phosphatase (A6PP).   
     
     
         2 . The method of  claim 1 , wherein the polymeric glucose carbohydrate is starch, cellodextrin, or glycogen. 
     
     
         3 . The method of  claim 1 , wherein the α-glucan phosphorylase is selected from the group consisting of:  Aquifex aeolicus, Thermocrinis minervae, Thermosulfidibacter takaii, Thermosulfurimonas dismutans, Thermococcus litoralis, Palaeococcus pacificus, Thermotoga neapolitana, Ruminiclostridium thermocellum, Pyrococcus abyssi, Thermococcus thioreducens, Deinococcus radiodurans, Sulfolobus acidocaldarius, Thermus caldophilus, Meiothermus silvanus, Oceanithermus profundus, Ardenticatena maritima, Thermococcus barophilus, Pseudothermotoga thermarum, Hydrogenobacter thermophilus, Thermus oshimai, Meiothermus ruber , and  Marinitoga piezophila  α-glucan phosphorylases. 
     
     
         4 . The method of  claim 1 , wherein the cellodextrin phosphorylase is selected from the group consisting of:  Clostridium thermocellum, Clostridium straminisolvens, Thermotoga  RQ2 ; Ignisphaera aggregans, Thermotoga maritima, Spirochaeta thermophila, Caldicellulosiruptor bescii, Dictyoglomus thermophilum, Thermoanaerobacterium thermosaccharolyticum, Thermosipho africanus, Caldisalinibacter kiritimatiensis, Defluviitalea phaphyphila, Caldicellulosiruptor kronotskyensis, Thermococcus sibiricus , and  Thermosphaera aggregans  cellodextrin phosphorylases. 
     
     
         5 . The method of  claim 1 , wherein the phosphoglucomutase is selected from the group consisting of:  Thermococcus kodakaraensis, Pyrococcus kukulkanii, Ammonifex degensii, Methanothermobacter wolfeii, Methanothermus fervidus, Sulfolobus acidocaldarius, Archaeoglobus fulgidus, Ferroglobus placidus, Geoglobus ahangari, Archaeoglobus veneficus, Archaeoglobus sulfaticallidus, Aciduliprofundum boonie, Clostridium thermocellum, Defluviitalea phaphyphila, Caminicella sporogenes, Caloranaerobacter ferrireducens, Thermosipho malanesiensis, Fervidobacterium pennivorans, Symbiobacterium thermophilum, Spirochaeta thermophila , and  Thermoanaerobacter wiegelii  phosphoglucomutases. 
     
     
         6 . The method of  claim 1  wherein the phosphoglucoisomerase is selected from the group consisting of:  Thermus thermophilus, Meiothermus timidus, Thermus filiformis, Marinithermus hydrothermalis, Thermosipho africanus, Sulfurihydrogenibium azorense, Persephonella marina, Marinitoga piezophila, Kosmotoga olearia, Thermotoga maritima, Geobacillus stearothermophilus, Anoxybacillus flavithermus, Thermosulfidibacter takaii, Fervidobacterium nodosum, Clostridium thermocellum, Thermoanaerobacterium thermosaccharolyticum, Methanococcus jannaschii, Methanotorris igneus, Methanocaldococcus villosus, Methanothermococcus okinawensis, Pseudothermotoga thermarum, Deferribacter desulfuricans , and  Thermovibrio ammonificans  phosphoglucoisomerases. 
     
     
         7 . The method of  claim 1 , wherein the allulose 6-phosphate epimerase is selected from the group consisting of:  Thermobacterium thermosaccharolyticum, Thermoanaerobacter brockii, Caldanaerobacter subterraneus, Deferribacter desulfuricans, Thermocrinis ruber, Hydrogenivirga  sp. 128-5-R1-1,  Brevibacillus thermoruber, Thermosipho atlanticus , and  Thermosulfidibacter takaii  allulose 6-phosphate epimerases. 
     
     
         8 . The method of  claim 1 , wherein the allulose 6-phosphate phosphatase is selected from the group consisting of:  Thermoanaerobacter wiegelii, Thermoanaerobacter ethanolicus, Thermus islandicus, Deinococcus geothermalis  DSM 11300 , Thermosphaera aggregans, Crenarchaeota archaeon, Pyrococcus horikoshii  Ot3,  Aquifex aeolicus, Ruminiclostridium thermocellum, Desulfotomaculum kuznetsovii, Caldanaerobacter subterraneus, Acidothermus cellulolyticus, Methanothermobacter thermautotrophicus, Thermobifida fusca, Thermotoga neapolitana, Petrotoga mobilis, and Thermodesulfatator indicus, Thermus thermophilus, Bacteroides vulgatus , and  Bacteroides fragilus  allulose 6-phosphate phosphatases. 
     
     
         9 . The method of  claim 1  wherein the allulose 6-phosphate phosphatase is specific to allulose 6-phosphate. 
     
     
         10 . The method of  claim 1 , wherein at least one of the enzymes is thermostable or at least two of the enzymes are thermostable. 
     
     
         11 . The method of  claim 1 , wherein the starch or glycogen is pretreated with α-amylase and a debranching enzyme to produce debranched maltodextrin. 
     
     
         12 . The method of  claim 11 , wherein the debranching enzyme is selected from isoamylases and pullulanases. 
     
     
         13 . The method of  claim 12 , wherein the isoamylases are selected from  Sulfolobus tokodaii, Metallosphaera hakonensis, Sphaerobacter thermophiles , and  Bacillus lentus  isoamylases. 
     
     
         14 . The method of  claim 12 , wherein the pullulanases are selected from  Fervidobacterium  pennavorans.  Thermotoga  sp. RQ5,  Bacillus  flavocaldarius,  Thermosipho africanus , and  Kosmotoga olearia  pullulanases. 
     
     
         15 . 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 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.   
     
     
         16 .- 21 . (canceled) 
     
     
         22 . 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 phosphoglucoisomerases, thermostable allulose 6-phosphate epimerases, and thermostable allulose 6-phosphate phosphatases 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;   (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.   
     
     
         23 .- 43 . (canceled) 
     
     
         44 . A cell lysate produced by the method of  claim 15 . 
     
     
         45 .- 68 . (canceled)