Enzymatic production of hexoses

Abstract

To provide methods of producing hexoses from saccharides by enzymatic processes.SOLUTION: The methods utilize fructose 6-phosphate and at least one enzymatic step to convert fructose 6-phosphate to a hexose.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Application No. 62 / 470,605, filed March 13, 2017, U.S. Application No. 62 / 470,620, filed March 13, 2017, U.S. Application No. 62 / 482,148, filed April 5, 2017, and U.S. Application No. 62 / 480,798, filed April 3, 2017, which are hereby incorporated by reference in their entireties.

[0002] FIELD OF THEINVENTION The present invention relates to the production of hexoses. More particularly, the present invention relates to a method for producing D-hexose (or hexoses) from sugars (e.g., polysaccharides, oligosaccharides, disaccharides, sucrose, D-glucose, and D-fructose), comprising converting fructose 6-phosphate to said hexoses by one or more enzymatic steps. [Background technology]

[0003] background Hexoses are monosaccharides with six carbon atoms. Hexoses can be classified according to their functional groups, with aldohexoses having an aldehyde at position 1 and ketohexoses having a ketone at position 2. Aldohexoses (or aldoses) include allose, altrose, glucose, gulose, galactose, idose, talose, and mannose. Ketohexoses (or ketoses) include psicose (allulose), fructose, tagatose, and sorbose. Various aspects of these aldohexoses and ketohexoses are mentioned in the following paragraphs.

[0004] For example, D-allose (hereinafter allose) is a low-calorie natural sweetener with a sweetness of about 80% that of sucrose, and is considered a non-caloric sweetener and bulking agent. Allose is a naturally occurring hexose that exists in small amounts in certain shrubs and algae. Allose has several potential medical and agricultural benefits, such as cryoprotection, antioxidant, antihypertensive, immunosuppressant, anti-inflammatory, antitumor, and anticancer activity. Allose also has similar functions to sucrose in food and beverages. Thus, allose clearly has various applications in the food and beverage industry. However, the high selling price of allose has limited its use as a sweetener.

[0005] Currently, allose is mainly produced by the enzymatic isomerization of D-psicose (WO2014069537). Overall, this method suffers from high raw material costs, the costly separation of allose from D-psicose, and the relatively low product yield (about 23%).

[0006] Altrose is another unnatural aldohexose and is the C-3 epimer of mannose. D-Altrose ((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexanal) can be used as a substrate to identify, differentiate, and characterize aldose isomerases such as L-fucose isomerase from Caldicellulosiruptor saccharolyticus and d-arabinose isomerase (d-AI) from Bacillus pallidus (B. pallidus) and Klebsiella pneumoniae. Recently, glycans such as oligosaccharides and polysaccharides that perform useful functions as bioactive substances have attracted attention in the field of fine chemicals such as pharmaceuticals and agricultural chemicals. Currently, the subject of research on the above glycans is limited to glycans composed of monosaccharides such as D-glucose, D-mannose, and D-galactose, which exist in large quantities in nature and are easily available to researchers. However, in the future, it is expected that various monosaccharides other than those present in nature will be required in research on the synthesis of glycans that perform more useful functions. Under such circumstances, it is very important and necessary to develop a method capable of producing D-altrose, a rare sugar that is difficult to obtain, in high yield while reducing the number of processing steps. U.S. Patent No. 5,410,038.

[0007] D-glucose is useful, for example, as an excipient, a chelating agent, a pharmaceutical intermediate, a glass and metal cleaning agent, a food additive, and as a detergent additive. U.S. Patent No. 5,215,591.

[0008] D-galactose (hereafter galactose) is a natural sweetener with a sweetness of about 33% that of sucrose and is considered a nutritive sweetener. Galactose is a naturally occurring hexose present in dairy products, legumes, grains, nuts, tubers, and vegetables. Galactose is used in the baking industry to limit sourness and acidity. It is also used as an energy source to increase endurance in the athletic supplement industry. In the pharmaceutical industry, galactose is an intermediate for several medicines and is also used as a cell metabolism modulator in optimizing the biological production of protein therapeutics. Furthermore, galactose has been shown to be effective as a control agent against plant diseases caused by certain plant pathogens, such as those that affect cucumber, carrot, potato, and tomato. Due to dietary concerns (e.g., veganism) and health concerns (e.g., BSE disease), the industry is looking to non-animal sources of galactose. Thus, galactose clearly has a variety of uses in the food, beverage, athletic, agricultural, and pharmaceutical industries. However, the high selling price of galactose has limited its use.

[0009] Galactose is primarily produced by hydrolysis of lactose (WO2005039299A3). This method is less desirable due to higher raw material costs and the expense of separating glucose from galactose. Alternatively, galactose can be produced by hydrolysis of plant-based biomass (WO2005001145A1). This method suffers from the high cost of separating galactose from multiple other sugars (e.g., xylose, arabinose, mannose, glucose, rhamnose) released during hydrolysis of biomass, and low yields (galactose is about 4.6% of the dry mass of common biomass sources).

[0010] Idose does not occur in nature, but its uronic acid, iduronic acid, is important. Iduronic acid is a component of the glycosaminoglycans dermatan sulfate and heparan sulfate. (https: / / en.wikipedia.org / wiki / Idose - accessed 3 / 7 / 18).

[0011] Talose is a non-natural aldohexose that is soluble in water and slightly soluble in methanol. Talose is the C-2 epimer of galactose and the C-4 epimer of mannose. Talose can be used as a substrate to identify, differentiate, and characterize Clostridia ribose-5-phosphate isomerase(s).

[0012] D-mannose (hereafter mannose) is a naturally occurring monosaccharide with a moderate sweetness that is present in many fruits, vegetables, plant materials, and even in the human body. Mannose has multiple health benefits and uses in pharmaceuticals. For example, mannose can be used to treat carbohydrate deficiency glycoprotein syndrome type 1b, and more generally urinary tract infections. In addition, mannose is a verified prebiotic, does not increase blood glucose levels, and exhibits anti-inflammatory properties. Furthermore, mannose has been shown to increase pork carcass yields and is a supplementary moisturizing agent that is widely used in skin care products. Thus, mannose has various applications in the pharmaceutical, cosmetic, beverage, food, dairy, confectionery, and livestock industries. However, the high selling price of mannose has limited its use in everyday products.

[0013] Mannose is primarily produced by extraction from plants. Common methods include acid hydrolysis, thermal hydrolysis, enzymatic hydrolysis, microbial fermentation hydrolysis, and combinations thereof. Less common methods include chemical and biological conversion. Overall, these methods are problematic due to harsh conditions, high capital expenditures, high raw material costs, costly separation of mannose from the isomerization reaction mixture, and relatively low product yields (15-35%).

[0014] D-Allulose (also known as D-psicose) (hereafter referred to as psicose) is a low-calorie natural sweetener that is 70% as sweet as sucrose but contains only 10% of the calories. Psicose is a naturally occurring hexose that occurs in small amounts in wheat and other plants. Psicose was approved as a food additive by the Food and Drug Administration (FDA) in 2012, and the FDA designated psicose as "Generally Recognized as Safe (GRAS)". However, its high retail price has limited its use as a sweetener. Psicose has numerous health benefits: it is low in calories (10% of sucrose), has a very low glycemic index of 1, is completely absorbed in the small intestine but not metabolized, is secreted in urine and feces, helps regulate blood sugar by inhibiting α-amylase, sucrase, and maltase, and has similar functions to sucrose in foods and beverages. Thus, psicose clearly has a variety of uses in the food and beverage industry.

[0015] Currently, psicose is mainly produced by enzymatic isomerization of fructose (WO2014049373). Overall, this method exhibits high raw material costs, high cost separation of psicose from fructose, and relatively low product yields.

[0016] Fructose is a simple ketone monosaccharide present in many plants, and is often combined with glucose to form the disaccharide sucrose. Industrially, fructose is derived from sugar cane, sugar beet, and corn. The main reasons for its industrial use in foods and beverages are its low cost as well as its high relative sweetness. Fructose is the sweetest of all naturally occurring carbohydrates. Fructose is also found in the manufactured sweetener high fructose corn syrup (HFCS), which is produced by treating corn syrup with enzymes to convert glucose to fructose. (https: / / en.wikipedia.org / wiki / Fructose#Physical_and_functional_properties-- accessed 3 / 7 / 18).

[0017] D-tagatose (hereafter, tagatose) is a low-calorie natural sweetener that is 92% as sweet as sucrose but contains only 38% of the calories. Tagatose is a naturally occurring hexose that is present in small amounts in fruits, cocoa, and dairy products. Tagatose was approved as a food additive by the Food and Drug Administration (FDA) in 2003, and the FDA designated tagatose as "Generally Recognized as Safe (GRAS)". However, due to the high selling price of tagatose, its use as a sweetener has been restricted. Tagatose has countless health benefits. Namely, tagatose is non-carcinogenic, low in calories, has a very low glycemic index of 3, can attenuate the glycemic index of glucose by 20%, can lower average blood glucose levels, helps prevent cardiovascular disease, stroke, and other vascular diseases by raising high-density lipoprotein (HDL) cholesterol, and is a verified prebiotic and antioxidant. Lu et al., Tagatose, a New Antidiabetic and Obesity Control Drug, Diabetes Obes. Metab. 10(2): 109-34 (2008). Thus, tagatose clearly has a variety of applications in the pharmaceutical, biotechnology, academic, food, beverage, nutraceutical, and processed food industries.

[0018] Tagatose is mainly produced by hydrolysis of lactose with lactase or acid to produce D-glucose and D-galactose (WO2011150556, CN103025894, US5002612, US6057135, and US8802843). D-galactose is then isomerized to D-tagatose either chemically with calcium hydroxide under alkaline conditions or enzymatically with L-arabinose isomerase under neutral pH conditions. The final product is isolated by a combination of filtration and ion exchange chromatography. The process is carried out in several tanks or bioreactors. Overall, the above process is disadvantageous due to the high cost of separation of other sugars (e.g., D-glucose, D-galactose, and unhydrolyzed lactose) and low product yield. Although several methods based on microbial cell fermentation are under development, none have yet proven to be a practical alternative due to their dependence on expensive raw materials (e.g., galactitol and D-psicose), low product yields, and costly separation.

[0019] Sorbose ((3R,4S,5R)-1,3,4,5,6-pentahydroxyhexan-2-one) is a ketohexose with a sweetness comparable to sucrose (sugar) and is a plant metabolite found to occur naturally in small amounts in grapes. D-Sorbose has been determined to be effective as an agent for controlling plant diseases caused by Pseudomonas syringae pv. Lachrymans and Ralstonia solanacearum. U.S. Patent Application Publication No. 2016 / 0037768.

[0020] There is a need to develop cost-effective synthetic pathways for the production of hexoses, such as the above-mentioned aldohexoses and aldoketoses, in high yields, where at least one step of the process involves an energetically favorable chemical reaction. There is also a need for production processes that allow multiple process steps to be carried out in one vessel or bioreactor and / or that avoid or eliminate costly separation steps. There is also a need for processes for the production of hexoses that can be carried out at relatively low concentrations of phosphate, where phosphate can be recycled and / or that do not require the use of adenosine triphosphate (ATP) as an added phosphate source. There is also a need for hexose production pathways that do not require the use of costly nicotinamide adenosine dinucleotide (NAD(P)(H)) coenzymes in any of the reaction steps. Summary of the Invention [Means for solving the problem]

[0021] Summary of the Invention The invention described herein generally relates to methods for producing hexoses from sugars by enzymatic conversion. The invention also relates to hexoses produced by any of the methods described herein.

[0022] More particularly, the present invention relates to a method for producing a hexose selected from allose, mannose, galactose, fructose, altrose, talose, sorbose, gulose and idose from sugars, comprising converting fructose 6-phosphate (F6P) to said hexose by catalysis with one or more enzymes selected from the group consisting of isomerases, epimerases and hexose-specific phosphatases, and mixtures thereof.

[0023] The method for producing allose of the present invention comprises the steps of: converting the F6P to psicose 6-phosphate (P6P) by catalyzing with psicose 6-phosphate 3-epimerase (P6PE); converting the F6P to allose 6-phosphate (A6P) by catalyzing allose 6-phosphate isomerase (A6PI); converting said A6P to allose by catalysis with allose 6-phosphate phosphatase (A6PP); Includes.

[0024] The method for producing mannose of the present invention comprises the steps of: converting said F6P to mannose 6-phosphate (M6P) by catalysis with mannose 6-phosphate isomerase (M6PI) or phosphoglucose / phosphomannose isomerase (PGPMI); converting said M6P to mannose by catalysis with mannose 6-phosphate phosphatase (M6PP); Includes.

[0025] The method for producing galactose of the present invention comprises the steps of: converting said F6P to tagatose 6-phosphate (T6P) by catalyzing with fructose 6-phosphate 4-epimerase (F6PE); converting said T6P to galactose 6-phosphate (Gal6P) by catalysis with galactose 6-phosphate isomerase (Gal6PI); converting the Gal6P to galactose by catalysis with galactose 6-phosphate phosphatase (Gal6PP); Includes.

[0026] The method for producing fructose of the present invention comprises the steps of: The method comprises converting said F6P to fructose by catalysis with fructose 6-phosphate phosphatase (F6PP).

[0027] The method for producing altrose of the present invention comprises the steps of: converting said F6P to P6P by catalyzing with P6PE; converting said P6P to altrose 6-phosphate (Alt6P) by catalyzing with altrose 6-phosphate isomerase (Alt6PI); converting the generated Alt6P to altrose by catalysis with altrose 6-phosphate phosphatase (Alt6PP); Includes.

[0028] The method for producing talose of the present invention comprises the steps of: converting said F6P to T6P by catalyzing with F6PE; converting said T6P to talose 6-phosphate (Tal6P) by catalysis with talose 6-phosphate isomerase (Tal6PI); converting the Tal6P to talose by catalysis with talose 6-phosphate phosphatase (Tal6PP); Includes.

[0029] The method for producing sorbose of the present invention comprises the steps of: converting said F6P to T6P by catalyzing with F6PE; converting the T6P to sorbose 6-phosphate (S6P) by catalyzing sorbose 6-phosphate epimerase (S6PE); converting said S6P to sorbose by catalysis with sorbose 6-phosphate phosphatase (S6PP); Includes.

[0030] The method for producing growth of the present invention is as follows: converting said F6P to T6P by catalyzing with F6PE; converting said S6P to glulose 6-phosphate (Gul6P) by catalyzing with glulose 6-phosphate isomerase (Gul6PI); and converting the Gul6P to Gulose by catalysis with Gul6PP. Includes.

[0031] The method for producing growth of the present invention is as follows: converting said F6P to T6P by catalyzing with F6PE; converting the T6P to sorbose 6-phosphate (S6P) by catalyzing sorbose 6-phosphate epimerase (S6PE); converting said S6P to idose 6-phosphate (I6P) by catalysis with idose 6-phosphate isomerase (I6PI); converting said I6P to idose by catalysis with idose 6-phosphate phosphatase (I6PP); Includes.

[0032] The method for producing hexose of the present invention may comprise a step of converting glucose 6-phosphate (G6P) to said F6P catalyzed by phosphoglucose isomerase (PGI). The method may also comprise a step of converting glucose 1-phosphate (G1P) to said G6P catalyzed by phosphoglucomutase (PGM). Furthermore, the method of the present invention may further comprise a step of converting a sugar to said G1P catalyzed by at least one enzyme, the sugar being selected from the group consisting of starch or a derivative thereof, cellulose or a derivative thereof, and sucrose.

[0033] The enzyme or enzymes used in the step of converting sugar to G1P in the production method of the present invention may be α-glucan phosphorylase (αGP), maltose phosphorylase, sucrose phosphorylase, cellodextrin phosphorylase, cellobiose phosphorylase, and / or cellulose phosphorylase, and mixtures thereof.When the sugar is starch or a starch derivative, the derivative may be selected from the group consisting of amylose, amylopectin, soluble starch, amylodextrin, maltodextrin, maltose, and glucose, and mixtures thereof.

[0034] Some of the methods of the present invention may further include a step of converting starch into a starch derivative, which is produced by enzymatic or acid hydrolysis of starch. 4-glucan transferase (4GT) may also be added to the method. 4GT can be used to increase the hexose yield by recycling the degradation products glucose, maltose, and maltotriose into longer malto-oligosaccharides, which can be phosphorolytically cleaved by αGP to produce G1P.

[0035] Where the method employs a starch derivative, the starch derivative may be produced by enzymatic hydrolysis of starch catalyzed by isoamylase, pullulanase, α-amylase, or a combination thereof.

[0036] The production method of the present invention may also include a step of converting fructose to said F6P, catalyzed by at least one enzyme, and optionally a step of converting sucrose to said fructose, catalyzed by at least one enzyme.

[0037] Furthermore, the method for producing hexose of the present invention may comprise a step of converting glucose to said G6P catalyzed by at least one enzyme, and optionally a step of converting sucrose to said glucose catalyzed by at least one enzyme.

[0038] The above steps in each of the methods for synthesizing hexose of the present invention may be carried out at a temperature in the range of about 40° C. to about 90° C. and at a pH in the range of about 5.0 to about 8.0. The above steps may be carried out for about 8 hours to about 48 hours.

[0039] The steps of the process of the invention may be carried out in a single bioreactor. The steps may also be carried out in multiple bioreactors arranged in series.

[0040] The enzymatic process steps of the invention may be carried out without ATP and / or without NAD(P)(H). The steps may be carried out at a phosphate concentration ranging from about 0.1 mM to about 150 mM. The phosphate used in the phosphorylation and dephosphorylation steps of the process of the invention may be recycled. At least one step of the process may comprise an energetically favorable chemical reaction.

[0041] The invention also relates to allose, mannose, galactose, fructose, altrose, talose, sorbose, gulose, and idose produced by these processes. [Brief description of the drawings]

[0042] Brief description of the drawings

[0043] [Figure 1-1] 1 is a schematic diagram showing the enzymatic pathway for the conversion of starch or its derived products to allose, using the following abbreviations: IA: isoamylase, PA: pullulanase, αGP: α-glucan phosphorylase or starch phosphorylase, MP: maltose phosphorylase, PGM: phosphoglucomutase, PPGK: polyphosphate glucokinase, PGI: phosphoglucoisomerase, P6PE: psicose 6-phosphate 3-epimerase, A6PI: allose 6-phosphate isomerase, A6PP: allose 6-phosphate phosphatase. [Figure 1-2] Same as Figure 1-1.

[0044] [Figure 2-1]1 is a schematic diagram showing the enzymatic pathway for the conversion of starch or its derived products to mannose, using the following abbreviations: IA: isoamylase, PA: pullulanase, αGP: α-glucan phosphorylase or starch phosphorylase, MP: maltose phosphorylase, PGM: phosphoglucomutase, PPGK: polyphosphate glucokinase, PGI: phosphoglucoisomerase, PGPMI: bifunctional phosphoglucose / phosphomannose isomerase, M6PI: mannose 6-phosphate isomerase, M6PP: mannose 6-phosphate phosphatase. [Figure 2-2] Same as Figure 2-1.

[0045] [Figure 3-1] Figure 1 is a schematic diagram showing the enzymatic pathway for the conversion of starch or its derived products to galactose, using the following abbreviations: IA: isoamylase, PA: pullulanase, αGP: α-glucan phosphorylase or starch phosphorylase, MP: maltose phosphorylase, PGM: phosphoglucomutase, PPGK: polyphosphate glucokinase, PGI: phosphoglucoisomerase, F6PE: fructose 6-phosphate 4-epimerase, Gal6PI: galactose 6-phosphate isomerase, Gal6PP: galactose 6-phosphate phosphatase. [Figure 3-2] Same as Figure 3-1.

[0046] [Figure 4-1] Figure 1 is a schematic diagram showing the enzymatic pathway for the conversion of starch or its derived products to galactose, using the following abbreviations: IA: isoamylase, PA: pullulanase, αGP: α-glucan phosphorylase or starch phosphorylase, MP: maltose phosphorylase, PGM: phosphoglucomutase, PPGK: polyphosphate glucokinase, PGI: phosphoglucoisomerase, F6PP: fructose 6-phosphate phosphatase. [Figure 4-2] Same as Figure 4-1.

[0047] [Diagram 5]FIG. 1 is a schematic diagram showing the enzymatic pathway for the conversion of sucrose to fructose, with the following abbreviations: SP: sucrose phosphorylase, PGM: phosphoglucomutase, PGI: phosphoglucoisomerase, F6PP: fructose 6-phosphate phosphatase.

[0048] [Figure 6-1] 1 is a schematic diagram showing the enzymatic pathway for the conversion of starch or its derived products to altrose, using the following abbreviations: IA: isoamylase, PA: pullulanase, αGP: α-glucan phosphorylase or starch phosphorylase, MP: maltose phosphorylase, PGM: phosphoglucomutase, PPGK: polyphosphate glucokinase, PGI: phosphoglucoisomerase, P6PE: psicose 6-phosphate epimerase, Alt6PI: altrose 6-phosphate isomerase, Alt6PP: altrose 6-phosphate phosphatase. [Figure 6-2] Same as Figure 6-1.

[0049] [Figure 7-1] 1 is a schematic diagram showing the enzymatic pathway for the conversion of starch or its derived products to talose, using the following abbreviations: IA: isoamylase, PA: pullulanase, αGP: α-glucan phosphorylase or starch phosphorylase, MP: maltose phosphorylase, PGM: phosphoglucomutase, PPGK: polyphosphate glucokinase, PGI: phosphoglucoisomerase, F6PE: fructose 6-phosphate epimerase, Tal6PI: talose 6-phosphate isomerase, Tal6PP: talose 6-phosphate phosphatase. [Figure 7-2] Same as Figure 7-1.

[0050] [Figure 8-1]1 is a schematic diagram showing the enzymatic pathway for the conversion of starch or its derived products to sorbose, using the following abbreviations: IA: isoamylase, PA: pullulanase, αGP: α-glucan phosphorylase or starch phosphorylase, MP: maltose phosphorylase, PGM: phosphoglucomutase, PPGK: polyphosphate glucokinase, PGI: phosphoglucoisomerase, F6PE: fructose 6-phosphate epimerase, S6PE: sorbose 6-phosphate epimerase, S6PP: sorbose 6-phosphate phosphatase. [Figure 8-2] Same as Figure 8-1.

[0051] [Figure 9-1] 1 is a schematic diagram showing the enzymatic pathway for the conversion of starch or its derived products to glose, using the following abbreviations: IA: isoamylase, PA: pullulanase, αGP: α-glucan phosphorylase or starch phosphorylase, MP: maltose phosphorylase, PGM: phosphoglucomutase, PPGK: polyphosphate glucokinase, PGI: phosphoglucoisomerase, F6PE: fructose 6-phosphate epimerase, S6PE: sorbose 6-phosphate epimerase, Gul6PI: glose 6-phosphate isomerase, Gul6PP: glose 6-phosphate phosphatase. [Figure 9-2] Same as Figure 9-1.

[0052] [Figure 10-1] 1 is a schematic diagram showing the enzymatic pathway for the conversion of starch or its derived products to idose, using the following abbreviations: IA: isoamylase, PA: pullulanase, αGP: α-glucan phosphorylase or starch phosphorylase, MP: maltose phosphorylase, PGM: phosphoglucomutase, PPGK: polyphosphate glucokinase, PGI: phosphoglucoisomerase, F6PE: fructose 6-phosphate epimerase, S6PE: sorbose 6-phosphate epimerase, I6PI: idose 6-phosphate isomerase, I6PP: idose 6-phosphate phosphatase. [Figure 10-2] Same as Figure 10-1.

[0053] [Figure 11] FIG. 1 shows Gibbs energies of reactions between intermediates, based on the Gibbs energies of formation for the conversion of glucose 1-phosphate to other hexoses. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] Description of the Invention The inventions described herein provide enzymatic pathways or processes for the synthesis of hexoses that provide high product yields and significantly reduce the costs of product separation and hexose production. Also described herein are the hexoses produced by these processes.

[0055] The method for producing hexose from sugar according to the present invention comprises the steps of: converting fructose 6-phosphate (F6P) to said hexose by catalysis with one or more enzymes; the hexose is selected from the group consisting of allose, mannose, galactose, fructose, altrose, talose, sorbose, gulose, and idose; The enzyme is selected from the group consisting of isomerases, epimerases, and hexose-specific phosphatases, and mixtures thereof.

[0056] One important advantage of the manufacturing method of the present invention is that multiple process steps can be carried out in a single bioreactor or reactor, or alternatively, the steps can be carried out in multiple bioreactors or reactors arranged in series.

[0057] The phosphate ions produced during the dephosphorylation step can then be recycled to the process step that converts sugars to G1P, especially if all process steps are carried out in a single bioreactor or reactor. The ability to recycle phosphate in the disclosed process allows for the use of non-stoichiometric amounts of phosphate, thereby keeping the phosphate concentration in the reaction low. This affects the overall pathway and overall rate of the process, but does not limit the activity of any individual enzyme, allowing for an overall efficient process for the production of hexose.

[0058] For example, the phosphoric acid concentration in the reaction solution in each of the present production methods may be in the range of about 0.1 mM to about 300 mM, about 0 mM to about 150 mM, about 1 mM to about 50 mM, preferably about 5 mM to about 50 mM, or more preferably about 10 mM to about 50 mM. For example, the phosphoric acid concentration in the reaction solution in each of the present production methods may be about 0.1 mM, about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, or about 55 mM.

[0059] Lower phosphate concentrations reduce production costs by reducing total phosphate, which in turn reduces the cost of phosphate removal, and also prevent phosphatase inhibition by high levels of free phosphate, reducing the potential for phosphate contamination.

[0060] Additionally, each of the methods disclosed herein can be performed without the addition of ATP as a phosphate source, i.e., without ATP. Each of the methods can be performed without the need for the addition of NAD(P)(H), i.e., without NAD(P)(H). Another advantage includes the fact that at least one step of the disclosed methods for producing hexoses involves an energetically favorable chemical reaction.

[0061] Examples of the enzymes used to convert sugars to G1P include α-glucan phosphorylase (αGP, EC 2.4.1.1), maltose phosphorylase (MP, EC 2.4.1.8), cellodextrin phosphorylase (CDP, EC 2.4.1.49), cellobiose phosphorylase (CBP, EC 2.4.1.20), cellulose phosphorylase, sucrose phosphorylase (SP, EC 2.4.1.7), and combinations thereof. The selection of the enzyme or combination of enzymes depends on the sugar used in the present production method.

[0062] The sugar used to generate G1P may be a polysaccharide, an oligosaccharide, and / or a disaccharide. For example, the sugar may be starch, one or more derivatives of starch, cellulose, one or more derivatives of cellulose, sucrose, one or more derivatives of sucrose, or a combination thereof.

[0063] Starch is the most widely used energy storage compound in nature, and is mostly stored in plant seeds. Natural starch contains linear amylose and branched amylopectin. Examples of starch derivatives include amylose, amylopectin, soluble starch, amylodextrin, maltodextrin, maltose, fructose, and glucose. Examples of cellulose derivatives include pretreated biomass, regenerated amorphous cellulose, cellodextrin, cellobiose, fructose, and glucose. Sucrose derivatives include fructose and glucose.

[0064] Methods for producing F6P from starch and its derivatives, cellulose and its derivatives, and sucrose and its derivatives are described, for example, in International Patent Application Publication No. WO2017 / 059278.

[0065] The starch derivatives can be produced by enzymatic hydrolysis of starch or by acid hydrolysis of starch, which is specifically catalyzed or promoted by isoamylase (IA, EC. 3.2.1.68), which hydrolyzes α-1,6-glucosidic bonds, pullulanases (PA, EC. 3.2.1.41), which hydrolyze α-1,6-glucosidic bonds, 4-α-glucanotransferase (4GT, EC. 2.4.1.25), which catalyzes the transglycosylation of short chain maltooligosaccharides to produce longer chain maltooligosaccharides, or α-amylase (EC 3.2.1.1), which cleaves α-1,4-glucosidic bonds.

[0066] Additionally, derivatives of cellulose can be produced by enzymatic hydrolysis of cellulose catalyzed by cellulase mixtures, acid, or biomass pretreatment.

[0067] The enzyme used to convert sugars to G1P may include αGP, in which, if the sugar contains starch, the G1P is produced from starch by αGP, and if the sugar contains soluble starch, amylodextrin, or maltodextrin, the G1P is produced from soluble starch, amylodextrin, or maltodextrin by αGP.

[0068] When the sugar comprises maltose and the enzyme comprises maltose phosphorylase, the G1P is generated from maltose by maltose phosphorylase.When the sugar comprises sucrose and the enzyme comprises sucrose phosphorylase, the G1P is generated from sucrose by sucrose phosphorylase.

[0069] When the sugar includes cellobiose and the enzyme includes cellobiose phosphorylase, the G1P can be produced from cellobiose by cellobiose phosphorylase.

[0070] When the sugar contains cellodextrin and the enzyme contains cellodextrin phosphorylase, the G1P can be produced from cellodextrin by cellodextrin phosphorylase.

[0071] When converting sugar to G1P, if the sugar contains cellulose and the enzyme contains cellulose phosphorylase, the G1P can be produced from cellulose by cellulose phosphorylase.

[0072] According to the present invention, hexose can also be produced from fructose. For example, this production method comprises the steps of: producing F6P from fructose and polyphosphate by catalysis with polyphosphate fructokinase (PPFK); converting F6P to T6P by catalysis with F6PE; Conversion of T6P to tagatose by catalysis with T6PP The fructose can be produced, for example, by the enzymatic conversion of sucrose.

[0073] Hexose can be produced from sucrose by, for example, the following enzymatic steps: generating G1P from sucrose and free phosphate by catalyzing with sucrose phosphorylase (SP); PGM-catalyzed conversion of G1P to G6P; converting G6P to F6P by catalysis with PGI; converting F6P to T6P by catalysis with F6PE; Conversion of T6P to tagatose by catalysis with T6PP The present invention provides an in vitro synthesis route comprising:

[0074] The phosphatase used in the production method of the present invention is specific to the above-mentioned hexoses. For example, allose 6-phosphate is converted to allose by allose 6-phosphate phosphatase, mannose 6-phosphate is converted to mannose by mannose 6-phosphate phosphatase, galactose 6-phosphate is converted to galactose by galactose 6-phosphate phosphatase, fructose 6-phosphate is converted to fructose by fructose 6-phosphate phosphatase, altrose 6-phosphate is converted to altrose by altrose 6-phosphate phosphatase, talose 6-phosphate is converted to talose by talose 6-phosphate phosphatase, sorbose 6-phosphate is converted to sorbose by sorbose 6-phosphate phosphatase, gulose 6-phosphate is converted to gulose by gulose 6-phosphate phosphatase, and idose 6-phosphate is converted to idose by idose 6-phosphate phosphatase. As used herein, specific means having a higher specific activity for the indicated hexose than for other hexoses, e.g., allose 6-phosphate phosphatase has a higher specific activity for allose 6-phosphate than for sorbose 6-phosphate or talose 6-phosphate.

[0075] The phosphate ions produced during the hexose dephosphorylation step can then be recycled to the sucrose conversion to G1P step. Additionally, the hexose yield can be increased by using PPFK and polyphosphate to generate F6P from fructose produced by phosphorolytic cleavage of sucrose by SP.

[0076] The process for producing hexose comprises the following steps: producing glucose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis; converting glucose to G6P by catalyzing at least one enzyme; producing fructose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis; converting fructose to G6P by catalysis with at least one enzyme; Examples of such polysaccharides and oligosaccharides are listed above. G6P can be produced from glucose and sodium polyphosphate by polyphosphate glucokinase.

[0077] The present invention provides a method for the conversion of sugars, such as polysaccharides and oligosaccharides in starch, cellulose, sucrose, and their derived products, to hexoses. A cell-free enzyme cocktail can be used to provide an artificial (non-natural), ATP-free enzymatic pathway for the conversion of starch, cellulose, sucrose, and their derived products to hexoses.

[0078] As shown above, several enzymes can be used to hydrolyze starch and increase the G1P yield. These enzymes include isoamylase, pullulanase, and α-amylase. Corn starch contains many branches that prevent the action of αGP. Isoamylase can be used to debranch starch and produce linear amylodextrins. Pre-treated starch with isoamylase can result in a higher concentration of F6P in the final product. Isoamylase and pullulanase cleave α-1,6-glycosidic bonds, which allows starch to be more completely degraded by α-glucan phosphorylase. α-amylase cleaves α-1,4-glycosidic bonds, so α-amylase can be used to break starch into fragments for more rapid conversion to hexoses and increased solubility.

[0079] Maltose phosphorylase (MP) can be used to increase hexose yields by phosphorolytic cleavage of the degradation product maltose to G1P and glucose, or 4-glucan transferase (4GT) can be used to increase hexose yields by recycling the degradation products glucose, maltose, and maltotriose to longer chain maltooligosaccharides that can be phosphorolytically cleaved by αGP to produce G1P.

[0080] Moreover, cellulose is the most abundant biological resource and is the major component of plant cell walls. Non-food lignocellulosic biomass contains cellulose, hemicellulose, and lignin, as well as other minor components. Pure cellulose, including Avicel (microcrystalline cellulose), regenerated amorphous cellulose, bacterial cellulose, filter paper, etc., can be produced through a series of processes. Partially hydrolyzed cellulose substrates include water-insoluble cellodextrins with a degree of polymerization greater than 7, water-soluble cellodextrins with a degree of polymerization between 3 and 6, cellobiose, glucose, and fructose.

[0081] Cellulose and its derived products can be converted to hexoses through a series of steps. The process comprises the following steps: producing G1P from cellodextrin and cellobiose and free phosphate by catalyzing with cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively; PGM-catalyzed conversion of G1P to G6P; Conversion of G6P to F6P by PGI catalysis In this method, phosphate ions can be recycled through the above steps of converting cellodextrin and cellobiose into G1P.

[0082] Several enzymes may be used to hydrolyze solid cellulose into water-soluble cellodextrins and cellobiose, including endoglucanases and cellobiohydrolases, but not including β-glucosidase (cellobiase).

[0083] Prior to cellulose hydrolysis and G1P production, cellulose and biomass may be pretreated to increase their reactivity and reduce the degree of polymerization of the cellulose chains. Methods for pretreatment of cellulose and biomass include dilute acid pretreatment, lignocellulose fractionation with cellulose solvents, fiber swelling with ammonia, aqueous ammonia soaking, ionic liquid treatment, and partial hydrolysis using concentrated acids including hydrochloric acid, sulfuric acid, phosphoric acid, and combinations thereof.

[0084] The hexose yield can be increased by adding polyphosphate and polyphosphate glucokinase (PPGK) to the production method of the present invention, thereby phosphorylating the degradation product glucose to G6P.

[0085] Hexose may be produced from glucose. The method for producing hexose includes: Producing G6P from glucose and polyphosphate by catalysis with polyphosphate glucokinase (PPGK); converting G6P to F6P by catalysis with PGI; may also include

[0086] Any suitable biologically compatible buffer known in the art may be used in each of the methods of the present invention, such as HEPES, PBS, BIS-TRIS, MOPS, DIPSO, Trizma, etc. The pH of the reaction buffer used in the methods of the present invention may range from 5.0 to 8.0. More preferably, the pH of the reaction buffer may range from about 6.0 to about 7.3. For example, the pH of the reaction buffer may be 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, or 7.3.

[0087] The reaction buffer may also contain metal cations. Examples of such metal ions include Mg 2+ and Zn 2+ As is known in the art, the desired metal cation can be introduced using an appropriate salt.

[0088] In each of the production methods of the present invention, the reaction temperature at which the process steps are carried out may range from 37 to 95° C. More preferably, the steps may be carried out at a temperature ranging from about 40° C. to about 90° C. The temperature may be, for example, about 40° C., about 45° C., 50° C., about 55° C., about 60° C., about 65° C., about 70° C., about 75° C., about 80° C., about 85° C., or about 90° C. Preferably, the reaction temperature is about 50° C.

[0089] The reaction time of each of the disclosed production methods may be adjusted as necessary and may be in the range of about 8 hours to about 48 hours. For example, the reaction time may be about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 26 hours, about 28 hours, about 30 hours, about 32 hours, about 34 hours, about 36 hours, about 38 hours, about 40 hours, about 42 hours, about 44 hours, about 46 hours, or about 48 hours. It is more preferable that the reaction time is about 24 hours.

[0090] Typically, the ratio of enzyme units used in each of the disclosed methods of production is 1:1 to 1:1:1:1:1 (depending on the number of catalytic reaction steps in the method). These ratios may be adjusted in any number of combinations to optimize product yield. For example, a ratio of 3:1:1:1:1 can be used to maximize the concentration of phosphorylated intermediates, which will increase the activity of downstream reactions. Conversely, a ratio of 1:1:1:1:3 can be used to ensure that a supply of phosphate is maintained for αGP, which will result in more efficient phosphorolytic cleavage of α-1,4-glycosidic bonds. For example, a ratio of 3:1:1:1:3 enzymes can be used to further increase the reaction rate. Thus, the ratio of enzymes, including other optional enzymes described below, can be varied to increase the efficiency of hexose production. For example, a particular enzyme may be present in an amount of about 2-fold, 3-fold, 4-fold, 5-fold, etc., relative to the amount of other enzymes.

[0091] Each of the manufacturing methods of the present invention can achieve high yields due to the highly favorable equilibrium constant for the overall reaction. For example, FIG. 11 shows the Gibbs energies of reaction between intermediates based on the Gibbs energy of formation for the conversion of glucose 1-phosphate to hexose. The Gibbs energies of reaction are given using http: / / equilibrator.weizmann.ac.il / . Theoretically, if the starting material is completely converted to the intermediate, a yield of up to 99% can be achieved.

[0092] The process of the present invention reduces production costs by using low-cost starting materials and eliminating costs associated with the separation of raw materials and products. Starch, cellulose, sucrose, and their derivatives are cheaper raw materials than, for example, lactose. When hexose is produced from lactose, glucose and other hexose(s) are separated by chromatography, which leads to higher production costs.

[0093] Furthermore, the step of dephosphorylation of hexose by the phosphatase according to the present invention is an irreversible phosphatase reaction regardless of the raw material, therefore, hexose is produced in a very high yield while effectively minimizing the subsequent cost of product separation.

[0094] In some embodiments of the invention, the phosphatase that converts A6P, M6P, F6P, or Gal6P to their respective unphosphorylated forms utilizes a divalent metal cofactor, preferably magnesium. a Rossmann-like fold domain for catalysis; Additionally, a C1 or C2 capping domain for substrate specificity; Additionally, a DxD signature in the first β-strand of the Rossmann-like fold, with the second Asp being a general acid / base catalyst for coordinating magnesium; Furthermore, a Thr or Ser at the end of the second β-strand of the Rossmann-like fold, which promotes the stability of the reaction intermediate, Furthermore, the N-terminal Lys of the C-terminus of the α-helix of the third β-strand of the Rossmann-like fold promotes the stability of the reaction intermediate. Furthermore, the GDxxxD, GDxxxxD, DD, or ED signature at the end of the fourth β-strand of the Rossmann-like fold for coordinating magnesium These features are known in the art and are referenced, for example, in Burroughs et al., Evolutionary Genomics of the HAD Superfamily: Understanding the Structural Adaptations and Catalytic Diversity in a Superfamily of Phosphoesterases and Allied Enzymes. J. Mol. Biol. 2006;361;1003-1034.

[0095] In contrast to cellular production methods, the present invention involves cell-free production of hexoses, which has relatively high reaction rates due to the elimination of cell membranes that often slow down transport of substrates into the cell / products out of the cell, and the present invention also does not include nutrient-rich fermentation media / cellular metabolic products in the final product.

[0096] Alose

[0097] One embodiment of the present invention comprises: converting fructose 6-phosphate (F6P) to psicose 6-phosphate (P6P) by catalyzing psicose 6-phosphate 3-epimerase (P6PE); converting P6P to allose 6-phosphate (A6P) by catalyzing allose 6-phosphate isomerase (A6PI); converting the produced A6P to allose by catalysis with allose 6-phosphate phosphatase; The method for producing allose comprises the steps of:

[0098] Examples of P6PE include, but are not limited to, the following proteins identified by UNIPROT identification numbers: D9TQJ4, A0A090IXZ8, and P32719. Of these, D9TQJ4 and A0A090IXZ8 are obtained from thermophilic organisms. P32719 is obtained from mesophilic organisms. P32719 has 53% identity to A0A090IXZ8 and 55% identity to D9TQJ4, and each protein catalyzes the epimerization of F6P to A6P. Additionally, A0A090IXZ8 has 45% identity to D9TQJ4. Conversely, other epimerase proteins identified by UNIPROT Identification Numbers A0A101D823, R1AXD6, A0A150LBU8, A0A023CQG9, and H1XWY2, which are 45% or less identical to D9TQJ4, do not catalyze the epimerization of F6P to A6P. Examples of P6PE also include any analogs that have at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity to any of the above Uniprot IDs.

[0099] Examples of A6PIs include, but are not limited to, Uniprot ID W4V2C8, having the amino acid sequence shown in SEQ ID NO: 1, and Uniprot ID Q67LX4, having the amino acid sequence shown in SEQ ID NO: 2. Both Uniprot IDs W4V2C8 and Q67LX4 catalyze the A6PI reaction and share 56% amino acid sequence identity. Thus, examples of A6PIs also include any analogs that have at least 55%, preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, at least 91%, at least 92%, at least 93%, or at least 94%, and even most preferably at least 96, 97, 98, 99, or 100% amino acid sequence identity to any of the above Uniprot IDs.

[0100] A6PI suitable for use in the method of converting P6P to A6P contains a Rossmann-like fold. Mesophilic A6PIs reported in the art (Mowbray et al., D-Ribose-5-Phosphate Isomerase B from Escherichia coli is Also a Functional D-Allose-6-phosphate Isomerase, While the Mycobacterium tuberculosis Enzyme is Not. J. Mol. Biol. 2008;382;667-679) share conserved residues with the thermophilic A6PIs disclosed in the present invention. In some embodiments of the present invention, the isomerase is a His (mesophilic residue 10) C-terminal to the first β-strand of the Rossmann-like fold for phosphate binding; In addition, the C-terminus of the α-helix of the fifth β-strand of the Rossmann-like fold is Arg (mesophilic residue 133), also for phosphate binding. In addition, a His (mesophilic residue 99) in the active site for ring-opening of the lactone; In addition, a Cys (mesophilic residue 66) in the active site acts as a catalytic base; In addition, a Thr in the active site (mesophilic residue 68) that acts as a catalytic acid; In addition, a GTG hydrophobic G motif (mesophilic residues 67–71) near the active site to stabilize the high-energy intermediate; In addition, there is a linkage between Asn (mesophilic residue 100) near the active site, which also stabilizes the high-energy intermediate. A6PI preferably contains all of these conserved residues, but is not limited to containing:

[0101] Examples of A6PP include, but are not limited to, the following proteins: Uniprot ID S9SDA3 having the amino acid sequence set forth in SEQ ID NO: 3, Uniprot ID Q9X0Y1 having the amino acid sequence set forth in SEQ ID NO: 4, Uniprot ID I3VT81 having the amino acid sequence set forth in SEQ ID NO: 5, Uniprot ID A0A132NF06 having the amino acid sequence set forth in SEQ ID NO: 6, and Uniprot ID D1C7G9 having the amino acid sequence set forth in SEQ ID NO: 7. Uniprot IDs S9SDA3 and I3VT81 both catalyze the A6PP reaction and share 30% amino acid sequence identity. Thus, examples of A6PP include any analogues that have an amino acid sequence identity to any of the above Uniprot IDs of at least 30%, preferably at least 35%, more preferably at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, at least 91%, at least 92%, at least 93%, or at least 94%, and even most preferably at least 96, 97, 98, 99, or 100%.

[0102] A6PP, which converts A6P to allose, a Rossmann-like fold domain for catalysis; A C1 capping domain, A DxD signature in the first β strand of the Rossmann-like fold; and Thr or Ser at the end of the second β-strand of the Rossmann-like fold; Lys at the N-terminus of the C-terminus of the α-helix of the third β-strand of the Rossmann-like fold; The ED signature at the end of the fourth β-strand of the Rossmann-like fold It is preferred that the compound contains

[0103] The method for producing allose of the present invention also includes a step of enzymatically converting glucose 6-phosphate (G6P) to F6P, which is catalyzed by phosphoglucoisomerase (PGI). In another embodiment, the method for producing allose further includes a step of converting glucose 1-phosphate (G1P) to G6P, which is catalyzed by phosphoglucomutase (PGM). In yet another embodiment, the method for producing allose also includes a step of converting sugar to said G1P, which is catalyzed by at least one enzyme.

[0104] Therefore, the method for producing allose of the present invention includes, for example, the following steps: (i) converting sugars to glucose 1-phosphate (G1P) using one or more enzymes; (ii) converting G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii) converting G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) converting F6P to P6P via P6PE; (v) converting P6P to A6P via A6PI; (vi) converting A6P to allose via A6PP; An example of the enzymatic process where the sugar is starch is shown in FIG.

[0105] Typically, the ratio of enzyme units used in each of the disclosed production methods is 1:1:1:1:1:1 (αGP:PGM:PGI:P6PE:A6PI:A6PP). These ratios may be adjusted in any number of combinations to optimize product yield. For example, a ratio of 3:1:1:1:1:1 can be used to maximize the concentration of phosphorylated intermediates, which will increase the activity of downstream reactions. Conversely, a ratio of 1:1:1:1:1:1:3 can be used to ensure that phosphate is maintained for αGP, which will result in more efficient phosphorolytic cleavage of α-1,4-glycosidic bonds. For example, a ratio of 3:1:1:1:1:3 enzymes can be used to further increase the reaction rate. Thus, the ratio of enzymes, including other optional enzymes described below, can be varied to increase the efficiency of allose production. For example, a particular enzyme may be present in an amount of about 2-fold, 3-fold, 4-fold, 5-fold, etc., relative to the amount of other enzymes.

[0106] Phosphate ions produced by dephosphorylation of A6P can then be recycled to the process step that converts sugar to G1P, especially when all process steps are carried out in a single bioreactor or reactor. The ability to recycle phosphate in the disclosed production method allows for the use of non-stoichiometric amounts of phosphate, which keeps the phosphate concentration in the reaction solution low. This affects the overall pathway and overall rate of the production method, but does not limit the activity of individual enzymes, allowing the overall efficiency of the allose production method.

[0107] For example, the phosphoric acid concentration in the reaction solution may be in the range of about 0.1 mM to about 300 mM, about 0.1 mM to about 150 mM, about 1 mM to about 50 mM, preferably about 5 mM to about 50 mM, or more preferably about 10 mM to about 50 mM. For example, the phosphoric acid concentration in the reaction solution may be about 0.1 mM, about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, or about 55 mM.

[0108] Lower phosphate concentrations reduce production costs by lowering total phosphate, which in turn reduces the cost of phosphate removal, and they also prevent inhibition of A6PP by high levels of free phosphate, reducing the possibility of phosphate contamination.

[0109] Furthermore, the method disclosed herein can be carried out without the addition of ATP as a phosphate source, i.e., without ATP. The method can also be carried out without the need to add NAD(P)(H), i.e., without NAD(P)(H). Another advantage is the fact that at least one step of the disclosed method for producing allose involves an energetically favorable reaction.

[0110] Allose can also be produced from fructose. For example, this production method includes the following steps: producing F6P from fructose and polyphosphate by catalysis with polyphosphate fructokinase (PPFK); converting F6P to P6P by catalysis with F6PE; converting P6P to A6P by catalysis with A6PI; Conversion of A6P to allose by catalysis with A6PP; The fructose can be produced, for example, by the enzymatic conversion of sucrose.

[0111] Allose can also be produced from sucrose. The production process involves the following enzymatic steps: generating G1P from sucrose and free phosphate by catalyzing with sucrose phosphorylase (SP); PGM-catalyzed conversion of G1P to G6P; converting G6P to F6P by catalysis with PGI; converting F6P to P6P by catalysis with P6PE; converting P6P to A6P by catalysis with A6PI; Conversion of A6P to allose by catalysis with A6PP; The present invention provides an in vitro synthesis route comprising:

[0112] The phosphate ions produced during the conversion of A6P to allose can then be recycled to the step of converting sucrose to G1P. Furthermore, the allose yield can be increased by using PPFK and polyphosphate to generate F6P from fructose produced by the phosphorolytic cleavage of sucrose by SP.

[0113] In a particular embodiment, the method for producing allose comprises the following steps: producing glucose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis; converting glucose to G6P by catalyzing at least one enzyme; producing fructose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis; converting fructose to F6P by catalysis with at least one enzyme; Examples of such polysaccharides and oligosaccharides are listed above.

[0114] In another embodiment, G6P is produced from glucose and sodium polyphosphate by polyphosphate glucokinase.

[0115] Several enzymes can be used to hydrolyze starch and increase the G1P yield. These enzymes include isoamylase, pullulanase, and α-amylase. Corn starch contains many branches that prevent the action of αGP. Isoamylase can be used to debranch starch and produce linear amylodextrins. Pre-treated starch with isoamylase can result in higher F6P concentrations in the final product. Isoamylase and pullulanase cleave α-1,6-glycosidic bonds, which allows starch to be more completely degraded by α-glucan phosphorylase. α-amylase cleaves α-1,4-glycosidic bonds, so α-amylase can be used to break starch into fragments for more rapid conversion to allose and increased solubility.

[0116] Maltose phosphorylase (MP) can be used to increase the allose yield by phosphorolytic cleavage of the degradation product maltose to G1P and glucose, or 4-glucan transferase (4GT) can be used to recycle the degradation products glucose, maltose, and maltotriose to longer chain maltooligosaccharides that can be phosphorolytically cleaved by αGP to produce G1P.

[0117] In a particular embodiment, cellulose and its derived products can be converted to allose through a series of steps. The process comprises the following steps: producing G1P from cellodextrin and cellobiose and free phosphate by catalyzing with cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively; PGM-catalyzed conversion of G1P to G6P; converting G6P to F6P by catalysis with PGI; converting F6P to P6P by catalysis with P6PE; converting P6P to A6P by catalysis with A6PI; Conversion of A6P to allose by catalysis with A6PP; In this method, phosphate ions can be recycled through the above steps of converting cellodextrin and cellobiose into G1P.

[0118] Several enzymes may be used to hydrolyze solid cellulose into water-soluble cellodextrins and cellobiose, including endoglucanases and cellobiohydrolases, but not including β-glucosidase (cellobiase).

[0119] In some embodiments, polyphosphate and polyphosphate glucokinase (PPGK) can be added to the process to increase the yield of allose by phosphorylating the degradation product glucose to G6P.

[0120] Allose may be produced from glucose. The production method comprises: Producing G6P from glucose and polyphosphate by catalysis with polyphosphate glucokinase (PPGK); converting G6P to F6P by catalysis with PGI; converting F6P to P6P by catalysis with P6PE; converting P6P to A6P by catalysis with A6PI; converting A6P to allose by catalysis with A6PP; Includes.

[0121] The method for producing allose of the present invention uses low-cost starting materials and reduces production costs by eliminating costs associated with the separation of raw materials and products. Starch, cellulose, sucrose, and some of their derivatives are cheaper raw materials than, for example, fructose. When allose is produced from psicose, the yield is lower than that of the present invention, and allose needs to be separated from psicose by chromatography, which leads to higher production costs.

[0122] In addition, the step of converting A6P to allose according to the present invention is an irreversible phosphatase reaction regardless of the raw material, so allose is produced in a very high yield and at the same time effectively minimizes the cost of subsequent product separation.

[0123] In contrast to cellular production methods, the present invention involves cell-free production of allose, which has a relatively high reaction rate due to the elimination of cell membranes that often slow down the transport of substrates into the cells / products out of the cells. Also in the present invention, the final product does not contain nutrient-rich fermentation medium / cellular metabolic products.

[0124] A particular embodiment of the present invention is allose produced by the method for producing allose described herein.

[0125] Since the functionality of allose is similar to sucrose, allose produced by the method of the present invention can be added to any beverage or foodstuff to produce a desired sweetness.

[0126] Allose produced by the manufacturing method disclosed herein may be used to synergize the effect of strong sweeteners. When allose is used in combination with one or more strong sweeteners, it can improve the sensory properties of sweetened products, such as texture, flavor, and aftertaste. It is common in food and beverage formulations to use low-calorie sweeteners, such as strong sweeteners, in various foods. However, for many consumers, products sold as diet or low-calorie type products with artificial sweetening amounts are not preferred. For many years, attempts have been made to improve the taste delivery of these diet or low-calorie products by adding small amounts of carbohydrates. Not only can allose produced by the method of the present invention improve the quality of food and beverage formulations, especially in diet / low-calorie beverages, but the use of allose can act synergistically with strong sweeteners, so that allose can replace a significant amount of strong sweeteners, even when allose is added at a concentration well below its measured sweetness threshold.

[0127] Allose produced by the process disclosed herein may be used in combination with other sweeteners, such as extracts of the Stevia rebaudiana Bertoni plant, in the production of low-calorie type food products, such as ice cream.

[0128] Allose produced by the manufacturing methods disclosed herein may be used as a frosting in pre-sweetened ready-to-eat (RTE) breakfast cereals and other food products in which D-allose partially or completely replaces sucrose or other commonly used sugars.

[0129] Allose produced by the manufacturing methods disclosed herein may be used as part of the sweetener for foods and beverages in combination with sugar alcohols such as erythritol, and nutritive sweeteners having significant caloric content such as fructose, sucrose, dextrose, maltose, trehalose, rhamnose, corn syrup, and fructooligosaccharides.

[0130] Allose produced by the methods disclosed herein may be used as part of a composition to enhance plant disease control.

[0131] Mannose

[0132] One embodiment of the present invention comprises: converting F6P to mannose 6-phosphate (M6P) by catalysis with mannose 6-phosphate isomerase (M6PI); converting said M6P to mannose by catalysis with mannose 6-phosphate phosphatase (M6PP); The method for producing mannose comprises the steps of:

[0133] Examples of M6PIs include, but are not limited to, Uniprot ID A0A1M6TLY7 having the amino acid sequence set forth in SEQ ID NO: 8, H1XQS6 having the amino acid sequence set forth in SEQ ID NO: 9, G2Q982 having the amino acid sequence set forth in SEQ ID NO: 10, and F8F1Z8 having the amino acid sequence set forth in SEQ ID NO: 11. Both Uniprot IDs G2Q982 and F8F1Z8 perform M6PI reactions and share 28% amino acid sequence identity. Thus, examples of M6PIs include any analogues that have an amino acid sequence identity to any of the above Uniprot IDs of at least 25%, preferably at least 30%, more preferably at least 35%, more preferably at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, at least 91%, at least 92%, at least 93%, or at least 94%, and even most preferably at least 96, 97, 98, 99, or 100%.

[0134] M6PI suitable for use in the method of converting F6P to M6P contains two domains with an antiparallel β-stranded core similar to the cupin fold and a third domain consisting only of α-helices. M6PI has been structurally characterized in the art (Sagurthi et al. Structures of mannose-6-phosphate isomerase from Salmonella typhimurium bound to metal atoms and substrate: implications for catalytic mechanism. Acta Cryst. 2009;D65;724-732) and shares conserved residues with the thermophilic M6PI described in the present invention. In some embodiments of the present invention, the isomerase comprises: Divalent metal cations, preferably Mg 2+ or Zn 2+ and, Additionally, there are Glu and two His residues (PDB 3H1M residues 134, 99, and 255, respectively) that are hypothesized to be used for metal binding; Additionally, Asp and Lys residues (PDB 3H1M residues 270 and 132, respectively) have been hypothesized to be for acid / base catalysis; Lys, Pro, and Ala residues hypothesized for phosphate binding (PDB 3H1M residues 132, 133, and 267, respectively) M6PI preferably contains all of these conserved residues, but is not limited to containing:

[0135] Examples of M6PP include, but are not limited to, the following proteins: Uniprot ID A0A1A6DSI3, having the amino acid sequence set forth in SEQ ID NO: 12, A0A1M4UN08, having the amino acid sequence set forth in SEQ ID NO: 13, and A0A1N6FCW3, having the amino acid sequence set forth in SEQ ID NO: 14. Uniprot IDs A0A1A6DSI3 and A0A1N6FCW3 both catalyze the M6PP reaction and share 35% amino acid sequence identity. Thus, examples of M6PP include any analogues that have an amino acid sequence identity to any of the above Uniprot IDs of at least 35%, more preferably at least 40%, preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, at least 91%, at least 92%, at least 93%, or at least 94%, and even most preferably at least 96, 97, 98, 99, or 100%.

[0136] M6PP, which converts M6P to mannose, a Rossmann-like fold domain for catalysis; A C1 capping domain, A DxD signature in the first β strand of the Rossmann-like fold; and Thr or Ser at the end of the second β-strand of the Rossmann-like fold; Lys at the N-terminus of the C-terminus of the α-helix of the third β-strand of the Rossmann-like fold; The GDxxxD signature at the end of the fourth β-strand of the Rossmann-like fold It is preferred that the compound contains

[0137] The method for producing mannose of the present invention also includes a step of enzymatically converting glucose 6-phosphate (G6P) to the F6P, which is catalyzed by phosphoglucoisomerase (PGI). In another embodiment, the method for producing mannose further includes a step of converting glucose 1-phosphate (G1P) to the G6P, which is catalyzed by phosphoglucomutase (PGM). In a further embodiment, the method for producing mannose includes the conversion of the G6P to F6P to M6P, which is catalyzed by bifunctional phosphoglucose / phosphomannose isomerase (PGPMI). In yet another embodiment, the method for producing mannose also includes a step of converting sugar to G1P, which is catalyzed by at least one enzyme.

[0138] The method for producing mannose of the present invention uses PGPMI to convert G6P or F6P to M6P. Examples of PGPMI include, but are not limited to, the following proteins: Uniprot ID D7CPH7 having the amino acid sequence set forth in SEQ ID NO: 15, A0A085L170 having the amino acid sequence set forth in SEQ ID NO: 16, and M1E6Z3 having the amino acid sequence set forth in SEQ ID NO: 17. Both Uniprot IDs A0A085L170 and M1E6Z3 catalyze the PGPMI reaction and share 28% amino acid sequence identity. Thus, examples of PGPMI include any analogues that have an amino acid sequence identity to any of the above Uniprot IDs of at least 25%, preferably at least 30%, more preferably at least 35%, more preferably at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, at least 91%, at least 92%, at least 93%, or at least 94%, and even most preferably at least 96, 97, 98, 99, or 100%.

[0139] PGPMI suitable for use in the above production methods to convert G6P or F6P to M6P contains two Rossmann-like folds. PGPMI has been structurally characterized in the art (Swan et al. A Novel Phosphoglucose Isomerase (PGI) / Phosphomannose Isomerase from the Crenarchaeon Pyrobaculum aerophilum Is a Member of the PGI Superfamily. J. Biol. Chem. 2004: 279;39838-39845) and shares conserved residues with the thermophilic PGPMI described in the present invention. In some embodiments of the present invention, the isomerase is A GGS motif (PDB 1TZB residues 46–48) in which Gly residues aid in substrate binding and Ser residues bind phosphate. In addition, the SYSG-XTX-ET-hydrophobic motif (PDB 1TZB residues 87–96) binds to phosphate. Additionally, it contains an Arg residue (PDB 1TZB residue 135) that stabilizes high-energy intermediates during catalysis; In addition, the EN signature (PDB 1TZB residues 203–204) indicates that Glu is essential for active site base proton transfer. In addition, the HN signature (PDB 1TZB residues 219–220) indicates that His is important for ring-opening / ring-closing of the substrate during catalysis; Additionally, a conserved Lys residue (PDB 1TZB residue 298) that is important for ring-opening / closing of the substrate during catalysis and The functions of the above conserved residues have been verified in a separate publication (Hansen et al. Bifunctional Phosphoglucose / Phosphomannose Isomerases from the Archaea Aeropyrum pernix and Thermoplasma acidophilum Constitute a Novel Enzyme Family within the Phosphoglucose Isomerase Superfamily. J. Biol. Chem. 2004;279;2262-2272). PGPMI preferably contains all of these conserved residues.

[0140] Therefore, the method for producing mannose of the present invention includes, for example, the following steps: (i) converting sugars to glucose 1-phosphate (G1P) using one or more enzymes; (ii) converting G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii) converting G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) converting F6P to M6P via mannose 6-phosphate isomerase (M6PI, EC 5.3.1.8); (v) converting G6P to M6P via a bifunctional phosphoglucose / phosphomannose isomerase (PGPMI, EC 5.3.1.8 and 5.3.1.9); (vi) converting M6P to mannose via M6PP; An example of this process in which the sugar is starch is shown in FIG.

[0141] Typically, the ratio of enzyme units used in the disclosed production methods is 1:1:1:1:1 (αGP:PGM:PGI:M6PI:M6PP) or 1:1:1:1 (αGP:PGM:PGPMI:M6PP). These ratios may be adjusted in any number of combinations to optimize product yield. For example, a ratio of 3:1:1:1:1 can be used to maximize the concentration of phosphorylated intermediates, which will increase the activity of downstream reactions. Conversely, a ratio of 1:1:1:1:3 can be used to ensure that a supply of phosphates for αGP is maintained, which will result in more efficient phosphorolytic cleavage of α-1,4-glycosidic bonds. For example, a ratio of 3:1:1:1:3 enzymes can be used to further increase the reaction rate. Thus, the ratio of enzymes, including other optional enzymes described below, can be varied to increase the efficiency of mannose production. For example, a particular enzyme may be present in an amount that is about 2-fold, 3-fold, 4-fold, 5-fold, etc., relative to the amount of another enzyme.

[0142] One of the key advantages of this production method is that multiple process steps can be carried out in a single bioreactor or reactor, or alternatively, the steps can be carried out in multiple bioreactors or reactors arranged in series.

[0143] The phosphate ions generated by the M6P dephosphorylation step can then be recycled to the process step of converting sugars to G1P, especially when all process steps are carried out in a single bioreactor or reactor. The ability to recycle phosphate in the disclosed process allows for the use of non-stoichiometric amounts of phosphate, thereby keeping the phosphate concentration in the reaction solution low. This affects the overall pathway and overall rate of the process, but does not limit the activity of any individual enzyme, allowing the overall efficiency of the mannose production process.

[0144] For example, the phosphoric acid concentration in the reaction solution may be in the range of about 0.1 mM to about 300 mM, about 0.1 mM to about 150 mM, about 1 mM to about 50 mM, preferably about 5 mM to about 50 mM, or more preferably about 10 mM to about 50 mM. For example, the phosphoric acid concentration in the reaction solution may be about 0.1 mM, about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, or about 55 mM.

[0145] Therefore, lower phosphate concentrations reduce production costs due to lower total phosphate, which in turn reduces the cost of phosphate removal, and also prevents inhibition of M6PP by high free phosphate, reducing the possibility of phosphate contamination.

[0146] Furthermore, the methods disclosed herein can be carried out without the addition of ATP as a phosphate source, i.e., without ATP. The methods can also be carried out without the need to add NAD(P)(H), i.e., without NAD(P)(H). Another advantage is the fact that at least one step of the disclosed methods for producing mannose involves an energetically favorable reaction.

[0147] Mannose can also be produced from fructose. This production method can be carried out, for example, by producing F6P from fructose and polyphosphate by catalysis with polyphosphate fructokinase (PPFK); converting F6P to M6P by catalysis with M6PI; Conversion of M6P to mannose by catalysis with M6PP; The fructose can be produced, for example, by enzymatic conversion of sucrose.

[0148] Mannose can also be produced from sucrose. The production process involves the following enzymatic steps: generating G1P from sucrose and free phosphate by catalyzing with sucrose phosphorylase (SP); PGM-catalyzed conversion of G1P to G6P; converting G6P to F6P by catalysis with PGI; converting F6P to M6P by catalysis with M6PI; Conversion of M6P to mannose by catalysis with M6PP; In the above steps, the conversion of G6P to F6P to M6P may alternatively be catalyzed by PGPMI.

[0149] The phosphate ions produced during the conversion of M6P to mannose can then be recycled to the step of converting sucrose to G1P. Furthermore, the mannose yield can be increased by using PPFK and polyphosphate to generate F6P from fructose produced by the phosphorolytic cleavage of sucrose by SP.

[0150] In some embodiments, the method for producing mannose comprises the following steps: producing glucose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis; converting glucose to G6P by catalyzing at least one enzyme; producing fructose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis; converting fructose to F6P by catalysis with at least one enzyme; Examples of such polysaccharides and oligosaccharides are listed above.

[0151] In another embodiment, G6P is produced from glucose and sodium polyphosphate by polyphosphate glucokinase.

[0152] The present disclosure provides methods for the conversion of sugars such as polysaccharides and oligosaccharides in starch, cellulose, sucrose, and their derived products, to mannose. In certain embodiments, an artificial (non-natural), ATP-free enzymatic pathway is provided for the conversion of starch, cellulose, sucrose, and their derived products to mannose using a cell-free enzyme cocktail.

[0153] As shown above, several enzymes can be used to hydrolyze starch and increase the G1P yield. These enzymes include isoamylase, pullulanase, and α-amylase. Corn starch contains many branches that prevent the action of αGP. Isoamylase can be used to debranch starch and produce linear amylodextrins. Pre-treated starch with isoamylase can result in a higher concentration of F6P in the final product. Isoamylase and pullulanase cleave α-1,6-glycosidic bonds, which allows starch to be more completely degraded by α-glucan phosphorylase. α-amylase cleaves α-1,4-glycosidic bonds, so α-amylase can be used to break down starch into fragments for more rapid conversion to mannose and increased solubility.

[0154] Mannose yields can be increased by using maltose phosphorylase (MP) to phosphorolytically cleave the degradation product maltose to G1P and glucose, or by using 4-glucan transferase (4GT) to recycle the degradation products glucose, maltose, and maltotriose to longer chain maltooligosaccharides that can be phosphorolytically cleaved by αGP to produce G1P.

[0155] In a particular embodiment, cellulose and its derived products can be converted to mannose through a series of steps. The production method comprises the following steps: producing G1P from cellodextrin and cellobiose and free phosphate by catalyzing with cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively; PGM-catalyzed conversion of G1P to G6P; converting G6P to F6P by catalysis with PGI; converting F6P to M6P by catalysis with M6PI; Conversion of M6P to mannose by catalysis with M6PP; Alternatively, in the above-mentioned pathway, the conversion of G6P to F6P to M6P may be catalyzed by PGPMI. In this preparation method, phosphate ions can be recycled through the above step of converting cellodextrin and cellobiose to G1P.

[0156] In some embodiments, polyphosphate and polyphosphate glucokinase (PPGK) can be added to the process to increase the yield of mannose by phosphorylating the breakdown product glucose to G6P.

[0157] In another embodiment, mannose can be produced from glucose. The method comprises: Producing G6P from glucose and polyphosphate by catalysis with polyphosphate glucokinase (PPGK); converting G6P to F6P by catalysis with PGI; converting F6P to M6P by catalysis with M6PI; converting M6P to mannose by catalysis with M6PP; Alternatively, the conversion of G6P to F6P to M6P may be catalyzed by PGPMI.

[0158] The production method of the present invention uses low-cost starting materials and reduces the production cost by eliminating the costs associated with the separation of raw materials and products.Starch, cellulose, sucrose, and some of their derivatives are cheaper raw materials than, for example, fructose.When mannose is produced from fructose, the yield is lower than that of the present invention, and mannose needs to be separated from fructose by chromatography, which leads to higher production costs.

[0159] Furthermore, the step of converting M6P to mannose according to the present invention is an irreversible phosphatase reaction regardless of the raw material, therefore, mannose is produced in very high yields while effectively minimizing the subsequent product separation costs.

[0160] In contrast to cellular production methods, the present invention involves cell-free production of mannose, which has a relatively high reaction rate due to the elimination of cell membranes that often slow down the transport of substrates into the cell / products out of the cell, and the present invention also does not include nutrient-rich fermentation media / cellular metabolic products in the final product.

[0161] A particular embodiment of the present invention is mannose produced by the methods for producing mannose described herein.

[0162] Mannose produced by the methods described herein can be used in a variety of applications in the pharmaceutical, cosmetic, beverage, food, dairy, confectionery, and livestock industries, as discussed above.

[0163] Furthermore, the mannose produced by the manufacturing method disclosed herein can be converted to mannitol by hydrogenation. Catalytic hydrogenation of mannose occurs in stoichiometric yield to give mannitol. U.S. Pat. No. 5,466,795. Mannitol is widely used in the manufacture of sugar-free chewing gum, confectionery, and pharmaceutical excipients. However, the production of high-purity mannose is very difficult and expensive. Ibid. Therefore, the mannose produced by the above manufacturing method can be converted to mannitol by catalytic hydrogenation.

[0164] Galactose

[0165] One embodiment of the present invention comprises: converting fructose 6-phosphate (F6P) to tagatose 6-phosphate (T6P) by catalyzing with fructose 6-phosphate 4-epimerase (F6PE); converting T6P to galactose 6-phosphate (Gal6P) by catalysis with galactose 6-phosphate isomerase (Gal6PI); converting the Gal6P to galactose by catalysis with galactose 6-phosphate phosphatase (Gal6PP); The present invention relates to a method for producing galactose, comprising the steps of:

[0166] Examples of F6PE include, but are not limited to, the following proteins: Uniprot IDs E8N0N6, E4SEH3, I0I507, H1XRG1, and B5YBD7. Uniprot IDs E8N0N6 and I0I507 both catalyze the F6PE reaction and share 27% amino acid sequence identity. Thus, examples of F6PE include any analogue that has an amino acid sequence identity to any of the above Uniprot IDs of at least 25%, preferably at least 30%, more preferably at least 35%, more preferably at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, at least 91%, at least 92%, at least 93%, or at least 94%, and even most preferably at least 96, 97, 98, 99, or 100%.

[0167] Gal6PI exists as a multimer of two subunits, LacA and LacB. Examples of Gal6PI include, but are not limited to, the following protein (LacA / LacB) subunit pair: Uniprot ID P23494 / P23495, having the amino acid sequences shown in SEQ ID NO:18 / SEQ ID NO:19. Examples of Gal6PI include any analogues that have an amino acid sequence identity of at least 25%, preferably at least 30%, more preferably at least 35%, more preferably at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, at least 91%, at least 92%, at least 93%, or at least 94%, and even most preferably at least 96, 97, 98, 99, or 100% to any of the above Uniprot IDs for subunit LacA, and Also included are any analogs that have an amino acid sequence identity to any of the IDs of at least 25%, at least 30%, more preferably at least 35%, more preferably at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, at least 91%, at least 92%, at least 93%, or at least 94%, and even most preferably at least 96, 97, 98, 99, or 100%.

[0168] Gal6PI suitable for use in the method of converting T6P to Gal6P contains a heterodimer ("A" and "B") consisting of subunits with a Rossmann-like αβα sandwich fold. Conserved residues have been discussed in the art (Jung et al. Crystal Structure and Substrate Specificity of D-Galactose-6-Phosphate Isomerase Complexed with Substrates. PLOS ONE. 2013;8;e72902). In some embodiments of the invention, the isomerase heterodimer comprises: Arg130 and Arg134 in "A" and His9 and Arg39 in "B" for binding to the phosphate group of the substrate; In addition, His96 in "A" for ring-opening of the substrate, In addition, Asn97 in "A" to stabilize the high-energy intermediate, In addition, Cys65 and Thr67 of "B" are involved in proton transfer. It contains, but is not limited to,

[0169] Examples of Gal6PP include, but are not limited to, Uniprot ID Q8A2F3, which has the amino acid sequence set forth in SEQ ID NO: 20. Examples of Gal6PP also include any analogs that have at least 25%, at least 30%, more preferably at least 35%, more preferably at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, at least 91%, at least 92%, at least 93%, or at least 94%, and even most preferably at least 96, 97, 98, 99, or 100% amino acid sequence identity to any of the above Uniprot IDs.

[0170] Gal6PP converts Gal6P to galactose. a Rossmann-like fold domain for catalysis; A C2 capping domain, A DxD signature in the first β strand of the Rossmann-like fold; and Thr or Ser at the end of the second β-strand of the Rossmann-like fold; The GDxxxD signature at the end of the fourth β-strand of the Rossmann-like fold It is preferred that the compound contains

[0171] The method for producing galactose of the present invention also includes a step of enzymatically converting glucose 6-phosphate (G6P) to F6P, which is catalyzed by phosphoglucoisomerase (PGI). In another embodiment, the method for producing galactose further includes a step of converting glucose 1-phosphate (G1P) to said G6P, which is catalyzed by phosphoglucomutase (PGM). In yet another embodiment, the method for producing galactose also includes a step of converting sugar to said G1P, which is catalyzed by at least one enzyme.

[0172] Thus, the method for producing galactose of the present invention comprises, for example, the following steps: (i) converting sugars to glucose 1-phosphate (G1P) using one or more enzymes; (ii) converting G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii) converting G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) converting F6P to T6P via F6PE; (v) converting T6P to Gal6P via Gal6PI (EC 5.3.1.26); (vi) converting Gal6P to galactose via Gal6PP; An example of this process in which the sugar is starch is shown in FIG.

[0173] Typically, the ratio of enzyme units used in the disclosed production methods is 1:1:1:1:1:1 (αGP:PGM:PGI:F6PE:Gal6PI:Gal6PP). These ratios may be adjusted in any number of combinations to optimize product yield. For example, a ratio of 3:1:1:1:1:1 can be used to maximize the concentration of phosphorylated intermediates, which will increase the activity of downstream reactions. Conversely, a ratio of 1:1:1:1:1:1:3 can be used to ensure that phosphate is maintained for αGP, which will result in more efficient phosphorolytic cleavage of α-1,4-glycosidic bonds. For example, a ratio of 3:1:1:1:1:3 enzymes can be used to further increase the reaction rate. Thus, the ratio of enzymes, including other optional enzymes described below, can be varied to increase the efficiency of galactose production. For example, a particular enzyme may be present in an amount of about 2-fold, 3-fold, 4-fold, 5-fold, etc., relative to the amount of other enzymes.

[0174] One of the key advantages of this production method is that multiple process steps can be carried out in a single bioreactor or reactor, or alternatively, the steps can be carried out in multiple bioreactors or reactors arranged in series.

[0175] The phosphate ions generated by dephosphorylation of Gal6P can then be recycled to the process step that converts sugar to G1P, especially when all process steps are carried out in a single bioreactor or reactor. The ability to recycle phosphate in the disclosed process allows for the use of non-stoichiometric amounts of phosphate, thereby keeping the phosphate concentration in the reaction low. This affects the overall pathway and overall rate of the process, but does not limit the activity of any individual enzyme, allowing the overall efficiency of the galactose production process.

[0176] For example, the phosphoric acid concentration in the reaction solution may be in the range of about 0.1 mM to about 300 mM, about 0.1 mM to about 150 mM, about 1 mM to about 50 mM, preferably about 5 mM to about 50 mM, or more preferably about 10 mM to about 50 mM. For example, the phosphoric acid concentration in the reaction solution may be about 0.1 mM, about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, or about 55 mM.

[0177] Therefore, lower phosphate concentrations reduce production costs due to lower total phosphate, which in turn reduces the cost of phosphate removal, and also prevent inhibition of Gal6PP by high concentrations of free phosphate, reducing the possibility of phosphate contamination.

[0178] Furthermore, the methods disclosed herein can be carried out without the addition of ATP as a phosphate source, i.e., without ATP. The methods can also be carried out without the need to add NAD(P)(H), i.e., without NAD(P)(H). Another advantage is the fact that at least one step of the disclosed methods for producing galactose involves an energetically favorable chemical reaction.

[0179] Galactose can also be produced from fructose. For example, this production method includes the following steps: producing F6P from fructose and polyphosphate by catalysis with polyphosphate fructokinase (PPFK); converting F6P to T6P by catalyzing with F6PE; converting T6P to Gal6P by catalysis with Gal6PI; Gal6PP-catalyzed conversion of Gal6P to galactose The fructose can be produced, for example, by the enzymatic conversion of sucrose.

[0180] Galactose can be produced from sucrose by the following enzymatic steps: generating G1P from sucrose and free phosphate by catalyzing with sucrose phosphorylase (SP); PGM-catalyzed conversion of G1P to G6P; converting G6P to F6P by catalysis with PGI; converting F6P to T6P by catalysis with F6PE; converting T6P to Gal6P by catalysis with Gal6PI; Gal6PP-catalyzed conversion of Gal6P to galactose The present invention provides an in vitro synthesis route comprising:

[0181] The phosphate ions produced during the conversion of Gal6P to galactose can then be recycled to the above step of converting sucrose to G1P. Furthermore, the galactose yield can be increased by using PPFK and polyphosphate to generate F6P from fructose produced by phosphorolytic cleavage of sucrose by SP.

[0182] In some embodiments, the method for producing galactose comprises the following steps: producing glucose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis; converting glucose to G6P by catalyzing at least one enzyme; producing fructose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis; converting fructose to F6P by catalysis with at least one enzyme; Examples of such polysaccharides and oligosaccharides are listed above.

[0183] In another embodiment, G6P is produced from glucose and sodium polyphosphate by polyphosphate glucokinase.

[0184] The present disclosure provides methods for the conversion of sugars such as polysaccharides and oligosaccharides in starch, cellulose, sucrose, and their derived products to galactose. In certain embodiments, an artificial (non-natural), ATP-free enzymatic pathway is provided for the conversion of starch, cellulose, sucrose, and their derived products to galactose using a cell-free enzyme cocktail.

[0185] As shown above, several enzymes can be used to hydrolyze starch and increase the G1P yield. These enzymes include isoamylase, pullulanase, and α-amylase. Corn starch contains many branches that prevent the action of αGP. Isoamylase can be used to debranch starch and produce linear amylodextrins. Pre-treated starch with isoamylase can result in a higher concentration of F6P in the final product. Isoamylase and pullulanase cleave α-1,6-glycosidic bonds, which allows starch to be more completely degraded by α-glucan phosphorylase. α-amylase cleaves α-1,4-glycosidic bonds, so α-amylase can be used to break starch into fragments for more rapid conversion to galactose and increased solubility.

[0186] Maltose phosphorylase (MP) can be used to increase galactose yield by phosphorolytic cleavage of the degradation product maltose to G1P and glucose, or 4-glucan transferase (4GT) can be used to increase galactose yield by recycling the degradation products glucose, maltose, and maltotriose to longer chain maltooligosaccharides that can be phosphorolytically cleaved by αGP to produce G1P.

[0187] In a particular embodiment, cellulose and its derived products can be converted to galactose through a series of steps. The process comprises the following steps: producing G1P from cellodextrin and cellobiose and free phosphate by catalyzing with cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively; PGM-catalyzed conversion of G1P to G6P; converting G6P to F6P by catalysis with PGI; converting F6P to T6P by catalysis with F6PE; converting T6P to Gal6P by catalysis with Gal6PI; Gal6PP-catalyzed conversion of Gal6P to galactose In this method, phosphate ions can be recycled through the above steps of converting cellodextrin and cellobiose into G1P.

[0188] In some embodiments, polyphosphate and polyphosphate glucokinase (PPGK) can be added to the process to increase the yield of galactose by phosphorylating the degradation product glucose to G6P.

[0189] In another embodiment, galactose can be produced from glucose. The method comprises: Producing G6P from glucose and polyphosphate by catalysis with polyphosphate glucokinase (PPGK); converting G6P to F6P by catalysis with PGI; converting F6P to T6P by catalysis with F6PE; converting T6P to Gal6P by catalysis with Gal6PI; converting Gal6P to galactose by catalysis with Gal6PP; Includes.

[0190] The present invention's manufacturing method reduces production costs by using low-cost starting materials and eliminating costs associated with the separation of raw materials and products. Starch, cellulose, sucrose, and some of their derivatives are cheaper raw materials than, for example, lactose. When galactose is produced from biomass or lactose, the yield is lower than in the present invention, and galactose needs to be separated from other sugars by chromatography, which leads to higher production costs. Furthermore, our manufacturing method does not involve animals.

[0191] The step of converting Gal6P to galactose according to the present invention is an irreversible phosphatase reaction regardless of the starting material, thus producing galactose in very high yields while effectively minimizing the cost of subsequent product isolation.

[0192] In contrast to cellular production methods, the present invention involves cell-free production of galactose, which has a relatively high reaction rate due to the elimination of cell membranes that often slow down the transport of substrates into the cell / products out of the cell, and the present invention also does not include nutrient-rich fermentation media / cellular metabolic products in the final product.

[0193] A particular embodiment of the present invention is galactose produced by the methods for producing galactose described herein.

[0194] Fructose

[0195] One embodiment of the present invention is a method for producing fructose comprising converting fructose 6-phosphate (F6P) to fructose by catalysis with fructose 6-phosphate phosphatase (F6PP).

[0196] A non-limiting example of an F6PP is Uniprot ID B8CWV3 having the amino acid sequence shown in SEQ ID NO: 21. Examples of F6PP also include any analogs having an amino acid sequence identity to the above Uniprot IDs of at least 25%, at least 30%, more preferably at least 35%, more preferably at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, at least 91%, at least 92%, at least 93%, or at least 94%, and even most preferably at least 96, 97, 98, 99, or 100%.

[0197] F6PP, which converts F6P to fructose, a Rossmann-like fold domain for catalysis; A C1 capping domain, A DxD signature in the first β strand of the Rossmann-like fold; and Thr or Ser at the end of the second β-strand of the Rossmann-like fold; Lys at the N-terminus of the C-terminus of the α-helix of the third β-strand of the Rossmann-like fold; The ED signature at the end of the fourth β-strand of the Rossmann-like fold It is preferred that the compound contains

[0198] The method for producing fructose of the present invention also includes a step of enzymatically converting glucose 6-phosphate (G6P) to F6P, which is catalyzed by phosphoglucoisomerase (PGI). In another embodiment, the method for producing fructose further includes a step of converting glucose 1-phosphate (G1P) to said G6P, which is catalyzed by phosphoglucomutase (PGM). In yet another embodiment, the method for producing fructose also includes a step of converting sugar to said G1P, which is catalyzed by at least one enzyme.

[0199] Thus, the method for producing fructose of the present invention may, for example, comprise the following steps: (i) converting sugars to glucose 1-phosphate (G1P) using one or more enzymes; (ii) converting G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii) converting G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) converting F6P to fructose using F6PP; may also include

[0200] Typically, the ratio of enzyme units used in the disclosed production methods is 1:1:1:1 (αGP:PGM:PGI:F6PP). These ratios may be adjusted in any number of combinations to optimize product yield. For example, a ratio of 3:1:1:1 may be used to maximize the concentration of phosphorylated intermediates, which will increase the activity of downstream reactions. Conversely, a ratio of 1:1:1:3 may be used to ensure that a supply of phosphates for αGP is maintained, which will result in more efficient phosphorolytic cleavage of α-1,4-glycosidic bonds. For example, a ratio of 3:1:1:3 enzymes may be used to further increase the reaction rate. Thus, the ratio of enzymes, including other optional enzymes described below, may be varied to increase the efficiency of fructose production. For example, a particular enzyme may be present in an amount of about 2-fold, 3-fold, 4-fold, 5-fold, etc., relative to the amount of other enzymes.

[0201] One of the key advantages of this production method is that multiple process steps can be carried out in a single bioreactor or reactor, or alternatively, the steps can be carried out in multiple bioreactors or reactors arranged in series.

[0202] Phosphate ions produced by dephosphorylation of F6P can then be recycled to the process step of converting sugar to G1P, especially when all process steps are carried out in a single bioreactor or reactor. The ability to recycle phosphate in the disclosed process allows for the use of non-stoichiometric amounts of phosphate, which keeps the phosphate concentration in the reaction solution low. This affects the overall pathway and overall rate of the process, but does not limit the activity of individual enzymes, making the overall fructose production process more efficient.

[0203] For example, the phosphoric acid concentration in the reaction solution may be in the range of about 0.1 mM to about 300 mM, about 0.1 mM to about 150 mM, about 1 mM to about 50 mM, preferably about 5 mM to about 50 mM, or more preferably about 10 mM to about 50 mM. For example, the phosphoric acid concentration in the reaction solution may be about 0.1 mM, about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, or about 55 mM.

[0204] Therefore, lower phosphate concentrations reduce production costs due to lower total phosphate, which in turn reduces the cost of phosphate removal, and also prevents inhibition of F6PP by high free phosphate, reducing the possibility of phosphate contamination.

[0205] Furthermore, the disclosed method can be carried out without the addition of ATP as a phosphate source, i.e., without ATP. The disclosed method can also be carried out without the need to add NAD(P)(H), i.e., without NAD(P)(H). Another advantage is the fact that at least one step of the disclosed method for producing fructose involves an energetically favorable chemical reaction.

[0206] Fructose can also be produced from sucrose via the F6P intermediate. This production process involves the following enzymatic steps: generating G1P from sucrose and free phosphate by catalyzing with sucrose phosphorylase (SP); PGM-catalyzed conversion of G1P to G6P; converting G6P to F6P by catalysis with PGI; converting F6P to fructose by catalysis with F6PP; An exemplary enzymatic pathway is shown in Figure 5.

[0207] The phosphate ions produced during the conversion of F6P to fructose can then be recycled to the step of converting sucrose to G1P.Furthermore, the fructose yield can be increased by using PPFK and polyphosphate to generate F6P from fructose produced by the phosphorolytic cleavage of sucrose by SP.

[0208] In some embodiments, a method for producing fructose comprises the following steps: producing glucose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis; converting glucose to G6P by catalyzing at least one enzyme; producing fructose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis; converting fructose to F6P by catalysis with at least one enzyme; Examples of such polysaccharides and oligosaccharides are listed above.

[0209] In another embodiment, G6P is produced from glucose and sodium polyphosphate by polyphosphate glucokinase.

[0210] The present disclosure provides a method for converting sugars such as polysaccharides and oligosaccharides in starch, cellulose, sucrose, and their derived products to fructose. In certain embodiments, an artificial (non-natural) ATP-free enzymatic pathway is provided for converting starch, cellulose, sucrose, and their derived products to fructose using a cell-free enzyme cocktail.

[0211] As shown above, several enzymes can be used to hydrolyze starch and increase the G1P yield. These enzymes include isoamylase, pullulanase, and α-amylase. Corn starch contains many branches that prevent the action of αGP. Isoamylase can be used to debranch starch and produce linear amylodextrin. Pre-treated starch with isoamylase can result in a higher concentration of F6P in the final product. Isoamylase and pullulanase cleave α-1,6-glycosidic bonds, which allows starch to be more completely degraded by α-glucan phosphorylase. α-amylase cleaves α-1,4-glycosidic bonds, so α-amylase can be used to break starch into fragments for more rapid conversion to fructose and increased solubility.

[0212] Maltose phosphorylase (MP) can be used to increase fructose yield by phosphorolytic cleavage of the degradation product maltose to G1P and glucose, or 4-glucan transferase (4GT) can be used to increase fructose yield by recycling the degradation products glucose, maltose, and maltotriose to longer chain maltooligosaccharides that can be phosphorolytically cleaved by αGP to produce G1P.

[0213] In certain embodiments, cellulose and its derived products can be converted to fructose through a series of steps. The process includes the following steps: producing G1P from cellodextrin and cellobiose and free phosphate by catalyzing with cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively; PGM-catalyzed conversion of G1P to G6P; converting G6P to F6P by catalysis with PGI; converting F6P to fructose by catalysis with F6PP; In this method, phosphate ions can be recycled through the above steps of converting cellodextrin and cellobiose into G1P.

[0214] In some embodiments, polyphosphate and polyphosphate glucokinase (PPGK) can be added to the process to increase the yield of fructose by phosphorylating the breakdown product glucose to G6P.

[0215] In another embodiment, fructose can be produced from glucose. The method comprises: Producing G6P from glucose and polyphosphate by catalysis with polyphosphate glucokinase (PPGK); converting G6P to F6P by catalysis with PGI; converting F6P to fructose by catalysis with F6PP; Includes.

[0216] The present invention's manufacturing method uses low-cost starting materials and reduces manufacturing costs by eliminating the costs associated with the separation of raw materials and products.Starch, cellulose, sucrose, and some of their derivatives are cheaper raw materials than, for example, lactose.When fructose is produced from biomass or lactose, the yield is lower than in the present invention, and fructose needs to be separated from other sugars by chromatography, which leads to higher manufacturing costs.In addition, the present inventors' manufacturing method does not involve animals.

[0217] The step of converting F6P to fructose according to the present invention is an irreversible phosphatase reaction regardless of the raw material, therefore, fructose is produced in a very high yield while effectively minimizing the subsequent product separation costs.

[0218] In contrast to cellular production methods, the present invention involves cell-free production of fructose, which has a relatively high reaction rate due to the elimination of cell membranes that often slow down the transport of substrates into the cell / products out of the cell, and the present invention also does not include nutrient-rich fermentation medium / cellular metabolic products in the final product.

[0219] A particular embodiment of the present invention is fructose produced by the methods for producing fructose described herein.

[0220] Altrose

[0221] One embodiment of the present invention comprises: converting fructose 6-phosphate (F6P) to psicose 6-phosphate (P6P) by catalyzing psicose 6-phosphate 3-epimerase (P6PE); converting P6P to altrose 6-phosphate (Alt6P) by catalysis with altrose 6-phosphate isomerase (Alt6PI); converting the generated Alt6P to altrose by catalysis with altrose 6-phosphate phosphatase; The method for producing altrose includes the steps of:

[0222] The method for producing altrose of the present invention also includes a step of enzymatically converting glucose 6-phosphate (G6P) to the F6P, which is catalyzed by phosphoglucoisomerase (PGI). In another embodiment, the method for producing altrose further includes a step of converting glucose 1-phosphate (G1P) to the G6P, which is catalyzed by phosphoglucomutase (PGM). In yet another embodiment, the method for producing altrose also includes a step of converting sugar to the G1P, which is catalyzed by at least one enzyme.

[0223] Therefore, the method for producing altrose of the present invention includes, for example, the following steps: (i) converting sugars to glucose 1-phosphate (G1P) using one or more enzymes; (ii) converting G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii) converting G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) converting F6P to P6P via P6PE; (v) converting P6P to Alt6P via Alt6PI; (vi) converting Alt6P to altrose via Alt6PP; An example of the enzymatic process where the sugar is starch is shown in FIG.

[0224] Typically, the ratio of enzyme units used in the disclosed production methods is 1:1:1:1:1:1 (αGP:PGM:PGI:P6PE:Alt6PI:Alt6PP). These ratios may be adjusted in any number of combinations to optimize product yield. For example, a ratio of 3:1:1:1:1:1 can be used to maximize the concentration of phosphorylated intermediates, which will increase the activity of downstream reactions. Conversely, a ratio of 1:1:1:1:1:1:3 can be used to ensure that phosphate is maintained for αGP, which will result in more efficient phosphorolytic cleavage of α-1,4-glycosidic bonds. For example, a ratio of 3:1:1:1:1:3 enzymes can be used to further increase the reaction rate. Thus, the ratio of enzymes, including other optional enzymes described below, can be varied to increase the efficiency of altose production. For example, a particular enzyme may be present in an amount of about 2-fold, 3-fold, 4-fold, 5-fold, etc., relative to the amount of other enzymes.

[0225] Phosphate ions produced by dephosphorylation of Alt6P can then be recycled to the process step that converts sugar to G1P, especially when all process steps are carried out in a single bioreactor or reactor. The ability to recycle phosphate in the disclosed production method allows the use of non-stoichiometric amounts of phosphate, which keeps the phosphate concentration in the reaction solution low. This affects the overall pathway and overall rate of the production method, but does not limit the activity of individual enzymes, making the production method of altose more efficient overall.

[0226] For example, the phosphoric acid concentration in the reaction solution may be in the range of about 0.1 mM to about 300 mM, about 0.1 mM to about 150 mM, about 1 mM to about 50 mM, preferably about 5 mM to about 50 mM, or more preferably about 10 mM to about 50 mM. For example, the phosphoric acid concentration in the reaction solution may be about 0.1 mM, about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, or about 55 mM.

[0227] Therefore, lower phosphate concentrations reduce production costs due to lower total phosphate, which in turn reduces the cost of phosphate removal, and also prevents inhibition of Alt6PP by high concentrations of free phosphate, reducing the possibility of phosphate contamination.

[0228] Furthermore, the method disclosed herein can be carried out without the addition of ATP as a phosphate source, i.e., without ATP. The method can also be carried out without the need to add NAD(P)(H), i.e., without NAD(P)(H). Another advantage is the fact that at least one step of the disclosed method for producing altrose includes an energetically favorable reaction.

[0229] Altrose can also be produced from fructose. For example, this production method includes: producing F6P from fructose and polyphosphate by catalysis with polyphosphate fructokinase (PPFK); converting F6P to P6P by catalysis with P6PE; converting P6P to Alt6P by catalysis with Alt6PI; Conversion of Alt6P to altrose by catalysis with Alt6PP; The fructose can be produced, for example, by the enzymatic conversion of sucrose.

[0230] Altrose can also be produced from sucrose. The production process involves the following enzymatic steps: generating G1P from sucrose and free phosphate by catalyzing with sucrose phosphorylase (SP); PGM-catalyzed conversion of G1P to G6P; converting G6P to F6P by catalysis with PGI; converting F6P to P6P by catalysis with F6PE; converting P6P to Alt6P by catalysis with Alt6PI; Conversion of Alt6P to altrose by catalysis with Alt6PP; The present invention provides an in vitro synthesis route comprising:

[0231] The phosphate ions produced during the conversion of Alt6P to altrose can then be recycled to the step of converting sucrose to G1P. Furthermore, altrose yield can be increased by using PPFK and polyphosphate to generate F6P from fructose produced by phosphorolytic cleavage of sucrose by SP.

[0232] In a particular embodiment, the method for producing altrose comprises the following steps: producing glucose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis; converting glucose to G6P by catalyzing at least one enzyme; producing fructose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis; converting fructose to F6P by catalysis with at least one enzyme; Examples of such polysaccharides and oligosaccharides are listed above.

[0233] In another embodiment, G6P is produced from glucose and sodium polyphosphate by polyphosphate glucokinase.

[0234] Several enzymes can be used to hydrolyze starch and increase the G1P yield. These enzymes include isoamylase, pullulanase, and α-amylase. Corn starch contains many branches that prevent the action of αGP. Isoamylase can be used to debranch starch and produce linear amylodextrins. Pre-treated starch with isoamylase can result in higher F6P concentrations in the final product. Isoamylase and pullulanase cleave α-1,6-glycosidic bonds, which allows starch to be more completely degraded by α-glucan phosphorylase. α-amylase cleaves α-1,4-glycosidic bonds, so α-amylase can be used to break starch into fragments for more rapid conversion to altrose and increased solubility.

[0235] Maltose phosphorylase (MP) can be used to increase the altrose yield by phosphorolytic cleavage of the degradation product maltose to G1P and glucose, or 4-glucan transferase (4GT) can be used to recycle the degradation products glucose, maltose, and maltotriose to longer chain maltooligosaccharides that can be phosphorolytically cleaved by αGP to produce G1P.

[0236] In a particular embodiment, cellulose and its derived products can be converted to altrose through a series of steps. The process comprises the following steps: producing G1P from cellodextrin and cellobiose and free phosphate by catalyzing with cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively; PGM-catalyzed conversion of G1P to G6P; converting G6P to F6P by catalysis with PGI; converting F6P to P6P by catalysis with P6PE; converting P6P to Alt6P by catalysis with Alt6PI; Conversion of Alt6P to altrose by catalysis with Alt6PP; In this method, phosphate ions can be recycled through the above steps of converting cellodextrin and cellobiose into G1P.

[0237] Several enzymes may be used to hydrolyze solid cellulose into water-soluble cellodextrins and cellobiose, including endoglucanases and cellobiohydrolases, but not including β-glucosidase (cellobiase).

[0238] In some embodiments, polyphosphate and polyphosphate glucokinase (PPGK) can be added to the process to increase the yield of altrose by phosphorylating the degradation product glucose to G6P.

[0239] Altrose may be produced from glucose. The production method comprises: Producing G6P from glucose and polyphosphate by catalysis with polyphosphate glucokinase (PPGK); converting G6P to F6P by catalysis with PGI; converting F6P to P6P by catalysis with P6PE; converting P6P to Alt6P by catalysis with A6PI; converting Alt6P to altrose by catalysis with Alt6PP; Includes.

[0240] The method for producing altrose of the present invention uses low-cost starting materials and reduces production costs by eliminating costs associated with the separation of raw materials and products. Starch, cellulose, sucrose, and some of their derivatives are cheaper raw materials than, for example, fructose. When altrose is produced from psicose, the yield is lower than that of the present invention, and altrose needs to be separated from psicose by chromatography, which leads to higher production costs.

[0241] In addition, the step of converting Alt6P to altrose according to the present invention is an irreversible phosphatase reaction regardless of the raw material, therefore altrose is produced in a very high yield and at the same time effectively minimizes the cost of subsequent product separation.

[0242] In contrast to cellular production methods, the present invention involves cell-free production of altrose, which has a relatively high reaction rate due to the elimination of cell membranes that often slow down the transport of substrates into cells / products out of cells.The present invention also does not include nutrient-rich fermentation medium / cellular metabolic products in the final product.

[0243] A particular embodiment of the present invention is altrose produced by the methods for producing altrose described herein.

[0244] Talos

[0245] One embodiment of the present invention comprises: converting fructose 6-phosphate (F6P) to tagatose 6-phosphate (T6P) by catalyzing with fructose 6-phosphate 4-epimerase (F6PE); converting T6P to talose 6-phosphate (Tal6P) by catalysis with talose 6-phosphate isomerase (Tal6PI); converting the produced Tal6P to talose by catalysis with talose 6-phosphate phosphatase (Tal6PP); A method for producing talose comprising the steps of:

[0246] Examples of F6PE include, but are not limited to, the following proteins: Uniprot IDs E8N0N6, E4SEH3, I0I507, H1XRG1, and B5YBD7. Uniprot IDs E8N0N6 and I0I507 both catalyze the F6PE reaction and share 27% amino acid sequence identity. Thus, examples of F6PE also include any analogs that have at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity to any of the above Uniprot IDs.

[0247] The method for producing talose of the present invention also includes a step of enzymatically converting glucose 6-phosphate (G6P) to the F6P, which is catalyzed by phosphoglucoisomerase (PGI). In another embodiment, the method for producing talose further includes a step of converting glucose 1-phosphate (G1P) to the G6P, which is catalyzed by phosphoglucomutase (PGM). In yet another embodiment, the method for producing talose also includes a step of converting sugars to the G1P, which is catalyzed by at least one enzyme.

[0248] Thus, the method for producing talose of the present invention may, for example, comprise the following steps: (i) converting sugars to glucose 1-phosphate (G1P) using one or more enzymes; (ii) converting G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii) converting G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) converting F6P to T6P via P6PE; (v) converting T6P to Tal6P via Tal6PI (EC 5.3.1.26); (vi) converting Tal6P to talose via Tal6PP; An example of the process where the sugar is starch is shown in FIG.

[0249] Typically, the ratio of enzyme units used in the disclosed production methods is 1:1:1:1:1:1 (αGP:PGM:PGI:F6PE:Tal6PI:Tal6PP). These ratios may be adjusted in any number of combinations to optimize product yield. For example, a ratio of 3:1:1:1:1:1:1 may be used to maximize the concentration of phosphorylated intermediates, which will increase the activity of downstream reactions. Conversely, a ratio of 1:1:1:1:1:1:3 may be used to ensure that a supply of phosphates for αGP is maintained, which will result in more efficient phosphorolytic cleavage of α-1,4-glycosidic bonds. For example, a ratio of 3:1:1:1:1:3 enzymes may be used to further increase the reaction rate. Thus, the ratio of enzymes, including other optional enzymes described below, may be varied to increase the efficiency of talose production. For example, a particular enzyme may be present in an amount of about 2-fold, 3-fold, 4-fold, 5-fold, etc., relative to the amount of other enzymes.

[0250] One of the key advantages of this production method is that multiple process steps can be carried out in a single bioreactor or reactor, or alternatively, the steps can be carried out in multiple bioreactors or reactors arranged in series.

[0251] The phosphate ions generated by the dephosphorylation of Tal6P can then be recycled to the process step that converts sugars to G1P, especially when all process steps are carried out in a single bioreactor or reactor. The ability to recycle phosphate in the disclosed production method allows the use of non-stoichiometric amounts of phosphate, which keeps the phosphate concentration in the reaction solution low. This affects the overall pathway and overall rate of the production method, but does not limit the activity of individual enzymes, allowing the overall efficiency of the production method of talose.

[0252] For example, the phosphoric acid concentration in the reaction solution may be in the range of about 0.1 mM to about 300 mM, about 0.1 mM to about 150 mM, about 1 mM to about 50 mM, preferably about 5 mM to about 50 mM, or more preferably about 10 mM to about 50 mM. For example, the phosphoric acid concentration in the reaction solution may be about 0.1 mM, about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, or about 55 mM.

[0253] Therefore, lower phosphate concentrations reduce production costs due to lower total phosphate, which in turn reduces the cost of phosphate removal, and also prevent inhibition of Tal6PP by high concentrations of free phosphate, reducing the possibility of phosphate contamination.

[0254] Furthermore, the method disclosed herein can be carried out without the addition of ATP as a phosphate source, i.e., without ATP. The method can also be carried out without the need to add NAD(P)(H), i.e., without NAD(P)(H). Another advantage is the fact that at least one step of the disclosed method for producing talose involves an energetically favorable reaction.

[0255] Talose can also be produced from fructose. For example, this production method includes the following steps: producing F6P from fructose and polyphosphate by catalysis with polyphosphate fructokinase (PPFK); converting F6P to P6P by catalysis with P6PE; converting P6P to Tal6P by catalysis with Tal6PI; Conversion of Tal6P to talose by Tal6PP catalysis The fructose can be produced, for example, by the enzymatic conversion of sucrose.

[0256] Talose can be produced from sucrose by the following enzymatic steps: generating G1P from sucrose and free phosphate by catalyzing with sucrose phosphorylase (SP); PGM-catalyzed conversion of G1P to G6P; converting G6P to F6P by catalysis with PGI; converting F6P to T6P by catalysis with F6PE; converting T6P to Tal6P by catalysis with Tal6PI; Conversion of Tal6P to talose by Tal6PP catalysis The present invention provides an in vitro synthesis route comprising:

[0257] The phosphate ions produced during the conversion of Tal6P to talose can then be recycled to the step of converting sucrose to G1P. Furthermore, the talose yield can be increased by using PPFK and polyphosphate to generate F6P from fructose produced by the phosphorolytic cleavage of sucrose by SP.

[0258] In some embodiments, the method for producing talose comprises the following steps: producing glucose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis; converting glucose to G6P by catalyzing at least one enzyme; producing fructose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis; converting fructose to F6P by catalysis with at least one enzyme; Examples of such polysaccharides and oligosaccharides are listed above.

[0259] In another embodiment, G6P is produced from glucose and sodium polyphosphate by polyphosphate glucokinase.

[0260] The present disclosure provides a method for converting sugars such as polysaccharides and oligosaccharides in starch, cellulose, sucrose, and their derived products to talose. In certain embodiments, an artificial (non-natural) ATP-free enzymatic pathway is provided for converting starch, cellulose, sucrose, and their derived products to talose using a cell-free enzyme cocktail.

[0261] As shown above, several enzymes can be used to hydrolyze starch and increase the G1P yield. These enzymes include isoamylase, pullulanase, and α-amylase. Corn starch contains many branches that prevent the action of αGP. Isoamylase can be used to debranch starch and produce linear amylodextrin. Pre-treated starch with isoamylase can result in a higher concentration of F6P in the final product. Isoamylase and pullulanase cleave α-1,6-glycosidic bonds, which allows starch to be more completely degraded by α-glucan phosphorylase. α-amylase cleaves α-1,4-glycosidic bonds, so α-amylase can be used to break starch into fragments for more rapid conversion to talose and increased solubility.

[0262] Maltose phosphorylase (MP) can be used to increase talose yield by phosphorolytic cleavage of the degradation product maltose to G1P and glucose, or 4-glucan transferase (4GT) can be used to recycle the degradation products glucose, maltose, and maltotriose to longer chain maltooligosaccharides that can be phosphorolytically cleaved by αGP to produce G1P.

[0263] In a particular embodiment, cellulose and its derived products can be converted to talose through a series of steps. The process comprises the following steps: producing G1P from cellodextrin and cellobiose and free phosphate by catalyzing with cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively; PGM-catalyzed conversion of G1P to G6P; converting G6P to F6P by catalysis with PGI; converting F6P to T6P by catalysis with P6PE; converting T6P to Tal6P by catalysis with Tal6PI; Conversion of Tal6P to talose by Tal6PP catalysis In this method, phosphate ions can be recycled through the above steps of converting cellodextrin and cellobiose into G1P.

[0264] In some embodiments, the yield of talose can be increased by adding polyphosphate and polyphosphate glucokinase (PPGK) to the process, thereby phosphorylating the degradation product glucose to G6P.

[0265] In another embodiment, talose can be produced from glucose. The method comprises the steps of: Producing G6P from glucose and polyphosphate by catalysis with polyphosphate glucokinase (PPGK); converting G6P to F6P by catalysis with PGI; converting F6P to T6P by catalysis with P6PE; converting T6P to Tal6P by catalysis with Tal6PI; converting Tal6P to talose by catalysis with Tal6PP; Includes.

[0266] The present invention's manufacturing method reduces production costs by using low-cost starting materials and eliminating costs associated with the separation of raw materials and products. Starch, cellulose, sucrose, and some of their derivatives are cheaper raw materials than, for example, lactose. When talose is produced from biomass or lactose, the yield is lower than in the present invention, and talose needs to be separated from other sugars by chromatography, which leads to higher production costs. Furthermore, our manufacturing method does not involve animals.

[0267] The step of converting Tal6P to talose according to the present invention is an irreversible phosphatase reaction regardless of the starting material, therefore talose is produced in very high yields while effectively minimizing the subsequent cost of product separation.

[0268] In contrast to cell-based production methods, the present invention involves cell-free production of talose, which has a relatively high reaction rate due to the elimination of cell membranes that often slow down the transport of substrates into cells / products out of cells. Also in the present invention, the final product does not contain nutrient-rich fermentation medium / cellular metabolic products.

[0269] A particular embodiment of the present invention is talose produced by the method for producing talose described herein.

[0270] Sorbose

[0271] One embodiment of the present invention comprises: converting fructose 6-phosphate (F6P) to tagatose 6-phosphate (T6P) by catalyzing with fructose 6-phosphate 4-epimerase (F6PE); converting T6P to sorbose 6-phosphate (S6P) by catalyzing sorbose 6-phosphate epimerase (S6PE); converting the S6P produced above into sorbose by catalysis with sorbose 6-phosphate phosphatase (S6PP); The method for producing sorbose comprises the steps of:

[0272] Examples of F6PE include, but are not limited to, the following proteins: Uniprot IDs E8N0N6, E4SEH3, I0I507, H1XRG1, and B5YBD7. Uniprot IDs E8N0N6 and I0I507 both catalyze the F6PE reaction and share 27% amino acid sequence identity. Thus, examples of F6PE also include any analogs that have at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity to any of the above Uniprot IDs.

[0273] The method for producing sorbose of the present invention also includes a step of enzymatically converting glucose 6-phosphate (G6P) to said F6P, which is catalyzed by phosphoglucoisomerase (PGI). In another embodiment, the method for producing sorbose further includes a step of converting glucose 1-phosphate (G1P) to said G6P, which is catalyzed by phosphoglucomutase (PGM). In yet another embodiment, the method for producing sorbose also includes a step of converting sugars to said G1P, catalyzed by at least one enzyme.

[0274] Thus, the method for producing sorbose of the present invention comprises, for example, the following steps: (i) converting sugars to glucose 1-phosphate (G1P) using one or more enzymes; (ii) converting G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii) converting G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) converting F6P to T6P via P6PE; (v) converting T6P to S6P via S6PE (EC 5.3.1.26); (vi) converting S6P to sorbose via S6PP; An example of the process is shown in FIG.

[0275] Typically, the ratio of enzyme units used in the disclosed production methods is 1:1:1:1:1:1 (αGP:PGM:PGI:F6PE:S6PE:S6PP). These ratios may be adjusted in any number of combinations to optimize product yield. For example, a ratio of 3:1:1:1:1:1:1 may be used to maximize the concentration of phosphorylated intermediates, which will increase the activity of downstream reactions. Conversely, a ratio of 1:1:1:1:1:1:3 may be used to ensure that a supply of phosphates for αGP is maintained, which will result in more efficient phosphorolytic cleavage of α-1,4-glycosidic bonds. For example, a ratio of 3:1:1:1:1:3 enzymes may be used to further increase the reaction rate. Thus, the ratio of enzymes, including other optional enzymes described below, may be varied to increase the efficiency of sorbose production. For example, a particular enzyme may be present in an amount of about 2-fold, 3-fold, 4-fold, 5-fold, etc., relative to the amount of other enzymes.

[0276] One of the key advantages of this production method is that multiple process steps can be carried out in a single bioreactor or reactor, or alternatively, the steps can be carried out in multiple bioreactors or reactors arranged in series.

[0277] The phosphate ions produced by the dephosphorylation of S6P can then be recycled to the process step that converts sugar to G1P, especially when all process steps are carried out in a single bioreactor or reactor. The ability to recycle phosphate in the disclosed process allows for the use of non-stoichiometric amounts of phosphate, thereby keeping the phosphate concentration in the reaction solution low. This affects the overall pathway and overall rate of the process, but does not limit the activity of any individual enzyme, allowing the process to be more efficient overall.

[0278] For example, the phosphoric acid concentration in the reaction solution may be in the range of about 0.1 mM to about 300 mM, about 0.1 mM to about 150 mM, about 1 mM to about 50 mM, preferably about 5 mM to about 50 mM, or more preferably about 10 mM to about 50 mM. For example, the phosphoric acid concentration in the reaction solution may be about 0.1 mM, about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, or about 55 mM.

[0279] Therefore, lower phosphate concentrations reduce production costs due to lower total phosphate, which in turn reduces the cost of phosphate removal, and also prevents inhibition of S6PP by high free phosphate, reducing the possibility of phosphate contamination.

[0280] Furthermore, the methods disclosed herein can be carried out without the addition of ATP as a source of phosphate, i.e., without ATP. The methods can also be carried out without the need to add NAD(P)(H), i.e., without NAD(P)(H). Another advantage is the fact that at least one step of the disclosed methods for producing sorbose involves an energetically favorable reaction.

[0281] Sorbose can also be produced from fructose. For example, this production method includes the following steps: producing F6P from fructose and polyphosphate by catalysis with polyphosphate fructokinase (PPFK); converting F6P to T6P by catalysis with F6PE; converting T6P to S6P by catalysis with S6PE; Conversion of S6P to sorbose by catalysis with S6PP; The fructose can be produced, for example, by the enzymatic conversion of sucrose.

[0282] Sorbose can be produced from sucrose by the following enzymatic steps: generating G1P from sucrose and free phosphate by catalyzing with sucrose phosphorylase (SP); PGM-catalyzed conversion of G1P to G6P; converting G6P to F6P by catalysis with PGI; converting F6P to T6P by catalysis with F6PE; converting T6P to S6P by catalysis with S6PE; Conversion of S6P to sorbose by catalysis with S6PP; The present invention provides an in vitro synthesis route comprising:

[0283] The phosphate ions produced during the conversion of S6P to sorbose can then be recycled to the step of converting sucrose to G1P. Furthermore, the sorbose yield can be increased by using PPFK and polyphosphate to generate F6P from fructose produced by the phosphorolytic cleavage of sucrose by SP.

[0284] In some embodiments, the method for producing sorbose comprises the following steps: producing glucose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis; converting glucose to G6P by catalyzing at least one enzyme; producing fructose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis; converting fructose to F6P by catalysis with at least one enzyme; Examples of such polysaccharides and oligosaccharides are listed above.

[0285] In another embodiment, G6P is produced from glucose and sodium polyphosphate by polyphosphate glucokinase.

[0286] The present disclosure provides methods for the conversion of sugars such as polysaccharides and oligosaccharides in starch, cellulose, sucrose, and their derived products to sorbose. In certain embodiments, an artificial (non-natural), ATP-free enzymatic pathway is provided for the conversion of starch, cellulose, sucrose, and their derived products to sorbose using a cell-free enzyme cocktail.

[0287] As shown above, several enzymes can be used to hydrolyze starch and increase the G1P yield. These enzymes include isoamylase, pullulanase, and α-amylase. Corn starch contains many branches that prevent the action of αGP. Isoamylase can be used to debranch starch and produce linear amylodextrins. Pre-treated starch with isoamylase can result in a higher concentration of F6P in the final product. Isoamylase and pullulanase cleave α-1,6-glycosidic bonds, which allows starch to be more completely degraded by α-glucan phosphorylase. α-amylase cleaves α-1,4-glycosidic bonds, so α-amylase can be used to break starch into fragments for more rapid conversion to sorbose and increased solubility.

[0288] Maltose phosphorylase (MP) can be used to increase sorbose yield by phosphorolytic cleavage of the degradation product maltose to G1P and glucose, or 4-glucan transferase (4GT) can be used to increase sorbose yield by recycling the degradation products glucose, maltose, and maltotriose to longer chain maltooligosaccharides that can be phosphorolytically cleaved by αGP to produce G1P.

[0289] In a particular embodiment, cellulose and its derived products can be converted to sorbose through a series of steps. The process comprises the following steps: producing G1P from cellodextrin and cellobiose and free phosphate by catalyzing with cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively; PGM-catalyzed conversion of G1P to G6P; converting G6P to F6P by catalysis with PGI; converting F6P to T6P by catalysis with F6PE; converting T6P to S6P by catalysis with S6PE; Conversion of S6P to sorbose by catalysis with S6PP; In this method, phosphate ions can be recycled through the above steps of converting cellodextrin and cellobiose into G1P.

[0290] In some embodiments, the yield of sorbose can be increased by adding polyphosphate and polyphosphate glucokinase (PPGK) to the process, thereby phosphorylating the breakdown product glucose to G6P.

[0291] In another embodiment, sorbose can be produced from glucose. The method comprises: Producing G6P from glucose and polyphosphate by catalysis with polyphosphate glucokinase (PPGK); converting G6P to F6P by catalysis with PGI; converting F6P to T6P by catalysis with P6PE; converting T6P to S6P by catalysis with S6PE; converting S6P to sorbose by catalysis with S6PP; Includes.

[0292] The production method of the present invention reduces production costs by using low-cost starting materials and eliminating costs associated with the separation of raw materials and products. Starch, cellulose, sucrose, and some of their derivatives are cheaper raw materials than, for example, lactose. If sorbose is produced from biomass or lactose, the yield is lower than in the present invention, and sorbose needs to be separated from other sugars by chromatography, which leads to higher production costs. Furthermore, our production method does not involve animals.

[0293] The step of converting S6P to sorbose according to the present invention is an irreversible phosphatase reaction regardless of the starting material, therefore producing sorbose in very high yields while effectively minimizing the subsequent product separation costs.

[0294] In contrast to cellular production methods, the present invention involves cell-free production of sorbose, which has a relatively high reaction rate due to the elimination of cell membranes that often slow down the transport of substrates into the cell / products out of the cell, and the present invention also does not include nutrient-rich fermentation media / cellular metabolic products in the final product.

[0295] A particular embodiment of the present invention is sorbose produced by the method for producing sorbose described herein.

[0296] Growth

[0297] One embodiment of the present invention comprises: converting fructose 6-phosphate (F6P) to tagatose 6-phosphate (T6P) by catalyzing with fructose 6-phosphate 4-epimerase (F6PE); converting T6P to sorbose 6-phosphate (S6P) by catalyzing sorbose 6-phosphate epimerase (S6PE); converting the S6P produced to glucose 6-phosphate (Gul6P) by catalyzing with glucose 6-phosphate isomerase; and converting the Gul6P to Gulose by catalysis with Gul6PP. The method for producing growth includes the steps of:

[0298] Examples of F6PE include, but are not limited to, the following proteins: Uniprot IDs E8N0N6, E4SEH3, I0I507, H1XRG1, and B5YBD7. Uniprot IDs E8N0N6 and I0I507 both catalyze the F6PE reaction and share 27% amino acid sequence identity. Thus, examples of F6PE also include any analogs that have at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity to any of the above Uniprot IDs.

[0299] The method for producing gulose of the present invention also includes a step of enzymatically converting glucose 6-phosphate (G6P) to said F6P, which is catalyzed by phosphoglucoisomerase (PGI). In another embodiment, the method for producing gulose further includes a step of converting glucose 1-phosphate (G1P) to said G6P, which is catalyzed by phosphoglucomutase (PGM). In yet another embodiment, the method for producing gulose also includes a step of converting sugars to said G1P, which is catalyzed by at least one enzyme.

[0300] Therefore, the method for producing growth of the present invention includes, for example, the following steps: (i) converting sugars to glucose 1-phosphate (G1P) using one or more enzymes; (ii) converting G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii) converting G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) converting F6P to T6P via F6PE; (v) converting T6P to S6P via S6PE (EC 5.3.1.26); (vi) converting S6P to Gul6P via Gul6PI; (vii) converting GulP to growth via Gul6PP; may also include

[0301] Typically, the ratio of enzyme units used in the disclosed production methods is 1:1:1:1:1:1:1:1 (αGP:PGM:PGI:F6PE, S6PE:Gul6PI:Gul6PP). These ratios may be adjusted in any number of combinations to optimize product yield. For example, a ratio of 3:1:1:1:1:1:1:1 can be used to maximize the concentration of phosphorylated intermediates, which will increase the activity of downstream reactions. Conversely, a ratio of 1:1:1:1:1:1:1:3 can be used to ensure that a supply of phosphates for αGP is maintained, which will result in more efficient phosphorolytic cleavage of α-1,4-glycosidic bonds. For example, an enzyme ratio of 3:1:1:1:1:1:3 can be used to further increase the reaction rate. Thus, the ratio of enzymes, including other optional enzymes described below, can be varied to increase the efficiency of growth production. For example, a particular enzyme may be present in an amount that is about 2-fold, 3-fold, 4-fold, 5-fold, etc., relative to the amount of another enzyme.

[0302] One of the key advantages of this production method is that multiple process steps can be carried out in a single bioreactor or reactor, or alternatively, the steps can be carried out in multiple bioreactors or reactors arranged in series.

[0303] The phosphate ions generated by the dephosphorylation of S6P can then be recycled to the process step that converts sugars to G1P, especially when all process steps are carried out in a single bioreactor or reactor. The ability to recycle phosphate in the disclosed production method allows for the use of non-stoichiometric amounts of phosphate, thereby keeping the phosphate concentration in the reaction solution low. This affects the overall pathway and overall rate of the production method, but does not limit the activity of individual enzymes, allowing the production method of growth to be more efficient overall.

[0304] For example, the phosphoric acid concentration in the reaction solution may be in the range of about 0.1 mM to about 300 mM, about 0.1 mM to about 150 mM, about 1 mM to about 50 mM, preferably about 5 mM to about 50 mM, or more preferably about 10 mM to about 50 mM. For example, the phosphoric acid concentration in the reaction solution may be about 0.1 mM, about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, or about 55 mM.

[0305] Therefore, lower phosphate concentrations reduce production costs due to lower total phosphate, which in turn reduces the cost of phosphate removal, and also prevents inhibition of S6PP by high free phosphate, reducing the possibility of phosphate contamination.

[0306] Furthermore, the methods disclosed herein can be carried out without the addition of ATP as a phosphate source, i.e., without ATP. The methods can also be carried out without the need to add NAD(P)(H), i.e., without NAD(P)(H). Another advantage is the fact that at least one step of the disclosed growth production method involves an energetically favorable reaction.

[0307] Gulose can also be produced from fructose. For example, this production method includes the following steps: producing F6P from fructose and polyphosphate by catalysis with polyphosphate fructokinase (PPFK); converting F6P to T6P by catalysis with F6PE; converting T6P to S6P by catalysis with S6PE; converting S6P to Gul6P by catalysis with Gul6PI; Gul6PP catalyzes the conversion of Gul6P to growth. The fructose can be produced, for example, by the enzymatic conversion of sucrose.

[0308] Gulose can be produced from sucrose. The production process involves the following enzymatic steps: generating G1P from sucrose and free phosphate by catalyzing with sucrose phosphorylase (SP); PGM-catalyzed conversion of G1P to G6P; converting G6P to F6P by catalysis with PGI; converting F6P to T6P by catalysis with F6PE; converting T6P to S6P by catalysis with S6PE; converting S6P to Gul6P by catalysis with Gul6PI; Gul6PP catalyzes the conversion of Gul6P to growth. The present invention provides an in vitro synthesis route comprising:

[0309] The phosphate ions produced during the conversion of S6P to sorbose can then be recycled to the step of converting sucrose to G1P.Furthermore, growth yields can be increased by using PPFK and polyphosphate to generate F6P from fructose produced by the phosphorolytic cleavage of sucrose by SP.

[0310] In some embodiments, the method for producing growth comprises the following steps: producing glucose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis; converting glucose to G6P by catalyzing at least one enzyme; producing fructose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis; converting fructose to F6P by catalysis with at least one enzyme; Examples of such polysaccharides and oligosaccharides are listed above.

[0311] In another embodiment, G6P is produced from glucose and sodium polyphosphate by polyphosphate glucokinase.

[0312] The present disclosure provides a method for converting sugars, such as polysaccharides and oligosaccharides in starch, cellulose, sucrose, and their derived products, to growth. In certain embodiments, an artificial (non-natural) ATP-free enzymatic pathway is provided for converting starch, cellulose, sucrose, and their derived products to growth using a cell-free enzyme cocktail.

[0313] As shown above, several enzymes can be used to hydrolyze starch and increase the G1P yield. These enzymes include isoamylase, pullulanase, and α-amylase. Corn starch contains many branches that prevent the action of αGP. Isoamylase can be used to debranch starch and produce linear amylodextrins. Pre-treated starch with isoamylase can result in higher F6P concentrations in the final product. Isoamylase and pullulanase cleave α-1,6-glycosidic bonds, which allows starch to be more completely degraded by α-glucan phosphorylase. α-amylase cleaves α-1,4-glycosidic bonds, so α-amylase can be used to break starch into fragments for more rapid conversion to growth and increased solubility.

[0314] Growth yields can be increased by using maltose phosphorylase (MP) to phosphorolytically cleave the degradation product maltose to G1P and glucose, or by using 4-glucan transferase (4GT) to recycle the degradation products glucose, maltose, and maltotriose to longer chain maltooligosaccharides that can be phosphorolytically cleaved by αGP to produce G1P.

[0315] In certain embodiments, cellulose and its derived products can be converted to growth through a series of steps. The process includes the following steps: producing G1P from cellodextrin and cellobiose and free phosphate by catalyzing with cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively; PGM-catalyzed conversion of G1P to G6P; converting G6P to F6P by catalysis with PGI; converting F6P to T6P by catalysis with F6PE; converting T6P to S6P by catalysis with S6PE; Conversion of S6P to sorbose by catalysis with S6PP; In this method, phosphate ions can be recycled through the above steps of converting cellodextrin and cellobiose into G1P.

[0316] In some embodiments, polyphosphate and polyphosphate glucokinase (PPGK) can be added to the process to phosphorylate the breakdown product glucose to G6P, thereby increasing the yield of growth.

[0317] In another embodiment, growth can be produced from glucose. Producing G6P from glucose and polyphosphate by catalysis with polyphosphate glucokinase (PPGK); converting G6P to F6P by catalysis with PGI; converting F6P to T6P by catalysis with F6PE; converting T6P to S6P by catalysis with S6PE; converting S6P to Gul6P by catalysis with Gul6PI; converting Gul6P to growth by catalyzing with Gul6PP; Includes.

[0318] The present invention's manufacturing method reduces production costs by using low-cost starting materials and eliminating costs associated with the separation of raw materials and products. Starch, cellulose, sucrose, and some of their derivatives are cheaper raw materials than, for example, lactose. When growth is produced from biomass or lactose, the yield is lower than in the present invention, and growth must be separated from other sugars by chromatography, which leads to higher production costs. Furthermore, our manufacturing method does not involve animals.

[0319] The step of converting S6P to growth according to the present invention is an irreversible phosphatase reaction regardless of the starting material, therefore growth is produced in very high yields while effectively minimizing the subsequent product separation costs.

[0320] In contrast to cellular production methods, the present invention involves cell-free production of growth, which has relatively high reaction rates due to the elimination of cell membranes that often slow down transport of substrates into the cell / products out of the cell, and the present invention also does not include nutrient-rich fermentation media / cellular metabolic products in the final product.

[0321] A particular embodiment of the present invention is a growth produced by the method for producing a growth described herein.

[0322] Idos

[0323] One embodiment of the present invention comprises: converting fructose 6-phosphate (F6P) to tagatose 6-phosphate (T6P) by catalyzing with fructose 6-phosphate 4-epimerase (F6PE); converting T6P to sorbose 6-phosphate (S6P) by catalyzing sorbose 6-phosphate epimerase (S6PE); converting the S6P produced to idose 6-phosphate (I6P) by catalysis with idose 6-phosphate isomerase; converting said I6P to idose by catalysis with idose 6-phosphate phosphatase (I6PP); The method for producing idose includes the steps of:

[0324] Examples of F6PE include, but are not limited to, the following proteins: Uniprot IDs E8N0N6, E4SEH3, I0I507, H1XRG1, and B5YBD7. Uniprot IDs E8N0N6 and I0I507 both catalyze the F6PE reaction and share 27% amino acid sequence identity. Thus, examples of F6PE also include any analogs that have at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity to any of the above Uniprot IDs.

[0325] The method for producing idose of the present invention also includes a step of enzymatically converting glucose 6-phosphate (G6P) to said F6P, which is catalyzed by phosphoglucoisomerase (PGI). In another embodiment, the method for producing idose further includes a step of converting glucose 1-phosphate (G1P) to said G6P, which is catalyzed by phosphoglucomutase (PGM). In yet another embodiment, the method for producing idose also includes a step of converting sugar to said G1P, which is catalyzed by at least one enzyme.

[0326] Thus, the method for producing idose of the present invention may, for example, comprise the following steps: (i) converting sugars to glucose 1-phosphate (G1P) using one or more enzymes; (ii) converting G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii) converting G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) converting F6P to T6P via F6PE; (v) converting T6P to S6P via S6PE (EC 5.3.1.26); (vi) converting S6P to I6P via I6PI; (vii) converting I6P to idose via I6PP; may also include

[0327] Typically, the ratio of enzyme units used in the disclosed production methods is 1:1:1:1:1:1:1:1 (αGP:PGM:PGI:F6PE:S6PE:I6PI:I6PP). These ratios may be adjusted in any number of combinations to optimize product yield. For example, a ratio of 3:1:1:1:1:1:1:1 can be used to maximize the concentration of phosphorylated intermediates, which will increase the activity of downstream reactions. Conversely, a ratio of 1:1:1:1:1:1:1:3 can be used to ensure that a supply of phosphates for αGP is maintained, which will result in more efficient phosphorolytic cleavage of the α-1,4-glycosidic bond. For example, an enzyme ratio of 3:1:1:1:1:1:3 can be used to further increase the reaction rate. Thus, the ratio of enzymes, including other optional enzymes described below, can be varied to increase the efficiency of idose production. For example, a particular enzyme may be present in an amount that is about 2-fold, 3-fold, 4-fold, 5-fold, etc., relative to the amount of another enzyme.

[0328] One of the key advantages of this production method is that multiple process steps can be carried out in a single bioreactor or reactor, or alternatively, the steps can be carried out in multiple bioreactors or reactors arranged in series.

[0329] The phosphate ions generated by the dephosphorylation of S6P can then be recycled to the process step that converts sugars to G1P, especially when all process steps are carried out in a single bioreactor or reactor. The ability to recycle phosphate in the disclosed process allows for the use of non-stoichiometric amounts of phosphate, which keeps the phosphate concentration in the reaction solution low. This affects the overall pathway and overall rate of the process, but does not limit the activity of any individual enzyme, allowing the overall efficiency of the idose production process.

[0330] For example, the phosphoric acid concentration in the reaction solution may be in the range of about 0.1 mM to about 300 mM, about 0.1 mM to about 150 mM, about 1 mM to about 50 mM, preferably about 5 mM to about 50 mM, or more preferably about 10 mM to about 50 mM. For example, the phosphoric acid concentration in the reaction solution may be about 0.1 mM, about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, or about 55 mM.

[0331] Therefore, lower phosphate concentrations reduce production costs due to lower total phosphate, which in turn reduces the cost of phosphate removal, and also prevents inhibition of S6PP by high free phosphate, reducing the possibility of phosphate contamination.

[0332] Furthermore, the methods disclosed herein can be carried out without the addition of ATP as a phosphate source, i.e., without ATP. The methods can also be carried out without the need to add NAD(P)(H), i.e., without NAD(P)(H). Another advantage is the fact that at least one step of the disclosed methods for producing idose involves an energetically favorable reaction.

[0333] Idose can also be produced from fructose. For example, this production method includes the following steps: producing F6P from fructose and polyphosphate by catalysis with polyphosphate fructokinase (PPFK); converting F6P to T6P by catalysis with F6PE; converting T6P to S6P by catalysis with S6PE; converting S6P to I6P by catalysis with I6PI; Conversion of I6P to idose by catalysis with I6PP The fructose can be produced, for example, by the enzymatic conversion of sucrose.

[0334] Idose can be produced from sucrose by the following enzymatic steps: generating G1P from sucrose and free phosphate by catalyzing with sucrose phosphorylase (SP); PGM-catalyzed conversion of G1P to G6P; converting G6P to F6P by catalysis with PGI; converting F6P to T6P by catalysis with F6PE; converting T6P to S6P by catalysis with S6PE; converting S6P to I6P by catalysis with I6PI; Conversion of I6P to idose by catalysis with I6PP The present invention provides an in vitro synthesis route comprising:

[0335] The phosphate ions produced during the conversion of S6P to sorbose can then be recycled to the step of converting sucrose to G1P. Furthermore, the idose yield can be increased by using PPFK and polyphosphate to generate F6P from fructose produced by the phosphorolytic cleavage of sucrose by SP.

[0336] In some embodiments, the method for producing idose comprises the following steps: producing glucose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis; converting glucose to G6P by catalyzing at least one enzyme; producing fructose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis; converting fructose to F6P by catalysis with at least one enzyme; Examples of such polysaccharides and oligosaccharides are listed above.

[0337] In another embodiment, G6P is produced from glucose and sodium polyphosphate by polyphosphate glucokinase.

[0338] The present disclosure provides methods for the conversion of sugars, such as polysaccharides and oligosaccharides in starch, cellulose, sucrose, and their derived products, to idose. In certain embodiments, an artificial (non-natural), ATP-free enzymatic pathway is provided for the conversion of starch, cellulose, sucrose, and their derived products to idose using a cell-free enzyme cocktail.

[0339] As shown above, several enzymes can be used to hydrolyze starch and increase the G1P yield. These enzymes include isoamylase, pullulanase, and α-amylase. Corn starch contains many branches that prevent the action of αGP. Isoamylase can be used to debranch starch and produce linear amylodextrins. Pre-treated starch with isoamylase can result in a higher concentration of F6P in the final product. Isoamylase and pullulanase cleave α-1,6-glycosidic bonds, which allows starch to be more completely degraded by α-glucan phosphorylase. α-amylase cleaves α-1,4-glycosidic bonds, so α-amylase can be used to break starch into fragments for more rapid conversion to idose and increased solubility.

[0340] Maltose phosphorylase (MP) can be used to increase the idose yield by phosphorolytic cleavage of the degradation product maltose to G1P and glucose, or 4-glucantransferase (4GT) can be used to recycle the degradation products glucose, maltose, and maltotriose to longer chain maltooligosaccharides that can be phosphorolytically cleaved by αGP to produce G1P.

[0341] In a particular embodiment, cellulose and its derived products can be converted to idose through a series of steps. The process comprises the following steps: producing G1P from cellodextrin and cellobiose and free phosphate by catalyzing with cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively; PGM-catalyzed conversion of G1P to G6P; converting G6P to F6P by catalysis with PGI; converting F6P to T6P by catalysis with F6PE; converting T6P to S6P by catalysis with S6PE; Conversion of S6P to sorbose by catalysis with S6PP; In this method, phosphate ions can be recycled through the above steps of converting cellodextrin and cellobiose into G1P.

[0342] In some embodiments, the yield of idose can be increased by adding polyphosphate and polyphosphate glucokinase (PPGK) to the process, thereby phosphorylating the degradation product glucose to G6P.

[0343] In another embodiment, idose can be produced from glucose. The method comprises: Producing G6P from glucose and polyphosphate by catalysis with polyphosphate glucokinase (PPGK); converting G6P to F6P by catalysis with PGI; converting F6P to T6P by catalysis with F6PE; converting T6P to S6P by catalysis with S6PE; converting S6P to I6P by catalysis with I6PI; converting I6P to idose by catalysis with I6PP; Includes.

[0344] The present invention's manufacturing method reduces production costs by using low-cost starting materials and eliminating costs associated with the separation of raw materials and products. Starch, cellulose, sucrose, and some of their derivatives are cheaper raw materials than, for example, lactose. When idose is produced from biomass or lactose, the yield is lower than in the present invention, and idose needs to be separated from other sugars by chromatography, which leads to higher production costs. Furthermore, our manufacturing method does not involve animals.

[0345] The step of converting S6P to idose according to the present invention is an irreversible phosphatase reaction regardless of the starting material, thus producing idose in very high yields while effectively minimizing the subsequent product separation costs.

[0346] In contrast to cellular production methods, the present invention involves cell-free production of idose, which has a relatively high reaction rate due to the elimination of cell membranes that often slow down the transport of substrates into the cell / products out of the cell, and the present invention also does not include nutrient-rich fermentation media / cellular metabolic products in the final product.

[0347] A particular embodiment of the present invention is idose produced by the method for producing idose described herein.

[0348] Tagatose

[0349] The method for producing tagatose is converting F6P to T6P by catalyzing the epimerase; converting said T6P to tagatose by phosphatase catalysis; Includes.

[0350] Suitable epimerases for use in the method of converting F6P to T6P include F6PE. Examples of F6PE include, but are not limited to, the following proteins: Uniprot ID E8N0N6, E4SEH3, I0I507, H1XRG1, and B5YBD7. Uniprot IDs E8N0N6 and I0I507 both catalyze the F6PE reaction and share 27% amino acid sequence identity. Thus, examples of F6PE also include any analogs that have at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity to any of the above Uniprot IDs.

[0351] In the production method, T6PP, a phosphatase that converts T6P to tagatose (D-tagatose), may be used. Examples of T6PP include, but are not limited to, the following proteins: Uniprot ID O29805, D2RHV2, and F2KMK2. Both Uniprot IDs O29805 and F2KMK2 catalyze the F6PE reaction and share 67% amino acid sequence identity. Thus, examples of T6PP also include any analog that has at least 65%, preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to any of the above Uniprot IDs.

[0352] The method for producing tagatose also includes a step of enzymatically converting glucose 6-phosphate (G6P) to the F6P, which is catalyzed by phosphoglucose isomerase (PGI).The method for producing tagatose further includes a step of converting glucose 1-phosphate (G1P) to the G6P, which is catalyzed by phosphoglucomutase (PGM).Furthermore, the method for producing tagatose also includes a step of converting sugar to the G1P, which is catalyzed by at least one enzyme.

[0353] Thus, the method for producing tagatose may, for example, comprise the following steps: (i) converting sugars to glucose 1-phosphate (G1P) using one or more enzymes; (ii) converting G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii) converting G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) converting F6P to T6P via fructose 6-phosphate epimerase (F6PE); (v) converting T6P to tagatose via tagatose 6-phosphate phosphatase (T6PP); Includes.

[0354] Typically, the ratio of enzyme units used in the present production method is 1:1:1:1:1 (αGP:PGM:PGI:F6PE:T6PP). These ratios may be adjusted in any number of combinations to optimize product yield. For example, a ratio of 3:1:1:1:1 can be used to maximize the concentration of phosphorylated intermediates, which will increase the activity of downstream reactions. Conversely, a ratio of 1:1:1:1:3 can be used to ensure that phosphate is maintained for αGP, which will result in more efficient phosphorolytic cleavage of α-1,4-glycosidic bonds. For example, a ratio of 3:1:1:1:3 enzymes can be used to further increase the reaction rate. Thus, the ratio of enzymes, including other optional enzymes described below, can be varied to increase the efficiency of tagatose production. For example, a particular enzyme may be present in an amount of about 2-fold, 3-fold, 4-fold, 5-fold, etc., relative to the amount of other enzymes.

[0355] Tagatose can also be produced from fructose. For example, this production method includes the following steps: producing F6P from fructose and polyphosphate by catalysis with polyphosphate fructokinase (PPFK); converting F6P to T6P by catalysis with F6PE; Conversion of T6P to tagatose by catalysis with T6PP The fructose can be produced, for example, by the enzymatic conversion of sucrose.

[0356] Tagatose can be produced from sucrose by the following enzymatic steps: generating G1P from sucrose and free phosphate by catalyzing with sucrose phosphorylase (SP); PGM-catalyzed conversion of G1P to G6P; converting G6P to F6P by catalysis with PGI; converting F6P to T6P by catalysis with F6PE; Conversion of T6P to tagatose by catalysis with T6PP The present invention provides an in vitro synthesis route comprising:

[0357] The phosphate ions produced during the conversion of T6P to tagatose can then be recycled to the step of converting sucrose to G1P. Furthermore, the yield of tagatose can be increased by using PPFK and polyphosphate to produce F6P from fructose produced by the phosphorolytic cleavage of sucrose by SP.

[0358] The method for producing tagatose comprises the following steps: producing glucose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis; converting glucose to G6P by catalyzing at least one enzyme; producing fructose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis; converting fructose to G6P by catalysis with at least one enzyme; Examples of such polysaccharides and oligosaccharides are listed above.

[0359] Cellulose and its derived products can be converted to tagatose through a series of steps. The process comprises the following steps: producing G1P from cellodextrin and cellobiose and free phosphate by catalyzing with cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively; PGM-catalyzed conversion of G1P to G6P; converting G6P to F6P by catalysis with PGI; converting F6P to T6P by catalysis with F6PE; Conversion of T6P to tagatose by catalysis with T6PP In this method, phosphate ions can be recycled by the above step of converting cellodextrin and cellobiose to G1P.

[0360] Tagatose can be produced from glucose. The production method is as follows: Producing G6P from glucose and polyphosphate by catalysis with polyphosphate glucokinase (PPGK); converting G6P to F6P by catalysis with PGI; converting F6P to T6P by catalysis with F6PE; converting T6P to tagatose by catalysis with T6PP; Includes.

[0361] Psicose

[0362] The method for producing psicose is as follows: converting fructose 6-phosphate (F6P) to psicose 6-phosphate (P6P) by catalyzing an epimerase (e.g., psicose 6-phosphate 3-epimerase, P6PE); converting the produced P6P to psicose by catalysis with a phosphatase (e.g., psicose 6-phosphate phosphatase, P6PP); Includes.

[0363] Examples of P6PEs include, but are not limited to, the following proteins: UNIPROT Identification Nos. D9TQJ4, A0A090IXZ8, and P32719. Uniprot IDs A0A090IXZ8 and D9TQJ4 both catalyze the P6PE reaction and share 45% amino acid sequence identity. Thus, examples of P6PEs also include any analogs that have at least 45%, preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96, 97, 98, 99, or 100% amino acid sequence identity to any of the above Uniprot IDs.

[0364] Examples of P6PP include, but are not limited to, the following proteins: Uniprot IDs A3DC21, Q5LGR4, and Q89ZR1. Both Uniprot IDs A3DC21 and Q89ZR1 catalyze the P6PP reaction and share 45% amino acid sequence identity. Thus, examples of P6PP also include any analogs that have at least 45%, preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96, 97, 98, 99, or 100% amino acid sequence identity to any of the above Uniprot IDs.

[0365] The method for producing psicose also includes a step of enzymatically converting glucose 6-phosphate (G6P) to the F6P, which is catalyzed by phosphoglucose isomerase (PGI). The method for producing psicose further includes a step of converting glucose 1-phosphate (G1P) to the G6P, which is catalyzed by phosphoglucomutase (PGM). Furthermore, the method for producing psicose also includes a step of converting sugar to the G1P, which is catalyzed by at least one enzyme.

[0366] Therefore, the method for producing psicose includes, for example, the following steps: (i) converting sugars to glucose 1-phosphate (G1P) using one or more enzymes; (ii) converting G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii) converting G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) converting F6P to P6P via psicose 6-phosphate epimerase (P6PE); (v) converting P6P to psicose via psicose 6-phosphate phosphatase (P6PP); Includes.

[0367] Typically, the ratio of enzyme units used in the present production method is 1:1:1:1:1 (αGP:PGM:PGI:P6PE:P6PP). These ratios may be adjusted in any number of combinations to optimize product yield. For example, a ratio of 3:1:1:1:1 can be used to maximize the concentration of phosphorylated intermediates, which will increase the activity of downstream reactions. Conversely, a ratio of 1:1:1:1:3 can be used to ensure that phosphate is maintained for αGP, which will result in more efficient phosphorolytic cleavage of α-1,4-glycosidic bonds. For example, a ratio of 3:1:1:1:3 enzymes can be used to further increase the reaction rate. Thus, the ratio of enzymes, including other optional enzymes described below, can be varied to increase the efficiency of tagatose production. For example, a particular enzyme may be present in an amount of about 2-fold, 3-fold, 4-fold, 5-fold, etc., relative to the amount of other enzymes.

[0368] Psicose can also be produced from fructose. For example, this production method includes the following steps: producing F6P from fructose and polyphosphate by catalysis with polyphosphate fructokinase (PPFK); converting F6P to P6P by catalysis with P6PE; Conversion of P6P to psicose by catalysis with P6PP. The fructose can be produced, for example, by the enzymatic conversion of sucrose.

[0369] Psicose can be produced from sucrose. The production process involves the following enzymatic steps: generating G1P from sucrose and free phosphate by catalyzing with sucrose phosphorylase (SP); PGM-catalyzed conversion of G1P to G6P; converting G6P to F6P by catalysis with PGI; converting F6P to P6P by catalysis with P6PE; Conversion of P6P to psicose by catalysis with P6PP. Includes.

[0370] The phosphate ions produced during the conversion of P6P to psicose can then be recycled to the step of converting sucrose to G1P. Furthermore, the psicose yield can be increased by using PPFK and polyphosphate to produce F6P from fructose produced by the phosphorolytic cleavage of sucrose by SP.

[0371] The method for producing psicose includes the following steps: producing glucose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis; converting glucose to G6P by catalyzing at least one enzyme; producing fructose from polysaccharides and oligosaccharides by enzymatic or acid hydrolysis; converting fructose to G6P by catalysis with at least one enzyme; Examples of such polysaccharides and oligosaccharides are listed above.

[0372] Cellulose and its derived products can be converted to psicose through a series of steps. The process comprises the following steps: producing G1P from cellodextrin and cellobiose and free phosphate by catalyzing with cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively; PGM-catalyzed conversion of G1P to G6P; converting G6P to F6P by catalysis with PGI; converting F6P to P6P by catalysis with P6PE; Conversion of P6P to psicose by catalysis with P6PP. In this method, phosphate ions can be recycled by the above step of converting cellodextrin and cellobiose to G1P.

[0373] Psicose can be produced from glucose. This production method includes the following steps: Producing G6P from glucose and polyphosphate by catalysis with polyphosphate glucokinase (PPGK); converting G6P to F6P by catalysis with PGI; converting F6P to P6P by catalysis with P6PE; converting P6P to psicose by catalysis with P6PP; Includes.

[0374] Working Example Materials and Methods

[0375] Chemicals

[0376] Unless otherwise noted, all chemicals, including corn starch, soluble starch, maltodextrin, glucose, and filter paper, were of reagent grade or higher and were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Fisher Scientific (Pittsburgh, PA, USA). Restriction enzymes, T4 ligase, and Phusion DNA polymerase were purchased from New England Biolabs (Ipswich, MA, USA). Oligonucleotides were synthesized by either Integrated DNA Technologies (Coralville, IA, USA) or Eurofins MWG Operon (Huntsville, AL, USA). The regenerated amorphous cellulose used for the purification of the enzyme was prepared from Avicel PH105 (FMC BioPolymer, Philadelphia, PA, USA) by dissolving and regenerating it as described in Ye et al., Fusion of a family 9 cellulose-binding module improves catalytic potential of Clostridium thermocellum cellodextrin phosphorylase on insoluble cellulose. Appl. Microbiol. Biotechnol. 2011;92:551-560. Escherichia coli Sig10 (Sigma-Aldrich, St. Louis, MO, USA) was used as the host cell for DNA manipulation, and E. coli BL21(DE3) (Sigma-Aldrich, St. Louis, MO, USA) was used as the host cell for recombinant protein expression. 100 mg L -1 of ampicillin or 50 mgL -1ZYM-5052 medium containing either 100% ethanol or 100% kanamycin was used for E. coli cell growth and recombinant protein expression. Cellulase (catalog number: C2730) and pullulanase (catalog number: P1067) from Trichoderma reesei were purchased from Sigma-Aldrich (St. Louis, MO, USA) and manufactured by Novozymes (Franklinton, NC, USA). Maltose phosphorylase (catalog number: M8284) was purchased from Sigma-Aldrich.

[0377] Recombinant enzyme production and purification

[0378] E. coli BL21(DE3) strain carrying the protein expression plasmid was cultured at 100 mg / L. -1 of ampicillin or 50 mgL -1 The cells were incubated in a 1 L Erlenmeyer flask with 100 mL of ZYM-5052 medium containing either 0.1 mM NaCl or 0.1 mM kanamycin. The cells were grown for 16-24 hours at 37°C with rotary shaking at 220 rpm. The cells were harvested by centrifugation at 12°C and washed once with either 20 mM phosphate buffered saline (pH 7.5) containing 50 mM NaCl and 5 mM MgCl2 (heat precipitated and cellulose binding module) or 20 mM phosphate buffered saline (pH 7.5) containing 300 mM NaCl and 5 mM imidazole (Ni purified). The cell pellet was resuspended in the same buffer and lysed by sonication (Fisher Scientific Sonic Dismembrator Model 500; 5 sec pulse on, 10 sec off, 50% amplitude for a total of 21 min). After centrifugation, the protein of interest in the supernatant was purified.

[0379] Three different techniques were used to purify various recombinant proteins. His-tagged proteins were purified using Ni Sepharose 6 Fast Flow resin (GE Life Sciences, Marlborough, MA, USA). Fusion proteins containing a cellulose-binding module (CBM) and a self-cleaving intein were purified by high-affinity adsorption to large surface area regenerated amorphous cellulose. Extremely thermostable enzymes were purified using heat precipitation at 70–95°C for 5–30 min. The purity of the above recombinant proteins was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0380] Enzymes used and their activity assays

[0381] α-Glucan phosphorylase (αGP) from Thermotoga maritima (Uniprot ID G4FEH8) was used. Activity was assayed at 50°C in 50 mM sodium phosphate buffer (pH 7.2) containing 1 mM MgCl2 and 30 mM maltodextrin. The reaction was stopped by filtration of the enzyme using Vivaspin 2 concentrators (10,000 MWCO) (Vivaproducts, Inc., Littleton, MA, USA). Glucose 1-phosphate (G1P) was measured using a Glucose Hexokinase / G6PDH Assay Kit (Sigma Aldrich, Cat. No. GAHK20-1KT) supplemented with 25 U / mL phosphoglucomutase. Units (U) are reported as μm / min.

[0382] Phosphoglucomutase (PGM) from Thermococcus kodakaraensis (Uniprot ID Q68BJ6) was used. Activity was measured at 50°C in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2 and 5 mM G1P. The reaction was stopped by filtration of the enzyme using a Vivaspin 2 concentrator (10,000 MWCO). The product glucose 6-phosphate (G6P) was measured using a hexokinase / G6PDH assay kit (Sigma Aldrich, catalog number GAHK20-1KT).

[0383] Phosphoglucoisomerase (PGI) from two different sources was used: from Clostridium thermocellum (Uniprot ID A3DBX9) and from Thermus thermophilus (Uniprot ID Q5SLL6). Activity was measured at 50°C in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2 and 10 mM G6P. Reactions were stopped by filtration of the enzyme using Vivaspin 2 concentrators (10,000 MWCO). The product fructose 6-phosphate (F6P) was measured using a fructose 6-phosphate kinase (F6PK) / pyruvate dehydrogenase (PK) / lactate dehydrogenase (LD) coupled enzyme assay, in which a decrease in absorbance at 340 nm indicates the production of F6P. The 200 μL reaction mixture contained 50 mM HEPES (pH 7.2), 5 mM MgCl2, 10 mM G6P, 1.5 mM ATP, 1.5 mM phosphoenolpyruvate, 200 μM NADH, 0.1 U PGI, 5 U PK, and 5 U LD.

[0384] Recombinant cellodextrin phosphorylase and cellobiose phosphorylase from C. thermocellum are described in Ye et al., Spontaneous high-yield production of hydrogen from cellulosic materials and water catalyzed by enzyme cocktails. ChemSusChem 2009;2:149-152. Their activities were assayed as described.

[0385] Recombinant polyphosphate glucokinase from Thermobifida fusca YX has been described in Liao et al., One-step purification and immobilization of thermophilic polyphosphate glucokinase from Thermobifida fusca YX: glucose-6-phosphate generation without ATP. Appl. Microbiol. Biotechnol. 2012;93:1109-1117. Its activity was assayed as described.

[0386] Recombinant isoamylase from Sulfolobus tokodaii is described in Cheng et al., Doubling power output of starch biobattery treated by the most thermostable isoamylase from an archaeon Sulfolobus tokodaii. Scientific Reports 2015;5:13184. Its activity was assayed as described.

[0387] Recombinant 4-α-glucanoltransferase from Thermococcus litoralis is described in Jeon et al. 4-α-Glucanotransferase from the Hyperthermophilic Archaeon Thermococcus Litoralis. Eur. J. Biochem. 1997;248:171-178. Its activity was measured as described.

[0388] Sucrose phosphorylase from Thermoanaerobacterium thermosaccharolyticum (Uniprot ID D9TT09) was used (Verhaeghe et al. The quest for a thermostable sucrose phosphorylase reveals sucrose 6'-phosphate phosphorylase as a novel specificity. Appl Microbiol Biotechnol. 2014 Aug;98(16):7027-37). Its activity was measured in 50 mM HEPES buffer (pH 7.5) containing 10 mM sucrose and 12 mM organophosphate. Glucose 1-phosphate (G1P) was measured using a glucose hexokinase / G6PDH assay kit supplemented with 25 U / mL phosphoglucomutase, as was α-glucan phosphorylase.

[0389] Psicose 6-phosphate 3-epimerase (P6PE) (Uniprot ID D9TQJ4) from Thermoanaerobacterium thermosaccharolyticum was used. Activity was measured at 50 °C in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2, 500 μM CoCl2, 1 U / mL P6PP, and 10 mM F6P. The reaction was stopped by filtration of the enzyme using a Vivaspin 2 concentrator (10,000 MWCO). The product psicose 6-phosphate (P6P) was measured by detecting the release of free phosphate using psicose 6-phosphate phosphatase. To detect the release of free phosphate, 500 μL of a solution containing 0.1 M zinc acetate and 2 mM ammonium molybdate (pH 5) was added to 50 μL of the reaction solution. The solution was mixed, followed by the addition of 125 μl of 5% ascorbic acid (pH 5). The solution was mixed and then incubated at 30° C. for 20 minutes. The absorbance was read at 850 nm to measure the release of free phosphate. Psicose was then confirmed by HPLC using an Agilent HiPlex H column (samples and controls were measured at 0.6 mL / min 5 mM H2SO4 and 65° C.).

[0390] Allose 6-phosphate isomerase (A6PI) from Clostridium thermocellum (Uniprot ID W4V2C8) was used, which has the amino acid sequence shown in SEQ ID NO: 1. Activity was measured at 50°C in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2, 500 μM CoCl2, 1 U / mL P6PE, 1 U / mL A6PP, and 10 mM F6P. The reaction was stopped by filtration of the enzyme using a Vivaspin 2 concentrator (10,000 MWCO). The product allose 6-phosphate (A6P) was measured using allose 6-phosphate phosphatase and detecting the release of free phosphate as described for P6PE. Allose was confirmed by HPLC in the same manner as for psicose. Another A6PI may be used, for example A6PI from Symbiobacterium thermophilum (Uniprot ID Q67LX4) which has the amino acid sequence shown in SEQ ID NO: 2.

[0391] Allose 6-phosphate phosphatase (A6PP) (Uniprot ID S9SDA3) from Rubellimicrobium thermophilum, having the amino acid sequence shown in SEQ ID NO: 3, was used. Activity was measured at 50°C in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2, 500 μM CoCl2, 1 U / mL P6PE, 1 U / mL A6PI, and 10 mM F6P. The reaction was stopped by filtration of the enzyme using a Vivaspin 2 concentrator (10,000 MWCO). The product allose was measured by detecting the release of free phosphate as described for P6PE. Allose was confirmed by HPLC in the same manner as for psicose. Other A6PPs may also be used, such as A6PP derived from Thermotoga maritima having the amino acid sequence set forth in SEQ ID NO: 4 (Uniprot ID Q9X0Y1), A6PP derived from Thermoanaerobacterium saccharolyticum having the amino acid sequence set forth in SEQ ID NO: 5 (Uniprot ID I3VT81), A6PP derived from Streptomyces thermoautotrophicus having the amino acid sequence set forth in SEQ ID NO: 6 (Uniprot ID A0A132NF06), and A6PP derived from Sphaerobacter thermophilus having the amino acid sequence set forth in SEQ ID NO: 7 (Uniprot ID D1C7G9).

[0392] Mannose 6-phosphate isomerase (M6PI) from Pseudonocardia thermophila (Uniprot ID A0A1M6TLY7) with the amino acid sequence shown in SEQ ID NO: 8 was used. Activity was measured at 50°C in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2, 1 U / mL PGI, 1 U / mL M6PP, and 10 mM F6P. The reaction was stopped by filtration of the enzyme using a Vivaspin 2 concentrator (10,000 MWCO). The product mannose 6-phosphate (M6P) was measured using mannose 6-phosphate phosphatase (M6PP) and detecting the release of free phosphate as described for P6PE. Mannose was confirmed by HPLC in the same manner as for psicose. Other M6PIs may also be used, such as M6PI derived from Caldithrix abyssi (Uniprot ID H1XQS6) having the amino acid sequence set forth in SEQ ID NO: 9, M6PI derived from Myceliophthora thermophila (Uniprot ID G2Q982) having the amino acid sequence set forth in SEQ ID NO: 10, and M6PI derived from Treponema caldarium (Uniprot ID F8F1Z8) having the amino acid sequence set forth in SEQ ID NO: 11.

[0393] Mannose 6-phosphate phosphatase (M6PP) from Tepidimonas fonticaldi (Uniprot ID A0A1A6DSI3) was used, which has the amino acid sequence shown in SEQ ID NO: 12. Activity was measured at 50°C in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2 and 10 mM mannose 6-phosphate. The reaction was stopped by filtration of the enzyme using a Vivaspin 2 concentrator (10,000 MWCO). The product mannose was measured by detecting the release of free phosphate as described for P6PE. Mannose was confirmed by HPLC in the same manner as for psicose. Other M6PPs may also be used, such as M6PP from Thermomonas hydrothermalis (Uniprot ID A0A1M4UN08), which has the amino acid sequence shown in SEQ ID NO: 13, and M6PP from Sulfurivirga caldicuralii (Uniprot ID A0A1N6FCW3), which has the amino acid sequence shown in SEQ ID NO: 14.

[0394] Bifunctional phosphoglucose / phosphomannose isomerase (PGPMI) (Uniprot ID D7CPH7) from Syntrophothermus lipocalidus with the amino acid sequence shown in SEQ ID NO: 15 was used. Activity was measured at 50°C in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2, 1 U / mL M6PP, and 10 mM G6P. The reaction was stopped by filtration of the enzyme using a Vivaspin 2 concentrator (10,000 MWCO). The product M6P was measured using M6PP by detecting the release of free phosphate as described for P6PE. Mannose was confirmed by HPLC in the same manner as for psicose. Other PGPMIs may also be used, such as PGPMI from Schleiferia thermophila having the amino acid sequence shown in SEQ ID NO: 16 (Uniprot ID A0A085L170) and PGPMI from Thermodesulfobium narugense having the amino acid sequence shown in SEQ ID NO: 17 (Uniprot ID M1E6Z3).

[0395] Galactose 6-phosphate isomerase (Gal6PI) from Lactococcus lactis (obligate dimer; Uniprot IDs P23494 and P23495 with the amino acid sequences shown in SEQ ID NOs: 18 and 19, respectively) is used (van Rooijen et al. Molecular Cloning, Characterization, and Nucleotide Sequence of the Tagatose 6-Phosphate Pathway Gene Cluster of the Lactose Operon of Lactococcus Zactis. J. Biol. Chem. 1991;266:7176-7181). Activity is measured at 37° C. in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2, 1 U / mL fructose 6-phosphate 4-epimerase (F6PE), 1 U / mL galactose 6-phosphate phosphatase (Gal6PP), and 10 mM fructose 6-phosphate. The reaction is stopped by filtration of the enzyme using Vivaspin 2 concentrators (10,000 MWCO). The product galactose 6-phosphate (gal6P) is measured using Gal6PP and detecting the release of free phosphate as described for P6PE. Galactose is confirmed by HPLC in the same manner as for psicose.

[0396] Galactose 6-phosphate phosphatase (Gal6PP) (Uniprot ID Q8A2F3) from Bacteroides thetaiotaomicron, having the amino acid sequence shown in SEQ ID NO: 20, was used. Activity was measured at 50°C in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2 and 10 mM galactose 6-phosphate. The reaction was stopped by filtration of the enzyme using a Vivaspin 2 concentrator (10,000 MWCO). The product galactose was measured by detecting the release of free phosphate as described for P6PE. Galactose was confirmed by HPLC in the same manner as for psicose.

[0397] Fructose 6-phosphate phosphatase (F6PP) from Halothermothrix orenii (Uniprot ID B8CWV3) was used, which has the amino acid sequence shown in SEQ ID NO: 21. Activity was measured at 50°C in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2 and 10 mM fructose 6-phosphate. The reaction was stopped by filtration of the enzyme using a Vivaspin 2 concentrator (10,000 MWCO). The product fructose was measured by detecting the release of free phosphate as described for P6PE. Fructose was confirmed by HPLC in the same manner as for psicose.

[0398] Tagatose 6-phosphate phosphatase (T6PP) from Archaeoglobus fugidis (Uniprot ID A0A075WB87) was used. Activity was measured at 50°C in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2 and 10 mM T6P. The reaction was stopped by filtration of the enzyme using Vivaspin 2 concentrators (10,000 MWCO). Tagatose production was measured by detecting the release of free phosphate as described for F6PE.

[0399] Psicose 6-phosphate phosphatase (P6PP) from Clostridium thermocellum (UNIPROT ID A3DC21) was used. Activity was measured at 50°C in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2, 80 μM CoCl2, 1 U / mL P6PE, and 10 mM F6P. The reaction was stopped by filtration of the enzyme using Vivaspin 2 concentrators (10,000 MWCO). The product psicose was measured by detecting the release of free phosphate as described for P6PE.

[0400] The enzyme units used in each of the following examples may be increased or decreased to adjust reaction time as desired. For example, if it is desired to perform Example 9 in 8 hours instead of 24 hours, the units of the enzyme would be increased by approximately 3-fold. Conversely, if it is desired to perform Example 9 in 48 hours instead of 24 hours, the enzyme units can be decreased by approximately 2-fold. These examples illustrate how the amount of enzyme units can be used to increase or decrease reaction time while maintaining constant productivity.

[0401] All Products

[0402] Example 1

[0403] To verify the technical feasibility of enzymatic biosynthesis of fructose 6-phosphate from starch, three enzymes were recombinantly expressed: α-glucan phosphorylase from T. maritima (Uniprot ID G4FEH8), phosphoglucomutase from Thermococcus kodakaraensis (Uniprot ID Q68BJ6), and phosphoglucoisomerase from Clostridium thermocellum (Uniprot ID A3DBX9). The recombinant proteins were overexpressed in E. coli BL21(DE3) and purified as described above.

[0404] A 0.20 mL reaction mixture containing 10 g / L soluble starch, 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.5 mM ZnCl2, 0.01 U αGP, 0.01 U PGM, and 0.01 U PGI was incubated at 50° C. for 24 hours. The reaction was stopped by filtration of the enzyme using a Vivaspin 2 concentrator (10,000 MWCO). The product fructose 6-phosphate (F6P) was measured using a fructose 6-phosphate kinase (F6PK) / pyruvate dehydrogenase (PK) / lactate dehydrogenase (LD) coupled enzyme assay, in which a decrease in absorbance at 340 nm indicates the production of F6P, as described above. The final concentration of F6P after 24 hours was 3.6 g / L.

[0405] Example 2

[0406] A similar test to that in Example 1 (except for reaction temperature) was carried out at 40-80°C. It was found that 10 g / L soluble starch produced 0.9 g / L F6P at 40°C and 3.6 g / L F6P at 80°C after 40 hours of reaction. These results suggest that for this enzyme combination, increasing the reaction temperature increases the F6P yield, but that too high a temperature may impair some enzyme activity.

[0407] Example 3

[0408] It was found that an enzyme ratio of approximately 1:1:1 αGP:PGM:PGI at 80° C. produced F6P rapidly. It was noted that the enzyme ratio did not significantly affect the final F6P concentration if the reaction time was long enough. However, the enzyme ratio does affect the reaction rate and the overall cost of enzymes used in the system.

[0409] Example 4

[0410] A 0.20 mL reaction mixture containing 10 g / L maltodextrin, 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.5 mM ZnCl2, 0.01 U αGP, 0.01 U PGM, and 0.01 U PGI was incubated at 50° C. for 24 hours. The reaction was stopped by filtration of the enzyme using a Vivaspin 2 concentrator (10,000 MWCO). The product fructose 6-phosphate (F6P) was measured using a fructose 6-phosphate kinase (F6PK) / pyruvate dehydrogenase (PK) / lactate dehydrogenase (LD) coupled enzyme assay, in which a decrease in absorbance at 340 nm indicates the production of F6P, as described above. The final concentration of F6P after 24 hours was 3.6 g / L.

[0411] Example 5

[0412] To test the production of F6P from Avicel, cellulose was hydrolyzed at 50°C using Sigma cellulase. To remove β-glucosidase from commercial cellulase, 10 filter paper digestion units / mL of cellulase was mixed with 10 g / L of Avicel in an ice-water bath for 10 minutes. After centrifugation at 4°C, the supernatant containing β-glucosidase was decanted. The Avicel combined with cellulase containing endoglucanases and cellobiohydrolases was resuspended in citrate buffer (pH 4.8) and hydrolyzed at 50°C for 3 days. The cellulose hydrolysate was mixed with 5 U / mL cellodextrin phosphorylase, 5 U / L cellobiose phosphorylase, 5 U / mL αGP, 5 U / mL PGM, and 5 U / mL PGI in 100 mM HEPES buffer (pH 7.2) containing 10 mM phosphate, 5 mM MgCl2, and 0.5 mM ZnCl2. The reaction was carried out at 60°C for 72 hours, and a high concentration of F6P was observed (small amounts of glucose and no cellobiose were present). F6P was detected using the coupled enzyme assay described above. Glucose was detected using the hexokinase / G6PDH assay kit described above.

[0413] Example 6

[0414] To increase the F6P yield from Avicel, Avicel was pretreated with concentrated phosphoric acid to generate amorphous cellulose (RAC) as described by Zhang et al. A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid: evidence from enzymatic hydrolysis and supramolecular structure. Biomacromolecules 2006;7:644-648. To remove β-glucosidase from commercial cellulase, 10 filter paper dissolution units / mL of cellulase was mixed with 10 g / L of RAC in an ice-water bath for 5 min. After centrifugation at 4°C, the supernatant containing β-glucosidase was decanted. The RAC bound to cellulase containing endoglucanase and cellobiohydrolase was resuspended in citrate buffer (pH 4.8) and hydrolyzed at 50°C for 12 h. The RAC hydrolysate was mixed with 5 U / mL cellodextrin phosphorylase, 5 U / L cellobiose phosphorylase, 5 U / mL αGP, 5 U / mL PGM, and 5 U / mL PGI in 100 mM HEPES buffer (pH 7.2) containing 10 mM phosphate, 5 mM MgCl2, and 0.5 mM ZnCl2. The reaction was carried out at 60°C for 72 hours. No enzyme was added to convert glucose to F6P, so high concentrations of F6P and glucose were recovered. F6P was detected using the coupled enzyme assay described above. Glucose was detected using the hexokinase / G6PDH assay kit described above.

[0415] Example 7

[0416] To further increase the F6P yield from RAC, polyphosphate glucokinase and polyphosphate were added. To remove β-glucosidase from commercial cellulase, 10 filter paper digestion units / mL of cellulase was mixed with 10 g / L of RAC in an ice-water bath for 5 min. After centrifugation at 4°C, the supernatant containing β-glucosidase was decanted. The RAC bound to cellulase containing endoglucanase and cellobiohydrolase was resuspended in citrate buffer (pH 4.8) and incubated at 50°C for 12 h for hydrolysis. The RAC hydrolysate was mixed with 5 U / mL polyphosphate glucokinase, 5 U / mL cellodextrin phosphorylase, 5 U / L cellobiose phosphorylase, 5 U / mL αGP, 5 U / mL PGM, and 5 U / mL PGI in 100 mM HEPES buffer (pH 7.2) containing 50 mM polyphosphate, 10 mM phosphate, 5 mM MgCl2, and 0.5 mM ZnCl2. The reaction was carried out at 50°C for 72 hours. High concentrations of F6P were observed, with only small amounts of glucose present at this stage. F6P was detected using the coupled enzyme assay described above. Glucose was detected using the hexokinase / G6PDH assay kit described above.

[0417] Example 8

[0418] To determine the concentration range of phosphate buffered saline (PBS), 0.20 mL reaction mixtures containing 50 g / L maltodextrin, 6.25 mM, 12.5 mM, 25 mM, 37.5 mM, or 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.1 U αGP, 0.1 U PGM, and 0.1 U PGI were incubated at 50° C. for 6 hours. The reaction time was kept short to ensure that the reaction did not go to completion, so that differences in efficiency could be clearly seen. The amount of F6P produced was quantified using a fructose 6-phosphate kinase (F6PK) / pyruvate dehydrogenase (PK) / lactate dehydrogenase (LD) coupled enzyme assay, in which a decrease in absorbance at 340 nm indicates the production of F6P. Yields of 4.5 g / L, 5.1 g / L, 5.6 g / L, 4.8 g / L, or 4.9 g / L of F6P were obtained for reactions containing either 6.25 mM, 12.5 mM, 25 mM, 37.5 mM, or 50 mM phosphate buffered saline (pH 7.2), respectively (Table 1). These results indicate that a concentration of 25 mM PBS (pH 7.2) was ideal for these particular reaction conditions. It is important to note that even when using 6.25 mM PBS at pH 7.2, significant turnover occurs due to phosphate recycling. This indicates that the disclosed phosphate recycling method can keep phosphate concentrations low even at industrial volumetric productivity levels (e.g., 200-300 g / L maltodextrin). [Table 1]

[0419] Example 9

[0420] To determine the pH range of the cascade reaction, 0.20 mL of reaction mixture containing 50 g / L maltodextrin, 50 mM phosphate buffered saline (pH 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, or 7.3), 5 mM MgCl2, 0.02 U αGP, 0.02 U PGM, and 0.02 U PGI was incubated at 50° C. for 16 hours. The small number of units ensured that the reaction did not go to completion, so that differences in efficiency could be clearly seen. The amount of F6P produced was quantified as in Example 12. Yields of F6P of 4.0 g / L, 4.1 g / L, 4.2 g / L, 4.1 g / L, 4.4 g / L, 4.1 g / L, 3.8 g / L, or 4.0 g / L were obtained for reactions containing 50 mM phosphate buffered saline at pH 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, or 7.3, respectively (Table 2). These results indicate that although the system functions over a wide pH range, a pH of 6.8 was ideal for these particular reaction conditions. [Table 2]

[0421] Alose

[0422] Example 10

[0423] To verify the production of allose from F6P, 10 g / L of F6P was mixed with 1 U / mL of P6PE, 1 U / mL of A6PI, and 1 U / mL of A6PP in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2 and 500 μM of CoCl2. The reaction was incubated at 50°C for 3 hours. The conversion of F6P to allose was confirmed by HPLC (Agilent 1100 series) using an Agilent Hi-Plex H column and a refractive index detector. The above samples and controls were measured in 5 mM H2SO4 at 0.6 mL / min and 65°C.

[0424] Example 11

[0425] To verify the production of allose from maltodextrin, a 0.20 mL reaction mixture containing 20 g / L maltodextrin, 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 500 μM CoCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U P6PE, 0.05 U A6PI, and 0.05 U A6PP was incubated at 50° C. for 24 hours. The reaction was stopped by filtration of the enzyme using a Vivaspin 2 concentrator (10,000 MWCO). Allose was confirmed by HPLC as described in Example 10.

[0426] Example 12

[0427] A reaction mixture containing 200 g / L maltodextrin, 10 mM acetate buffer (pH 5.5), 5 mM MgCl2, and 0.1 g / L isoamylase was incubated at 80° C. for 24 hours. This was used to prepare another reaction mixture containing 20 g / L isoamylase-treated maltodextrin, 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 500 μM CoCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U P6PE, 0.05 U A6PI, and 0.05 U A6PP, which was then incubated at 50° C. for 24 hours. The production of allose was confirmed in the same manner as in Example 10.

[0428] Example 13

[0429] To further increase the allose yield from maltodextrin, 0.05 U of 4-glucan transferase (4GT) was added to the reaction mixture described in Example 11.

[0430] A 0.2 mL reaction mixture containing 20 g / L isoamylase-treated maltodextrin (see Example 12), 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 500 μM CoCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U P6PE, 0.05 U A6PI, 0.05 U A6PP, and 0.05 U 4GT was incubated at 50° C. for 24 hours. Allose production was confirmed as in Example 10.

[0431] Example 14

[0432] To further increase the allose yield from maltodextrin, 0.05 U of maltose phosphorylase is added to the reaction mixture described in Example 11.

[0433] Example 15

[0434] To further increase the allose yield from maltodextrin, 0.05 U of polyphosphate glucokinase and 75 mM polyphosphate are added to the reaction mixture described in Example 11.

[0435] Example 16

[0436] To produce allose from fructose, a reaction mixture containing 10 g / L fructose, 50 mM Tris buffer (pH 7.0), 75 mM polyphosphate, 5 mM MgCl2, 500 μM CoCl2, 0.05 U fructose polyphosphate kinase, 0.05 U P6PE, 0.05 U A6PI, and 0.05 U A6PP is incubated at 50° C. for 24 hours. The amount of allose produced is quantified as in Example 10.

[0437] Example 17

[0438] To produce allose from glucose, a reaction mixture containing 10 g / L glucose, 50 mM Tris buffer (pH 7.0), 75 mM polyphosphate, 5 mM MgCl2, 500 μM CoCl2, 0.05 U glucose polyphosphate kinase, 0.05 U PGI, 0.05 U P6PE, 0.05 U A6PI, and 0.05 U A6PP is incubated at 50° C. for 24 hours. The amount of allose produced is quantified as in Example 10.

[0439] Example 18

[0440] To produce allose from sucrose, a reaction mixture containing 10 g / L sucrose, 50 mM phosphate buffered saline (pH 7.0), 5 mM MgCl2, 500 μM CoCl2, 0.05 U sucrose phosphorylase, 0.05 U PGM, 0.05 U PGI, 0.05 U P6PE, 0.05 U A6PI, and 0.05 U A6PP is incubated at 50° C. for 24 hours. The amount of allose produced is quantified as in Example 10.

[0441] Example 19

[0442] To further increase the yield of allose from sucrose, 75 mM polyphosphate and 0.05 U of polyphosphate fructokinase are added to the reaction mixture of Example 18. The amount of allose produced is quantified as in Example 10.

[0443] Mannose

[0444] Example 20

[0445] To verify the production of mannose from F6P, 10 g / L of F6P was mixed with 1 U / mL of M6PI / PGPMI and 1 U / mL of M6PP in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2. The reaction was incubated at 50°C for 3 hours. The conversion of F6P to mannose was confirmed by HPLC (Agilent 1100 series) using an Agilent Hi-Plex H column and a refractive index detector. The above samples and controls were measured in 5 mM H2SO4 at 0.6 mL / min and 65°C.

[0446] Example 21

[0447] To verify the production of mannose from maltodextrin, a 0.20 mL reaction mixture containing 20 g / L maltodextrin, 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U M6PI / PGPMI (no PGI required for PGPMI), and 0.05 U M6PP was incubated at 50° C. for 24 hours. The reaction was stopped by filtration of the enzyme using a Vivaspin 2 concentrator (10,000 MWCO). Mannose was confirmed by HPLC as described in Example 20.

[0448] Example 22

[0449] A reaction mixture containing 200 g / L maltodextrin, 10 mM acetate buffer (pH 5.5), 5 mM MgCl2, and 0.1 g / L isoamylase was incubated at 80° C. for 24 hours. This was used to prepare another reaction mixture containing 20 g / L isoamylase-treated maltodextrin, 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U M6PI / PGPMI (PGI is not required in the case of PGPMI), and 0.05 U M6PP, which was incubated at 50° C. for 24 hours. Mannose production was confirmed in the same manner as in Example 20.

[0450] Example 23

[0451] To further increase the mannose yield from maltodextrin, 0.05 U of 4-glucan transferase (4GT) was added to the reaction described in Example 21.

[0452] A 0.2 mL reaction mixture containing 20 g / L isoamylase-treated maltodextrin (see Example 22), 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U M6PI / PGPMI (PGI not required for PGPMI), 0.05 U M6PP, and 0.05 U 4GT was incubated for 24 hours at 50° C. Mannose production was confirmed as in Example 20.

[0453] Example 24

[0454] To further increase the mannose yield from maltodextrin, 0.05 U of maltose phosphorylase is added to the reaction mixture described in Example 21.

[0455] Example 25

[0456] To further increase the mannose yield from maltodextrin, 0.05 U of polyphosphate glucokinase and 75 mM polyphosphate are added to the reaction mixture described in Example 21.

[0457] Example 26

[0458] To produce mannose from fructose, a reaction mixture containing 10 g / L fructose, 50 mM Tris buffer (pH 7.0), 75 mM polyphosphate, 5 mM MgCl2, 0.05 U fructose polyphosphate kinase, 0.05 U M6PI / PGPMI (PGI is not required in the case of PGPMI), and 0.05 U M6PP is incubated at 50° C. for 24 hours. The amount of mannose produced is quantified as in Example 20.

[0459] Example 27

[0460] To produce mannose from glucose, a reaction mixture containing 10 g / L glucose, 50 mM Tris buffer (pH 7.0), 75 mM polyphosphate, 5 mM MgCl2, 0.05 U glucose polyphosphate kinase, 0.05 U PGI, 0.05 U M6PI / PGPMI (PGI is not required in the case of PGPMI), and 0.05 U M6PP is incubated for 24 hours at 50° C. The amount of mannose produced is quantified as in Example 20.

[0461] Example 28

[0462] To produce mannose from sucrose, a reaction mixture containing 10 g / L sucrose, 50 mM phosphate buffered saline (pH 7.0), 5 mM MgCl2, 0.05 U sucrose phosphorylase, 0.05 U PGM, 0.05 U PGI, 0.05 U M6PI / PGPMI (PGI is not required in the case of PGPMI), and 0.05 U M6PP is incubated for 24 hours at 50° C. The amount of mannose produced is quantified as in Example 20.

[0463] Example 29

[0464] To further increase the yield of mannose from sucrose, 75 mM polyphosphate and 0.05 U of polyphosphate fructokinase are added to the reaction mixture of Example 28. The amount of mannose produced is quantified as in Example 20.

[0465] Galactose

[0466] Example 30

[0467] To verify the production of galactose from F6P, 10 g / L of F6P is mixed with 1 U / mL of Gal6PI and 1 U / mL of Gal6PP in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2. The reaction is incubated at 37°C for 3 hours. The conversion of F6P to galactose is confirmed by HPLC (Agilent 1100 series) using an Agilent Hi-Plex H column and a refractive index detector. The samples and controls are measured in 5 mM H2SO4 at 0.6 mL / min and 65°C.

[0468] Example 31

[0469] To verify the production of galactose from maltodextrin, 0.20 mL of reaction mixture containing 20 g / L maltodextrin, 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U Gal6PI, and 0.05 U Gal6PP is incubated at 37° C. for 24 hours. The reaction is stopped by filtration of the enzyme using a Vivaspin 2 concentrator (10,000 MWCO). Galactose is confirmed by HPLC as described in Example 30.

[0470] Example 32

[0471] A reaction mixture containing 200 g / L maltodextrin, 10 mM acetate buffer (pH 5.5), 5 mM MgCl2, and 0.1 g / L isoamylase is incubated at 80° C. for 24 hours. This is used to prepare another reaction mixture containing 20 g / L isoamylase-treated maltodextrin, 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U Gal6PI, and 0.05 U Gal6PP, which is incubated at 37° C. for 24 hours. Galactose production is confirmed as in Example 30.

[0472] Example 33

[0473] To further increase the galactose yield from maltodextrin, 0.05 U of 4-glucan transferase (4GT) is added to the reaction described in Example 31.

[0474] A 0.2 mL reaction mixture containing 20 g / L isoamylase-treated maltodextrin (see Example 12), 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U Gal6PI, 0.05 U Gal6PP, and 0.05 U 4GT is incubated for 24 hours at 37° C. Galactose production is confirmed as in Example 30.

[0475] Example 34

[0476] To further increase the galactose yield from maltodextrin, 0.05 U of maltose phosphorylase is added to the reaction mixture described in Example 31.

[0477] Example 35

[0478] To further increase the galactose yield from maltodextrin, 0.05 U of polyphosphate glucokinase and 75 mM polyphosphate are added to the reaction mixture described in Example 31.

[0479] Example 36

[0480] To produce galactose from fructose, a reaction mixture containing 10 g / L fructose, 50 mM Tris buffer (pH 7.0), 75 mM polyphosphate, 5 mM MgCl2, 0.05 U fructose polyphosphate kinase, 0.05 U Gal6PI, and 0.05 U Gal6PP is incubated for 24 hours at 37° C. The amount of galactose produced is quantified as in Example 30.

[0481] Example 37

[0482] To produce galactose from glucose, a reaction mixture containing 10 g / L glucose, 50 mM Tris buffer (pH 7.0), 75 mM polyphosphate, 5 mM MgCl2, 0.05 U glucose polyphosphate kinase, 0.05 U PGI, 0.05 U Gal6PI, and 0.05 U Gal6PP is incubated for 24 hours at 37° C. The amount of galactose produced is quantified as in Example 30.

[0483] Example 38

[0484] To produce galactose from sucrose, a reaction mixture containing 10 g / L sucrose, 50 mM phosphate buffered saline (pH 7.0), 5 mM MgCl2, 0.05 U sucrose phosphorylase, 0.05 U PGM, 0.05 U PGI, 0.05 U Gal6PI, and 0.05 U Gal6PP is incubated for 24 hours at 37° C. The amount of galactose produced is quantified as in Example 30.

[0485] Example 39

[0486] To further increase the yield of galactose from sucrose, 75 mM polyphosphate and 0.05 U of polyphosphate fructokinase are added to the reaction mixture of Example 38. The amount of galactose produced is quantified as in Example 30.

[0487] Example 40

[0488] To verify the production of galactose from Gal6P, 10 g / L Gal6P was mixed with 1 U / mL Gal6PP in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2. The reaction was incubated at 50° C. for 1 hour. The conversion of Gal6P to galactose is confirmed by detection of free phosphate. To detect the release of free phosphate, 500 μL of a solution containing 0.1 M zinc acetate and 2 mM ammonium molybdate (pH 5) was added to 50 μL of the reaction. The solution was mixed, followed by the addition of 125 μl of 5% ascorbic acid (pH 5). The solution was mixed and then incubated at 30° C. for 20 minutes. The absorbance at 850 nm was read to measure the release of free phosphate.

[0489] Fructose

[0490] Example 41

[0491] To verify the production of fructose from F6P, 10 g / L of F6P was mixed with 1 U / mL of F6PP in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2. The reaction was incubated at 50°C for 3 hours. The conversion of F6P to fructose was confirmed by HPLC (Agilent 1100 series) using an Agilent Hi-Plex H column and a refractive index detector. The samples and controls were measured in 5 mM H2SO4 at 0.6 mL / min and 65°C.

[0492] Example 42

[0493] To verify the production of fructose from maltodextrin, a 0.20 mL reaction mixture containing 20 g / L maltodextrin, 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, and 0.05 U F6PP was incubated at 50° C. for 24 hours. The reaction was stopped by filtration of the enzyme using a Vivaspin 2 concentrator (10,000 MWCO). Fructose was confirmed by HPLC as described in Example 41.

[0494] Example 43

[0495] A reaction mixture containing 200 g / L maltodextrin, 10 mM acetate buffer (pH 5.5), 5 mM MgCl2, and 0.1 g / L isoamylase was incubated at 80° C. for 24 hours. This was used to prepare another reaction mixture containing 20 g / L isoamylase-treated maltodextrin, 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, and 0.05 U F6PP, which was incubated at 50° C. for 24 hours. Fructose production was confirmed as in Example 41.

[0496] Example 44

[0497] To further increase the fructose yield from maltodextrin, 0.05 U of 4-glucan transferase (4GT) was added to the reaction described in Example 42.

[0498] A 0.2 mL reaction mixture containing 20 g / L isoamylase-treated maltodextrin (see Example 12), 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PP, and 0.05 U 4GT was incubated for 24 hours at 50° C. Fructose production was confirmed as in Example 41.

[0499] Example 45

[0500] To further increase the fructose yield from maltodextrin, 0.05 U of maltose phosphorylase is added to the reaction mixture described in Example 42.

[0501] Example 46

[0502] To further increase the fructose yield from maltodextrin, 0.05 U of polyphosphate glucokinase and 75 mM polyphosphate are added to the reaction mixture described in Example 42.

[0503] Example 47

[0504] To produce fructose from glucose, a reaction mixture containing 10 g / L glucose, 50 mM Tris buffer (pH 7.0), 75 mM polyphosphate, 5 mM MgCl2, 0.05 U glucose polyphosphate kinase, 0.05 U PGI, and 0.05 U F6PP is incubated for 24 hours at 50° C. The amount of fructose produced is quantified as in Example 41.

[0505] Example 48

[0506] To produce fructose from sucrose, a reaction mixture containing 10 g / L sucrose, 50 mM phosphate buffered saline (pH 7.0), 5 mM MgCl2, 0.05 U sucrose phosphorylase, 0.05 U PGM, 0.05 U PGI, and 0.05 U F6PP was incubated for 24 hours at 50° C. The amount of fructose produced was quantified as in Example 41.

[0507] Altrose

[0508] Example 49

[0509] To verify the production of altrose from F6P, 10 g / L of F6P is mixed with 1 U / mL of P6PE, 1 U / mL of altrose 6-phosphate isomerase (Alt6PI), and 1 U / mL of altrose 6-phosphate phosphatase (Alt6PP) in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2. The reaction is incubated at 50°C for 3 hours. The conversion of F6P to altrose is confirmed by HPLC (Agilent 1100 series) using an Agilent Hi-Plex H column and a refractive index detector. The samples and controls are measured in 5 mM H2SO4 at 0.6 mL / min and 65°C.

[0510] Example 50

[0511] To verify the production of altrose from maltodextrin, 0.20 mL of reaction mixture containing 20 g / L maltodextrin, 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U P6PE, 0.05 U Alt6PI, and 0.05 U Alt6PP is incubated at 50° C. for 24 hours. The reaction is stopped by filtration of the enzyme using a Vivaspin 2 concentrator (10,000 MWCO). The altrose is confirmed by HPLC as described in Example 49.

[0512] Example 51

[0513] A reaction mixture containing 200 g / L maltodextrin, 10 mM acetate buffer (pH 5.5), 5 mM MgCl2, and 0.1 g / L isoamylase is incubated at 80° C. for 24 hours. This is used to prepare another reaction mixture containing 20 g / L isoamylase-treated maltodextrin, 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U P6PE, 0.05 U Alt6PI, and 0.05 U Alt6PP, which is incubated at 50° C. for 24 hours. The production of altrose is confirmed as in Example 49.

[0514] Example 52

[0515] To further increase the altrose yield from maltodextrin, 0.05 U of 4-glucan transferase (4GT) is added to the reaction described in Example 50.

[0516] A 0.2 mL reaction mixture containing 20 g / L isoamylase-treated maltodextrin (see Example 50), 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U P6PE, 0.05 U Alt6PI, 0.05 U Alt6PP, and 0.05 U 4GT is incubated for 24 hours at 50° C. The production of altrose is confirmed as in Example 49.

[0517] Example 53

[0518] To further increase the altrose yield from maltodextrin, 0.05 U of maltose phosphorylase is added to the reaction mixture described in Example 50.

[0519] Example 54

[0520] To further increase the altrose yield from maltodextrin, 0.05 U of polyphosphate glucokinase and 75 mM polyphosphate are added to the reaction mixture described in Example 50.

[0521] Example 55

[0522] To produce altrose from fructose, a reaction mixture containing 10 g / L fructose, 50 mM Tris buffer (pH 7.0), 75 mM polyphosphate, 5 mM MgCl2, 0.05 U fructose polyphosphate kinase, 0.05 U P6PE, 0.05 U Alt6PI, and 0.05 U Alt6PP is incubated at 50° C. for 24 hours. The amount of altrose produced is quantified as in Example 49.

[0523] Example 56

[0524] To produce altrose from glucose, a reaction mixture containing 10 g / L glucose, 50 mM Tris buffer (pH 7.0), 75 mM polyphosphate, 5 mM MgCl2, 0.05 U glucose polyphosphate kinase, 0.05 U PGI, 0.05 U P6PE, 0.05 U Alt6PI, and 0.05 U Alt6PP is incubated at 50° C. for 24 hours. The amount of altrose produced is quantified as in Example 49.

[0525] Example 57

[0526] To produce altrose from sucrose, a reaction mixture containing 10 g / L sucrose, 50 mM phosphate buffered saline (pH 7.0), 5 mM MgCl2, 0.05 U sucrose phosphorylase, 0.05 U PGM, 0.05 U PGI, 0.05 U P6PE, 0.05 U Alt6PI, and 0.05 U Alt6PP is incubated for 24 hours at 50° C. The amount of altrose produced is quantified as in Example 49.

[0527] Example 58

[0528] To further increase the yield of altose from sucrose, 75 mM polyphosphate and 0.05 U of polyphosphate fructokinase are added to the reaction mixture of Example 56. The amount of altose produced is quantified as in Example 49.

[0529] Talos

[0530] Example 59

[0531] To verify the production of talose from F6P, 10 g / L of F6P is mixed with 1 U / mL of F6PE, 1 U / mL of talose 6-phosphate isomerase (Tal6PI), and 1 U / mL of talose 6-phosphate phosphatase (Tal6PP) in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2. The reaction is incubated at 50°C for 3 hours. The conversion of F6P to talose is confirmed by HPLC (Agilent 1100 series) using an Agilent Hi-Plex H column and a refractive index detector. The samples and controls are measured in 5 mM H2SO4 at 0.6 mL / min and 65°C.

[0532] Example 60

[0533] To verify the production of talose from maltodextrin, 0.20 mL of reaction mixture containing 20 g / L maltodextrin, 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U Tal6PI, and 0.05 U Tal6PP is incubated at 50° C. for 24 hours. The reaction is stopped by filtration of the enzyme using a Vivaspin 2 concentrator (10,000 MWCO). The talose is confirmed by HPLC as described in Example 59.

[0534] Example 61

[0535] A reaction mixture containing 200 g / L maltodextrin, 10 mM acetate buffer (pH 5.5), 5 mM MgCl2, and 0.1 g / L isoamylase is incubated at 80° C. for 24 hours. This is used to prepare another reaction mixture containing 20 g / L isoamylase-treated maltodextrin, 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U Tal6PI, and 0.05 U Tal6PP, which is incubated at 50° C. for 24 hours. The production of talose is confirmed as in Example 59.

[0536] Example 62

[0537] To further increase the talose yield from maltodextrin, 0.05 U of 4-glucan transferase (4GT) is added to the reaction described in Example 60.

[0538] A 0.2 mL reaction mixture containing 20 g / L isoamylase-treated maltodextrin (see Example 60), 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U Tal6PI, 0.05 U Tal6PP, and 0.05 U 4GT is incubated for 24 hours at 50° C. The production of talose is confirmed as in Example 59.

[0539] Example 63

[0540] To further increase the talose yield from maltodextrin, 0.05 U of maltose phosphorylase is added to the reaction mixture described in Example 59.

[0541] Example 64

[0542] To further increase the talose yield from maltodextrin, 0.05 U of polyphosphate glucokinase and 75 mM polyphosphate are added to the reaction mixture described in Example 60.

[0543] Example 65

[0544] To produce talose from fructose, a reaction mixture containing 10 g / L fructose, 50 mM Tris buffer (pH 7.0), 75 mM polyphosphate, 5 mM MgCl2, 0.05 U fructose polyphosphate kinase, 0.05 U F6PE, 0.05 U Tal6PI, and 0.05 U Tal6PP is incubated at 50° C. for 24 hours. The amount of talose produced is quantified as in Example 59.

[0545] Example 66

[0546] To produce talose from glucose, a reaction mixture containing 10 g / L glucose, 50 mM Tris buffer (pH 7.0), 75 mM polyphosphate, 5 mM MgCl2, 0.05 U glucose polyphosphate kinase, 0.05 U PGI, 0.05 U F6PE, 0.05 U Tal6PI, and 0.05 U Tal6PP is incubated at 50° C. for 24 hours. The amount of talose produced is quantified as in Example 59.

[0547] Example 67

[0548] To produce talose from sucrose, a reaction mixture containing 10 g / L sucrose, 50 mM phosphate buffered saline (pH 7.0), 5 mM MgCl2, 0.05 U sucrose phosphorylase, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U Tal6PI, and 0.05 U Tal6PP is incubated for 24 hours at 50° C. The amount of talose produced is quantified as in Example 59.

[0549] Example 68

[0550] To further increase the yield of talose from sucrose, 75 mM polyphosphate and 0.05 U of polyphosphate fructokinase are added to the reaction mixture of Example 66. The amount of talose produced is quantified as in Example 59.

[0551] Sorbose

[0552] Example 69

[0553] To verify the production of sorbose from F6P, 10 g / L of F6P is mixed with 1 U / mL of F6PE, 1 U / mL of sorbose 6-phosphate 3-epimerase (S6PE), and 1 U / mL of sorbose 6-phosphate phosphatase (S6PP) in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2. The reaction is incubated at 50°C for 3 hours. The conversion of F6P to sorbose is confirmed by HPLC (Agilent 1100 series) using an Agilent Hi-Plex H column and a refractive index detector. The samples and controls are measured in 5 mM H2SO4 at 0.6 mL / min and 65°C.

[0554] Example 70

[0555] To verify the production of sorbose from maltodextrin, a 0.20 mL reaction mixture containing 20 g / L maltodextrin, 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE, and 0.05 U S6PP is incubated at 50° C. for 24 hours. The reaction is stopped by filtration of the enzyme using a Vivaspin 2 concentrator (10,000 MWCO). Sorbose is confirmed by HPLC as described in Example 68.

[0556] Example 71

[0557] A reaction mixture containing 200 g / L maltodextrin, 10 mM acetate buffer (pH 5.5), 5 mM MgCl2, and 0.1 g / L isoamylase is incubated at 80° C. for 24 hours. This is used to prepare another reaction mixture containing 20 g / L isoamylase-treated maltodextrin, 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE, and 0.05 U S6PP, which is incubated at 50° C. for 24 hours. The production of sorbose is confirmed as in Example 69.

[0558] Example 72

[0559] To further increase the yield of sorbose from maltodextrin, 0.05 U of 4-glucan transferase (4GT) is added to the reaction described in Example 70.

[0560] A 0.2 mL reaction mixture containing 20 g / L isoamylase-treated maltodextrin (see Example 70), 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE, 0.05 U S6PP, and 0.05 U 4GT is incubated for 24 hours at 50° C. The production of sorbose is confirmed as in Example 69.

[0561] Example 73

[0562] To further increase the yield of sorbose from maltodextrin, 0.05 U of maltose phosphorylase is added to the reaction mixture described in Example 70.

[0563] Example 74

[0564] To further increase the yield of sorbose from maltodextrin, 0.05 U of polyphosphate glucokinase and 75 mM polyphosphate are added to the reaction mixture described in Example 69.

[0565] Example 75

[0566] To produce sorbose from fructose, a reaction mixture containing 10 g / L fructose, 50 mM Tris buffer (pH 7.0), 75 mM polyphosphate, 5 mM MgCl2, 0.05 U fructose polyphosphate kinase, 0.05 U F6PE, 0.05 U S6PE, and 0.05 U S6PP is incubated for 24 hours at 50° C. The amount of sorbose produced is quantified as in Example 69.

[0567] Example 76

[0568] To produce sorbose from glucose, a reaction mixture containing 10 g / L glucose, 50 mM Tris buffer (pH 7.0), 75 mM polyphosphate, 5 mM MgCl2, 0.05 U glucose polyphosphate kinase, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE, and 0.05 U S6PP is incubated for 24 hours at 50° C. The amount of sorbose produced is quantified as in Example 69.

[0569] Example 77

[0570] To produce sorbose from sucrose, a reaction mixture containing 10 g / L sucrose, 50 mM phosphate buffered saline (pH 7.0), 5 mM MgCl2, 0.05 U sucrose phosphorylase, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE, and 0.05 U S6PP is incubated for 24 hours at 50° C. The amount of sorbose produced is quantified as in Example 69.

[0571] Example 78

[0572] To further increase the yield of sorbose from sucrose, 75 mM polyphosphate and 0.05 U of polyphosphate fructokinase are added to the reaction mixture of Example 76. The amount of sorbose produced is quantified as in Example 69.

[0573] Growth

[0574] Example 79

[0575] To verify the production of gulose from F6P, 10 g / L of F6P is mixed with 1 U / mL of F6PE, 1 U / mL of S6PE, 1 U / mL of gulose 6-phosphate isomerase (Gul6PI), and 1 U / mL of gulose 6-phosphate phosphatase (Gul6PP) in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2. The reaction is incubated at 50°C for 3 hours. The conversion of F6P to gulose is confirmed by HPLC (Agilent 1100 series) using an Agilent Hi-Plex H column and a refractive index detector. The above samples and controls are measured in 5 mM H2SO4 at 0.6 mL / min and 65°C.

[0576] Example 80

[0577] To verify the production of growth from maltodextrin, a 0.20 mL reaction mixture containing 20 g / L maltodextrin, 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE, 0.05 U Gul6PI, and 0.05 U Gul6PP is incubated at 50° C. for 24 hours. The reaction is stopped by filtration of the enzyme using a Vivaspin 2 concentrator (10,000 MWCO). Growth is confirmed by HPLC as described in Example 79.

[0578] Example 81

[0579] A reaction mixture containing 200 g / L maltodextrin, 10 mM acetate buffer (pH 5.5), 5 mM MgCl2, and 0.1 g / L isoamylase is incubated at 80° C. for 24 hours. This is used to prepare another reaction mixture containing 20 g / L isoamylase-treated maltodextrin, 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE, 0.05 U Gul6PI, and 0.05 U Gul6PP, which is incubated at 50° C. for 24 hours. Growth generation is confirmed as in Example 79.

[0580] Example 82

[0581] To further increase the growth yield from maltodextrin, 0.05 U of 4-glucan transferase (4GT) is added to the reaction described in Example 80.

[0582] A 0.2 mL reaction mixture containing 20 g / L isoamylase-treated maltodextrin (see Example 80), 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE, 0.05 U Gul6PI, 0.05 U Gul6PP, and 0.05 U 4GT is incubated for 24 hours at 50° C. Growth is confirmed as in Example 79.

[0583] Example 83

[0584] To further increase the growth yield from maltodextrin, 0.05 U of maltose phosphorylase is added to the reaction mixture described in Example 80.

[0585] Example 84

[0586] To further increase the growth yield from maltodextrin, 0.05 U of polyphosphate glucokinase and 75 mM polyphosphate are added to the reaction mixture described in Example 80.

[0587] Example 85

[0588] To produce gulose from fructose, a reaction mixture containing 10 g / L fructose, 50 mM Tris buffer (pH 7.0), 75 mM polyphosphate, 5 mM MgCl2, 0.05 U fructose polyphosphate kinase, 0.05 U F6PE, 0.05 U S6PE, 0.05 U Gul6PI, and 0.05 U Gul6PP is incubated for 24 hours at 50° C. The amount of gulose produced is quantified as in Example 79.

[0589] Example 86

[0590] To produce gulose from glucose, a reaction mixture containing 10 g / L glucose, 50 mM Tris buffer (pH 7.0), 75 mM polyphosphate, 5 mM MgCl2, 0.05 U glucose polyphosphate kinase, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE, 0.05 U Gul6PI, and 0.05 U Gul6PP is incubated for 24 hours at 50° C. The amount of gulose produced is quantified as in Example 79.

[0591] Example 87

[0592] To produce gulose from sucrose, a reaction mixture containing 10 g / L sucrose, 50 mM phosphate buffered saline (pH 7.0), 5 mM MgCl2, 0.05 U sucrose phosphorylase, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE, 0.05 U Gul6PI, and 0.05 U Gul6PP is incubated for 24 hours at 50° C. The amount of gulose produced is quantified as in Example 79.

[0593] Example 88

[0594] To further increase the yield of gulose from sucrose, 75 mM polyphosphate and 0.05 U of polyphosphate fructokinase are added to the reaction mixture of Example 86. The amount of gulose produced is quantified as in Example 79.

[0595] Idos

[0596] Example 89

[0597] To verify the production of idose from F6P, 10 g / L of F6P is mixed with 1 U / mL of F6PE, 1 U / mL of S6PE, 1 U / mL of idose 6-phosphate isomerase (I6PI), and 1 U / mL of idose 6-phosphate phosphatase (I6PP) in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2. The reaction is incubated at 50°C for 3 hours. The conversion of F6P to idose is confirmed by HPLC (Agilent 1100 series) using an Agilent Hi-Plex H column and a refractive index detector. The above samples and controls are measured in 5 mM H2SO4 at 0.6 mL / min and 65°C.

[0598] Example 90

[0599] To verify the production of idose from maltodextrin, a 0.20 mL reaction mixture containing 20 g / L maltodextrin, 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE, 0.05 U I6PI, and 0.05 U I6PP is incubated at 50° C. for 24 hours. The reaction is stopped by filtration of the enzyme using a Vivaspin 2 concentrator (10,000 MWCO). Idose is confirmed by HPLC as described in Example 89.

[0600] Example 91

[0601] A reaction mixture containing 200 g / L maltodextrin, 10 mM acetate buffer (pH 5.5), 5 mM MgCl2, and 0.1 g / L isoamylase is incubated at 80° C. for 24 hours. This is used to prepare another reaction mixture containing 20 g / L isoamylase-treated maltodextrin, 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE, 0.05 U I6PI, and 0.05 U I6PP, which is incubated at 50° C. for 24 hours. The production of idose is confirmed as in Example 89.

[0602] Example 92

[0603] To further increase the idose yield from maltodextrin, 0.05 U of 4-glucan transferase (4GT) is added to the reaction described in Example 90.

[0604] A 0.2 mL reaction mixture containing 20 g / L isoamylase-treated maltodextrin (see Example 90), 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE, 0.05 U I6PI, 0.05 U I6PP, and 0.05 U 4GT is incubated for 24 hours at 50° C. The production of idose is confirmed as in Example 89.

[0605] Example 93

[0606] To further increase the idose yield from maltodextrin, 0.05 U of maltose phosphorylase is added to the reaction mixture described in Example 90.

[0607] Example 94

[0608] To further increase the idose yield from maltodextrin, 0.05 U of polyphosphate glucokinase and 75 mM polyphosphate are added to the reaction mixture described in Example 90.

[0609] Example 95

[0610] To produce idose from fructose, a reaction mixture containing 10 g / L fructose, 50 mM Tris buffer (pH 7.0), 75 mM polyphosphate, 5 mM MgCl2, 0.05 U fructose polyphosphate kinase, 0.05 U F6PE, 0.05 U S6PE, 0.05 U I6PI, and 0.05 U I6PP is incubated at 50° C. for 24 hours. The amount of idose produced is quantified as in Example 89.

[0611] Example 96

[0612] To produce idose from glucose, a reaction mixture containing 10 g / L glucose, 50 mM Tris buffer (pH 7.0), 75 mM polyphosphate, 5 mM MgCl2, 0.05 U glucose polyphosphate kinase, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE, 0.05 U I6PI, and 0.05 U I6PP is incubated for 24 hours at 50° C. The amount of idose produced is quantified as in Example 89.

[0613] Example 97

[0614] To produce idose from sucrose, a reaction mixture containing 10 g / L sucrose, 50 mM phosphate buffered saline (pH 7.0), 5 mM MgCl2, 0.05 U sucrose phosphorylase, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U S6PE, 0.05 U I6PI, and 0.05 U I6PP is incubated for 24 hours at 50° C. The amount of idose produced is quantified as in Example 89.

[0615] Example 98

[0616] To further increase the yield of idose from sucrose, 75 mM polyphosphate and 0.05 U of polyphosphate fructokinase are added to the reaction mixture of Example 96. The amount of idose produced is quantified as in Example 89.

[0617] Tagatose

[0618] Example 99

[0619] To verify the production of tagatose from F6P, 2 g / L of F6P was mixed with 1 U / mL of fructose 6-phosphate epimerase (F6PE) and 1 U / mL of tagatose 6-phosphate phosphatase (T6PP) in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2. The reaction was incubated at 50°C for 16 hours. 100% conversion of F6P to tagatose was confirmed by HPLC (Agilent 1100 series) using an Agilent Hi-Plex H column and a refractive index detector. The samples were run at 0.6 mL / min in 5 mM H2SO4.

[0620] Example 100

[0621] To verify the production of tagatose from maltodextrin, 0.20 mL of reaction mixture containing 20 g / L maltodextrin, 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, and 0.05 U T6PP was incubated at 50° C. for 24 hours. The reaction was stopped by filtration of the enzyme using a Vivaspin 2 concentrator (10,000 MWCO). Tagatose was detected and quantified using an Agilent 1100 series HPLC equipped with a refractive index detector and an Agilent Hi-Plex H column. The mobile phase was 5 mM H2SO4, and the mobile phase was run at 0.6 mL / min. A yield of 9.2 g / L of tagatose was obtained. This is equivalent to 92% of the theoretical yield due to the limited decomposition of maltodextrin in the absence of enzymes such as isoamylase or 4-glucan transferase. Standard samples of tagatose with various concentrations were used to quantify the yield of our experiments.

[0622] Example 101

[0623] A reaction mixture containing 200 g / L maltodextrin, 10 mM acetate buffer (pH 5.5), 5 mM MgCl2, and 0.1 g / L isoamylase was incubated at 80° C. for 24 hours. This was used to prepare another reaction mixture containing 20 g / L isoamylase-treated maltodextrin, 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, and 0.05 U T6PP, which was incubated at 50° C. for 24 hours. The amount of tagatose produced was quantified as in Example 99. Pretreatment of maltodextrin with isoamylase increased the yield of tagatose to 16 g / L, which is equivalent to 80% of the theoretical yield.

[0624] Example 102

[0625] To further increase the yield of tagatose from maltodextrin, 0.05 U of 4-glucan transferase (4GT) was added to the reaction mixture described in Example 100.

[0626] A 0.2 mL reaction mixture containing 20 g / L isoamylase-treated maltodextrin (see Example 9), 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U F6PE, 0.05 U T6PP, and 0.05 U 4GT was incubated at 50° C. for 24 hours. The amount of tagatose produced was quantified as in Example 9. The addition of 4GT to IA-treated maltodextrin increased the yield of tagatose to 17.7 g / L, which is equivalent to 88.5% of the theoretical yield.

[0627] Example 103

[0628] A 20 mL reaction mixture containing 50 g / L isoamylase-treated maltodextrin (see Example 99), 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 10 U αGP, 10 U PGM, 10 U PGI, 10 U F6PE, and 10 U T6PP was incubated at 50° C. for 24 hours. The amount of tagatose produced was quantified as in Example 8. The yield of tagatose was 37.6 g / L at 20 mL scale and 50 g / L maltodextrin, which is equivalent to 75% of the theoretical yield. These results indicate that there is no significant decrease in yield when scaling up to a larger reaction volume.

[0629] Example 104

[0630] To further increase the yield of tagatose from maltodextrin, 0.05 U of maltose phosphorylase is added to the reaction mixture described in Example 100.

[0631] Example 105

[0632] To further increase the yield of tagatose from maltodextrin, 0.05 U of polyphosphate glucokinase and 75 mM polyphosphate are added to the reaction mixture described in Example 99.

[0633] Example 106

[0634] To produce tagatose from fructose, the reaction mixture containing 10 g / L fructose, 50 mM Tris buffer (pH 7.0), 75 mM polyphosphate, 5 mM MgCl2, 0.05 U fructose polyphosphate kinase, 0.05 U F6PE, and 0.05 U T6PP is incubated at 50° C. for 24 hours. The amount of tagatose produced is quantified in the same manner as in Example 100.

[0635] Example 107

[0636] To produce tagatose from glucose, the reaction mixture containing 10g / L glucose, 50mM Tris buffer (pH 7.0), 75mM polyphosphate, 5mM MgCl2, 0.05U glucose polyphosphate kinase, 0.05U PGI, 0.05U F6PE and 0.05U T6PP is incubated at 50°C for 24 hours. The amount of tagatose produced is quantified in the same manner as in Example 100.

[0637] Example 108

[0638] To produce tagatose from sucrose, the reaction mixture containing 10g / L sucrose, 50mM phosphate buffered saline (pH 7.0), 5mM MgCl2, 0.05U sucrose phosphorylase, 0.05U PGM, 0.05U PGI, 0.05U F6PE and 0.05U T6PP is incubated at 50°C for 24 hours. The amount of tagatose produced is quantified in the same manner as in Example 100.

[0639] Example 109

[0640] In order to further increase the yield of tagatose from sucrose, 75 mM polyphosphate and 0.05 U polyphosphate fructokinase are added to the reaction mixture of Example 15. The amount of tagatose produced is quantified in the same manner as in Example 100.

[0641] Psicose

[0642] Example 110

[0643] To verify the production of psicose from F6P, 2 g / L of F6P was mixed with 1 U / mL of P6PE and 1 U / mL of P6PP in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl2 and 80 μM CoCl2. The reaction solution was incubated at 50° C. for 6 hours. 99% conversion of F6P to psicose was confirmed by HPLC (Agilent 1100 series) using an Agilent Hi-Plex H column and a refractive index detector. The above sample was measured in 5 mM H2SO4 at 0.6 mL / min.

[0644] Example 111

[0645] To verify the production of psicose from maltodextrin, 0.20 mL of reaction mixture containing 20 g / L maltodextrin, 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 80 μM CoCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U P6PE, and 0.05 U P6PP is incubated at 50° C. for 24 hours. The reaction is stopped by filtration of the enzyme using a Vivaspin 2 concentrator (10,000 MWCO). Psicose is detected and quantified using an Agilent 1100 series HPLC equipped with a refractive index detector and an Agilent Hi-Plex H column. The mobile phase is 5 mM H2SO4, and the mobile phase is run at 0.6 mL / min. Standard samples of psicose with different concentrations were used to quantify the yield of our experiments.

[0646] Example 112

[0647] A reaction mixture containing 200 g / L maltodextrin, 10 mM acetate buffer (pH 5.5), 5 mM MgCl2, 80 μM CoCl2, and 0.1 g / L isoamylase is incubated at 80° C. for 24 hours. This is used to prepare another reaction mixture containing 20 g / L isoamylase-treated maltodextrin, 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U P6PE, and 0.05 U P6PP, which is incubated at 50° C. for 24 hours. The amount of psicose produced is quantified in the same manner as in Example 111.

[0648] Example 113

[0649] A reaction mixture containing 200 g / L maltodextrin, 10 mM acetate buffer (pH 4.5), 5 mM MgCl2, and 1:200 diluted Novozymes D6 pullulanase is incubated for 4 hours at 50° C. This is used to make another reaction mixture containing 20 g / L pullulanase-treated maltodextrin, 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 80 μM CoCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U P6PE, and 0.05 U P6PP, which is incubated for 24 hours at 50° C. The amount of psicose produced is quantified as in Example 111.

[0650] Example 114

[0651] To further increase the psicose yield from maltodextrin, 0.05 U of 4-glucan transferase (4GT) is added to the reaction solution described in Example 111.

[0652] A 0.2 mL reaction mixture containing 20 g / L isoamylase-treated maltodextrin (see Example 9), 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 80 μM CoCl2, 0.05 U αGP, 0.05 U PGM, 0.05 U PGI, 0.05 U P6PE, 0.05 U P6PP, and 0.05 U 4GT is incubated for 24 hours at 50° C. The amount of psicose produced is quantified as in Example 111.

[0653] Example 115

[0654] To study scale-up, a 20 mL reaction mixture containing 50 g / L isoamylase-treated maltodextrin (see Example 10), 50 mM phosphate buffered saline (pH 7.2), 5 mM MgCl2, 80 μM CoCl2, 10 U αGP, 10 U PGM, 10 U PGI, 10 U P6PE, and 10 U P6PP was incubated at 50° C. for 24 hours. The amount of psicose produced was quantified in the same manner as in Example 111.

[0655] Example 116

[0656] To further increase the psicose yield from maltodextrin, 0.05 U of maltose phosphorylase is added to the reaction solution described in Example 110.

[0657] Example 117

[0658] To further increase the psicose yield from maltodextrin, 0.05 U of polyphosphate glucokinase and 75 mM polyphosphate are added to the reaction solution described in Example 111.

[0659] Example 118

[0660] To produce psicose from fructose, a reaction mixture containing 10 g / L fructose, 50 mM Tris buffer (pH 7.0), 75 mM polyphosphate, 5 mM MgCl2, 80 μM CoCl2, 0.05 U fructose polyphosphate kinase, 0.05 U P6PE, and 0.05 U P6PP is incubated at 50° C. for 24 hours. The amount of psicose produced is quantified in the same manner as in Example 111.

[0661] Example 119

[0662] To produce psicose from glucose, a reaction mixture containing 10 g / L glucose, 50 mM Tris buffer (pH 7.0), 75 mM polyphosphate, 5 mM MgCl2, 80 μM CoCl2, 0.05 U glucose polyphosphate kinase, 0.05 U PGI, 0.05 U P6PE, and 0.05 U P6PP is incubated for 24 hours at 50° C. The amount of psicose produced is quantified in the same manner as in Example 111.

[0663] Example 120

[0664] To produce psicose from sucrose, a reaction mixture containing 10 g / L sucrose, 50 mM phosphate buffered saline (pH 7.0), 5 mM MgCl2, 80 μM CoCl2, 0.05 U sucrose phosphorylase, 0.05 U PGM, 0.05 U PGI, 0.05 U P6PE, and 0.05 U P6PP is incubated for 24 hours at 50° C. The amount of psicose produced is quantified in the same manner as in Example 111.

[0665] Example 121

[0666] To further increase the yield of psicose from sucrose, 75 mM polyphosphate and 0.05 U of polyphosphate fructokinase are added to the reaction mixture of Example 20. The amount of psicose produced is quantified in the same manner as in Example 111.

[0667] The present invention encompasses substantially all of the embodiments and variations described hereinabove and with reference to the examples and drawings. Although various embodiments of the invention are disclosed herein, modifications and variations can be made within the scope of the invention in accordance with the ordinary general knowledge of those skilled in the art.

Claims

1. A method for producing hexose from sugar, comprising the steps of: converting fructose 6-phosphate (F6P) to said hexose by catalysis with one or more enzymes; the hexose is selected from the group consisting of allose, mannose, galactose, fructose, altrose, talose, sorbose, gulose, and idose; The enzyme is selected from the group consisting of isomerases, epimerases, and hexose-specific phosphatases, and mixtures thereof. The method.

2. the hexose is allose, converting the F6P into psicose 6-phosphate (P6P) by catalyzing with psicose 6-phosphate 3-epimerase (P6PE); converting the P6P to allose 6-phosphate (A6P) by catalyzing with allose 6-phosphate isomerase (A6PI); converting said A6P to allose by catalysis with allose 6-phosphate phosphatase (A6PP); The method of claim 1 , comprising:

3. the hexose is mannose, converting said F6P to mannose 6-phosphate (M6P) by catalysis with mannose 6-phosphate isomerase (M6PI) or phosphoglucose / phosphomannose isomerase (PGPMI); converting said M6P to mannose by catalysis with mannose 6-phosphate phosphatase (M6PP); The method of claim 1 , comprising:

4. the hexose is galactose; converting said F6P to tagatose 6-phosphate (T6P) by catalysis with fructose 6-phosphate 4-epimerase (F6PE); converting the T6P to galactose 6-phosphate (Gal6P) by catalysis with galactose 6-phosphate isomerase (Gal6PI); converting said Gal6P to galactose by catalysis with galactose 6-phosphate phosphatase (Gal6PP); The method of claim 1 , comprising:

5. the hexose is fructose, 2. The method of claim 1, comprising converting said F6P to fructose by catalysis with fructose 6-phosphate phosphatase (F6PP).

6. the hexose is altrose, converting the F6P into psicose 6-phosphate (P6P) by catalyzing with psicose 6-phosphate 3-epimerase (P6PE); converting said P6P to altrose 6-phosphate (Alt6P) by catalyzing with altrose 6-phosphate isomerase (Alt6PI); converting the generated Alt6P to altrose by catalyzing with altrose 6-phosphate phosphatase (Alt6PP); The method of claim 1 , comprising:

7. the hexose is talose, converting said F6P to tagatose 6-phosphate (T6P) by catalyzing with fructose 6-phosphate 4-epimerase (F6PE); converting the T6P to talose 6-phosphate (Tal6P) by catalysis with talose 6-phosphate isomerase (Tal6PI); converting said Tal6P to talose by catalysis with talose 6-phosphate phosphatase (Tal6PP); The method of claim 1 , comprising:

8. the hexose is sorbose, converting said F6P to tagatose 6-phosphate (T6P) by catalyzing with fructose 6-phosphate 4-epimerase (F6PE); converting the T6P to sorbose 6-phosphate (S6P) by catalyzing a sorbose 6-phosphate epimerase (S6PE); converting said S6P to sorbose by catalysis with sorbose 6-phosphate phosphatase (S6PP); The method of claim 1 , comprising:

9. the hexose is gulose, converting said F6P to tagatose 6-phosphate (T6P) by catalyzing with fructose 6-phosphate 4-epimerase (F6PE); converting the T6P to sorbose 6-phosphate (S6P) by catalyzing a sorbose 6-phosphate epimerase (S6PE); converting said S6P to gulose 6-phosphate (Gul6P) by catalyzing with gulose 6-phosphate isomerase (Gul6PI); converting said Gul6P to Gulose by catalysis with Gul6PP; The method of claim 1 , comprising:

10. the hexose is idose, converting said F6P to tagatose 6-phosphate (T6P) by catalyzing with fructose 6-phosphate 4-epimerase (F6PE); converting the T6P to sorbose 6-phosphate (S6P) by catalyzing a sorbose 6-phosphate epimerase (S6PE); converting said S6P to idose 6-phosphate (I6P) by catalyzing with idose 6-phosphate isomerase (I6PI); converting said I6P to idose by catalysis with idose 6-phosphate phosphatase (I6PP); The method of claim 1 , comprising:

11. 11. The method of any one of claims 1 to 10, further comprising the step of converting glucose 6-phosphate (G6P) to said F6P, said step being catalyzed by phosphoglucoisomerase (PGI).

12. 12. The method of claim 11, further comprising the step of converting glucose 1-phosphate (G1P) to said G6P, said step being catalyzed by phosphoglucomutase (PGM).

13. 13. The method of claim 12, further comprising a step of converting sugars to said G1P, said step being catalyzed by at least one enzyme, and said sugars being selected from the group consisting of starch or derivatives thereof, and sucrose.

14. The method of claim 13, wherein the at least one enzyme in the step of converting sugars to G1P is selected from the group consisting of α-glucan phosphorylase (αGP), maltose phosphorylase, and sucrose phosphorylase, and mixtures thereof.

15. 15. The method of claim 13 or 14, wherein the sugar is a starch or a derivative thereof selected from the group consisting of amylose, amylopectin, soluble starch, amylodextrin, maltodextrin, maltose, and glucose, and mixtures thereof.

16. 16. The method of claim 15 further comprising the step of converting the starch to a starch derivative produced by enzymatic hydrolysis of starch or acid hydrolysis of starch.

17. The method according to claim 15 or 16, wherein 4-glucan transferase (4GT) is added to the method.

18. 18. The method of any one of claims 13 to 17, wherein the starch derivative is produced by enzymatic hydrolysis of starch catalyzed by isoamylase, pullulanase, α-amylase, or a combination thereof.

19. the A6PI comprises an amino acid sequence having at least 55% sequence identity with SEQ ID NO: 1 or 2; The method of claim 2, wherein the A6PI catalyzes the conversion of the P6P to A6P.

20. The A6PI is A Rossmann-like fold for catalysis, the His C-terminus of the first β-strand of the Rossmann-like fold; an Arg C-terminal to the α-helix C-terminal of the fifth β-strand of the Rossmann-like fold; His in the active site; Cys and Thr in the active site; a GTG hydrophobic G motif near the active site; Asn near the active site 20. The method of claim 19, comprising the Rossmann-like fold having the formula:

21. The A6PP comprises an amino acid sequence having at least 30% sequence identity to any one of SEQ ID NOs: 3 to 7; 21. The method of any one of claims 2, 19, and 20, wherein the A6PP catalyzes the conversion of the A6P to allose.

22. The A6PP is a Rossmann-like fold domain for catalysis; A C1 capping domain; and A DxD signature in the first β-strand of the Rossmann-like fold; Thr or Ser at the end of the second β-strand of the Rossmann-like fold; Lys at the N-terminus of the α-helix C-terminus of the third β-strand of the Rossmann-like fold; the ED signature at the end of the fourth β-strand of the Rossmann-like fold; 22. The method of claim 21 , comprising:

23. The M6PI comprises an amino acid sequence having at least 25% sequence identity to any one of SEQ ID NOs: 8 to 11; The method of claim 3, wherein the M6PI catalyzes the conversion of the F6P to M6P.

24. 24. The method of claim 23, wherein the M6PI contains two domains with an antiparallel β-stranded core similar to the cuprin fold and a third domain consisting only of α-helices, and a divalent metal cation.

25. the PGPMI comprises an amino acid sequence having at least 25% sequence identity to any one of SEQ ID NOs: 15-17; 25. The method of any one of claims 3, 23, or 24, wherein the PGPMI catalyzes the conversion of the F6P to M6P.

26. The PGPMI is 2 Rossman folds, The GGS motif, SYSG-X-T-X-ET-hydrophobic motif; EN signature in which the presence of Glu results in active site base proton transfer; The presence of HIS leads to ring-opening / ring-closure of the substrate during catalytic reaction.

26. The method of claim 25, comprising:

27. The M6PP comprises an amino acid sequence having at least 30% sequence identity to any one of SEQ ID NOs: 12 to 14; 27. The method of any one of claims 3 and 23-26, wherein the M6PP catalyzes the conversion of the M6P to mannose.

28. The M6PP is a Rossmann-like fold domain for catalysis; A C1 capping domain; and A DxD signature in the first β-strand of the Rossmann-like fold; Thr or Ser at the end of the second β-strand of the Rossmann-like fold; Lys at the N-terminus of the α-helix C-terminus of the third β-strand of the Rossmann-like fold; A GDxxxD signature at the end of the fourth β-strand of the Rossmann-like fold; 28. The method of claim 27, comprising:

29. said F6PP comprises an amino acid sequence having at least 25% sequence identity with SEQ ID NO:21; The method of claim 5 , wherein the F6PP catalyzes the conversion of the F6P to fructose.

30. The F6PP is a Rossmann-like fold domain for catalysis; A C1 capping domain; and A DxD signature in the first β-strand of the Rossmann-like fold; Thr or Ser at the end of the second β-strand of the Rossmann-like fold; Lys at the N-terminus of the α-helix C-terminus of the third β-strand of the Rossmann-like fold; the ED signature at the end of the fourth β-strand of the Rossmann-like fold; 34. The method of claim 33, comprising:

31. said Gal6PI is a multimer of two subunits, Lac A and Lac B; said Lac A comprises an amino acid sequence having at least 25% sequence identity to SEQ ID NO: 18; said Lac B comprising an amino acid sequence having at least 25% sequence identity to SEQ ID NO:19; The Gal6PI catalyzes the conversion of the T6P to Gal6P. The method according to claim 4.

32. The Gal6PI is a heterodimer ("A" and "B"), Rossman-like αβα sandwich fold, Arg130 and Arg134 in "A" and His9 and Arg39 in "B" for binding the phosphate group of the substrate; His96 in "A" for ring opening of the substrate; Asn97 in "A" to stabilize a high energy intermediate; Cys65 and Thr67 in "B" for participating in proton transfer; 32. The method of claim 31 , wherein the heterodimer comprises a subunit having the formula:

33. the Gal6PP comprises an amino acid sequence having at least 25% sequence identity with SEQ ID NO: 20; The Gal6PP catalyzes the conversion of the Gal6P to galactose.

33. The method of any one of claims 4, 31 and 32.

34. The Gal6PP is a Rossmann-like fold domain for catalysis; A C2 capping domain; and A DxD signature in the first β-strand of the Rossmann-like fold; Thr or Ser at the end of the second β-strand of the Rossmann-like fold; A GDxxxD signature at the end of the fourth β-strand of the Rossmann-like fold; 34. The method of claim 33, comprising:

35. the hexose is fructose, the sugar is sucrose; The at least one enzyme is sucrose phosphorylase. The method of claim 14.

36. 29. The method of any one of claims 3 and 23-28, further comprising converting said G6P to F6P catalyzed by PGPMI, wherein F6P is converted to M6P by PGPMI.

37. 37. The method of any one of claims 1 to 36, wherein the process steps are carried out at a temperature in the range of about 40°C to about 70°C, at a pH in the range of about 5.0 to about 8.0, and / or for about 8 hours to about 48 hours.

38. 38. The method of any one of claims 1 to 37, wherein the process steps are carried out in a single bioreactor or in multiple bioreactors arranged in series.

39. 39. The method of any one of claims 1 to 38, wherein process steps are carried out without ATP, without NAD(P)(H), at a phosphate concentration of about 0.1 mM to about 150 mM, said phosphate is recycled, and / or at least one of said process steps comprises an energetically favorable chemical reaction.

40. A hexose produced by the method according to any one of claims 1 to 39, wherein the hexose is selected from the group consisting of allose, mannose, galactose, fructose, altrose, talose, sorbose, gulose, and idose.

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