RU2819709C2 - Enzymatic production of d-allulose - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed is a method of producing allulose, involving conversion of fructose-6-phosphate (F6P) to allulose-6-phosphate (A6P), catalysed by allulose-6-phosphate-3-epimerase (A6PE); and conversion of the obtained A6P into allulose, catalysed by allulose-6-phosphatase (A6PP). A6PE contains an amino acid sequence having at least 55% sequence identity to SEQ ID NO: 3 or 6, and A6PP comprises an amino acid sequence having at least 55% sequence identity to SEQ ID NO: 9.

EFFECT: invention provides efficient production of allulose.

17 cl, 7 dwg, 2 tbl, 21 ex

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[1] This application claims priority to US Serial No. 62/434,033, filed December 14, 2016, which is incorporated herein by reference.

FIELD OF THE INVENTION

[2] The invention relates to the production of D-allulose sugar. More specifically, the invention relates to methods for producing D-allulose by enzymatically converting saccharides (eg, polysaccharides, oligosaccharides, disaccharides, sucrose, D-glucose and D-fructose) into D-allulose.

BACKGROUND OF THE INVENTION

[3] D-allulose (also known as D-psicose) (hereinafter referred to as allulose) is a low-calorie, natural sweetener that has 70% of the sweetness of sucrose, but only 10% of the calories. It is a naturally occurring monosaccharide hexose that is present only in small quantities in wheat and other plants. Allulose was approved as a dietary supplement by the Food and Drug Administration (FDA) in 2012 and is classified as Generally Recognized as Safe (GRAS). However, due to the high selling prices of allulose, its use as a sweetener is limited. Allulose has many health benefits: it is low in calories (10% of sucrose); it has a very low glycemic index of 1; it is completely absorbed in the small intestine but is not metabolized and is instead secreted into urine and feces; it helps regulate blood sugar by inhibiting α-amylase, sucrase and maltase; and in foods and beverages it has functionality similar to sucrose. As such, allulose clearly has various applications in the food and beverage industry.

[4] Currently, allulose is produced mainly through the enzymatic isomerization of fructose (WO 2014049373). In general, the process suffers from increased feedstock costs, costly separation of allulose from fructose, and relatively low product yield.

[5] There is a need to develop a cost-effective synthetic route to produce allulose in high yield, where at least one step of the process involves an energetically favorable chemical reaction. In addition, there is a need for a production method in which the process steps can be carried out in a single tank or bioreactor. There is also a need for a process for producing allulose that can be carried out at a relatively low concentration of phosphate, where the phosphate can be recycled, and/or a process that does not require the use of adenosine triphosphate (ATP) as a source of phosphate. There is also a need for a route to allulose that does not require the use of the expensive coenzyme nicotinamide adenosine dinucleotide (NAD(H)) at any stage of the reaction.

SUMMARY OF THE INVENTION

[6] The inventions described herein relate to a process for producing allulose. Various aspects of the method include the conversion of fructose 6-phosphate (F6P) to allulose 6-phosphate (A6P) catalyzed by allulose 6-phosphate 3-epimerase (A6PE); and the conversion of A6P to allulose, catalyzed by allulose-6-phosphate phosphatase (A6PP). The inventions also relate to allulose produced by any of the methods described herein.

[7] In some aspects of the invention, the method for producing allulose also includes the step of converting glucose-6-phosphate (G6P) to F6P, where the step is catalyzed by phosphoglucoisomerase (PGI). In other aspects, the allulose synthesis method also includes the step of converting glucose-1-phosphate (G1P) to G6P, and this conversion step is catalyzed by phosphoglucomutase (PGM).

[8] In various aspects, a method for producing allulose may comprise converting a saccharide to G1P catalyzed by at least one enzyme; the conversion of G1P to G6P, catalyzed by phosphoglucomutase (PGM); the conversion of G6P to F6P, catalyzed by phosphoglucoisomerase (PGI); conversion of F6P to allulose 6-phosphate (A6P) catalyzed by A6PE; and converting the resulting A6P to allulose, catalyzed by A6PP.

[9] The saccharides used in any of the methods can be selected from the group consisting of starch or a derivative thereof, cellulose or a derivative thereof, and sucrose. The starch or derivative thereof may be amylose, amylopectin, soluble starch, amylodextrin, maltodextrin, maltose or glucose. In some aspects of the invention, a method for producing allulose includes converting starch into a starch derivative by enzymatic hydrolysis or by acid hydrolysis of starch. In other aspects, a starch derivative can be produced by enzymatic hydrolysis of starch catalyzed by isoamylase, pullulanase, α-amylase, or a combination of two or more of these enzymes. The method for producing allulose, in certain aspects, may also include the addition of 4-glucan transferase (4GT).

[10] In various aspects, a method for producing allulose may comprise converting fructose to F6P catalyzed by at least one enzyme; conversion of F6P to allulose 6-phosphate (A6P) catalyzed by A6PE; and converting the resulting A6P to allulose, catalyzed by A6PP. In other embodiments, a method for producing allulose comprises converting sucrose to fructose catalyzed by at least one enzyme; converting fructose to F6P catalyzed by at least one enzyme; conversion of F6P to allulose 6-phosphate (A6P) catalyzed by A6PE; and converting the resulting A6P to allulose, catalyzed by A6PP.

[11] In other aspects of the invention, G6P to be used in a process for producing allulose can be created by converting glucose to G6P catalyzed by at least one enzyme. Glucose can in turn be produced by the conversion of sucrose to glucose catalyzed by at least one enzyme.

[12] In other aspects of the invention, the allulose process steps are conducted without ATP, without NAD(H), at a phosphate concentration of from about 0 mM to about 150 mM, the phosphate is recycled, and/or at least one process step includes an energetically favorable chemical reaction.

BRIEF DESCRIPTION OF THE FIGURES

[13] These drawings illustrate certain aspects of some of the embodiments of the invention and should not be used as a limitation or definition of the invention.

[14] In FIG. 1 is a schematic diagram illustrating the enzymatic pathway for converting fructose 6-phosphate to allulose 6-phosphate and then to allulose.

[15] In FIG. 2 is a schematic diagram illustrating the enzymatic pathway for converting starch or its derivatives into allulose. The following abbreviations are used: αGP, α-glucan phosphorylase or starch phosphorylase; PGM, phosphoglucomutase; PGI, phosphoglucoisomerase; IA, isoamylase; PA, pullulanase; MP, maltose phosphorylase; PPGK, polyphosphate glucokinase.

[16] In FIG. Figure 3 shows the enzymatic pathway for converting cellulose or its derivative products into allulose. CDP, cellodextrin phosphorylase; CBP, cellobiose phosphorylase; PPGK, polyphosphate glucokinase; PGM, phosphoglucomutase; PGI, phosphoglucoisomerase.

[17] In FIG. 4 is a schematic diagram illustrating the enzymatic pathway for converting fructose to allulose. PPFK, polyphosphate fructokinase.

[18] In FIG. 5 is a schematic diagram illustrating the enzymatic pathway for converting glucose to allulose. PPGK, polyphosphate glucokinase; PGI, phosphoglucoisomerase.

[19] In FIG. 6 shows the enzymatic pathway for converting sucrose or its derivative products into allulose. SP, sucrose phosphorylase; PPFK, polyphosphate fructokinase; PGM, phosphoglucomutase; PGI, phosphoglucoisomerase.

[20] In FIG. 7 presents the Gibbs energy for the reaction between intermediates based on the Gibbs energy for formation in the conversion of glucose-1-phosphate to allulose.

DETAILED DESCRIPTION OF THE INVENTION

[21] The invention relates to enzymatic routes or methods for the synthesis of allulose with high product yield while significantly reducing the cost of isolating the product and the cost of producing allulose.

[22] The invention relates to a method for producing allulose, where the method includes the conversion of fructose-6-phosphate (F6P) to allulose-6-phosphate (A6P), catalyzed by epimerase, and the conversion of the resulting A6P to allulose, catalyzed by phosphatase (eg , allulose-6-phosphate phosphatase, A6PP). This method is generally shown in FIG. 1. In certain embodiments, the epimerase that catalyzes the conversion of F6P to A6P is allulose-6-phosphate-3-epimerase (A6PE).

[23] Epimerases that convert F6P to A6P can be used in the method of the invention. Epimerases are also capable of converting A6P to F6P. In some aspects of the invention, epimerases suitable for use in methods for converting F6P to A6P contain an amino acid sequence that has a degree of identity with the amino acid sequence of SEQ ID Nos. 3 or 6 of 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%, at least 91%, at least 92%, at least 93% or at least 94%, most preferably at least 95% and even most preferably at least 96, 97, 98, 99 or 100%. A suitable epimerase encodes a polynucleotide containing a nucleotide sequence that has a degree of identity with the nucleotide sequence of SEQ ID Nos. 1, 2, 4 and 5 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% and even most preferably at least 96, 97, 98, 99 or 100%.

[24] Examples of A6PE include, but are not limited to, the following proteins identified by UNIPROT ID numbers: D9TQJ4, A0A090IXZ8 and P32719. Among them, D9TQJ4 and A0A090IXZ8 are derived from thermophilic organisms. P32719 is obtained from a mesophilic organism. P32719 is 53% identical to A0A090IXZ8 and 55% identical to D9TQJ4, and each protein catalyzes the epimerization of F6P to A6P. Additionally, A0A090IXZ8 is 45% identical to D9TQJ4. In contrast, other epimerase proteins, identified by UNIPROT ID numbers: A0A101D823, R1AXD6, A0A150LBU8, A0A023CQG9, and H1XWY2, which share a degree of identity with D9TQJ4 of 45% or less, do not catalyze the epimerization of F6P to A6P.

[25] In some aspects of the invention, epimerases useful in methods for converting F6P to A6P use a divalent metal cofactor: preferably, but not limited to, cobalt. In further aspects of the invention, the epimerase comprises, but is not limited to, an (α/β) ₈ -barrel domain for catalysis; additionally, but not limited to, contains a phosphate binding domain containing Ser at the end of the 7th β-barrel strand, Ser at the end of the 8th β-barrel strand, and Gly in the active site loop; additionally, but not limited to, contains a metal-binding domain containing His in the 2nd and 3rd β-barrel strands; additionally, but not limited to, contains Asp in the 2nd and 7th β-strand of the barrel to act as an acid/base catalyst for 1,1 proton transfer, and additionally, but not limited to, contains a His-hydrophobic signature residue-Asp in the 2nd β-strand of the barrel, where His is involved in metal and Asp binding for acid/base catalysis. These features are known in the art and are mentioned, for example, in Chan et al., Structural Basis for Substrate Specificity in Phosphate Binding (beta/alpha)8-Barrels: D-Allulose 6-Phosphate 3-Epimerase from Escherichia coli K-12. Biochemistry. 2008; 47 (36); 9608-9617. Preferably, the epimerase for use in the methods of the invention contains an (α/β) _8- barrel domain for catalysis, Ser at the end of the 7th β-barrel strand, Ser at the end of the 8th β-barrel strand, Gly in the active site loop, His in the 2nd and 3rd barrel β-strands, Asp in the 2nd and 7th barrel β-strands, and the His-hydrophobic residue-Asp signature in the 2nd barrel β-strand.

[26] The methods of the invention use phosphatases that convert A6P to allulose (D-allulose). In some aspects of the invention, phosphatases suitable for the method for converting A6P to allulose contain an amino acid sequence that has a degree of identity with the amino acid sequence of SEQ ID No. 9 of 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 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 at least 96, 97, 98, 99 or 100%. A suitable epimerase encodes a polynucleotide containing a nucleotide sequence that has a degree of identity with the nucleotide sequence of SEQ ID Nos. 7 and 8 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% and even most preferably at least 96, 97, 98, 99 or 100% .

[27] Examples of A6PP include, but are not limited to, the following proteins, identified by UNIPROT ID numbers: A3DC21, Q5LGR4 and Q89ZR1. A3DC21 is 46% identical to Q5LGR4 and 45% identical to Q89ZR1, and each protein catalyzes the specific dephosphorylation of A6P to allulose. Conversely, other phosphatases from the halogen acid dehydrogenase superfamily, proteins identified by UNIPROT ID numbers: H0UQ29, Q67LU4, A0A0K6IPM3, C8WSJ0, A0A151YX61 and others, which are less than 45% identical to A3DC21, do not catalyze the specific dephosphorylation of A6P to allulose.

[28] Phosphatases for converting A6P to allulose suitable for use in the methods of the invention have specificity for allulose-6-phosphate. As used herein, allulose-6-phosphate specificity refers to having a higher specific activity for allulose-6-phosphate compared to glucose-1-phosphate, glucose-6-phosphate, or fructose-6-phosphate.

[29] Phosphatases use a divalent metal cofactor to convert A6P to allulose: preferably magnesium. In additional aspects of the invention, the phosphatase comprises, but is not limited to, a Rossmann-like fold domain for catalysis; additionally, but not limited to, contains a C1 capping domain for substrate specificity; additionally, but not limited to, contains a DxD signature in the 1st β-strand of the Rossmann-like fold for magnesium coordination, where the second Asp is the main acid/base catalyst; additionally, but not limited to, contains Thr or Ser at the end of the 2nd β-strand of the Rossmann-like fold, which contributes to the stability of reaction intermediates; additionally, but not limited to, contains a Lys at the N-terminus of the α-helix closer to the C-terminus relative to the 3rd β-strand of the Rossmann-like fold, which contributes to the stability of reaction intermediates; and additionally, but not limited to, contains the GDxxxD signature at the end of the 4th β-strand of the Rossmann-like fold for magnesium coordination. These traits are known in the art and are mentioned, 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. Preferably, the phosphatase for converting A6P to allulose used in the methods of the invention contains a Rossmann-like fold domain for catalysis, a C1 capping domain, a DxD signature in the 1st β-strand of the Rossmann-like fold, a Thr or Ser at the end of the 2- th β-strand of the Rossmann-like fold, Lys at the N-terminus of the α-helix closer to the C-terminus relative to the 3rd β-strand of the Rossmann-like fold, and the GDxxxD signature at the end of the 4th β-strand of the Rossmann-like fold.

[30] In some embodiments, the method for producing allulose in accordance with the invention also includes the step of enzymatically converting glucose-6-phosphate (G6P) to F6P, and this step is catalyzed by phosphoglucoisomerase (PGI). In other embodiments, the method for producing allulose further includes the step of converting glucose-1-phosphate (G1P) to G6P, where the step is catalyzed by phosphoglucomutase (PGM). In other embodiments, the method for producing allulose also includes the step of converting a saccharide to G1P, which is catalyzed by at least one enzyme.

[31] Therefore, the method for producing allulose in accordance with the invention may include, for example, the following steps: (i) converting a saccharide to glucose-1-phosphate (G1P) using one or more enzymes; (ii) conversion of G1P to G6P using phosphoglucomutase (PGM, EC 5.4.2.2); (iii) conversion of G6P to F6P using phosphoglucoisomerase (PGI, EC 5.3.1.9); (iv) conversion of F6P to A6P via A6PE and (v) conversion of A6P to allulose via A6PP. An example of a process where the saccharide is starch is shown in FIG. 2.

[32] Typically, the ratios of enzyme units used in the disclosed method are 1:1:1:1:1 (αGP:PGM:PGI:A6PE:A6PP). These ratios can 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, resulting in increased activity of downstream reactions. Conversely, a ratio of 1:1:1:1:3 can be used to maintain a steady supply of phosphate to αGP, leading to more efficient phosphorolytic cleavage of α-1,4-glycosidic bonds. Enzyme ratios such as 3:1:1:1:3 can be used to further increase the reaction rate. Therefore, the ratios of enzymes, including other optional enzymes discussed below, can be varied to increase the efficiency of allulose production. For example, a particular enzyme may be present in amounts approximately 2×, 3×, 4×, 5×, etc., relative to the amount of other enzymes.

[33] One of the important advantages of the methods is that the process steps can be carried out in a single bioreactor or reaction vessel. Alternatively, the steps can also be carried out in a plurality of bioreactors or reaction vessels that are arranged in series.

[34] The phosphate ions produced by dephosphorylation of A6P can then be reused in the process in the saccharide to G1P step, particularly when all process steps are carried out in a single bioreactor or reaction vessel. The ability to recycle phosphate in the disclosed methods allows for non-stoichiometric amounts of phosphate to be used, which keeps phosphate concentrations in the reaction low. This disrupts the overall pathway and overall speed of the methods, but does not limit the activity of individual enzymes and allows for the overall efficiency of the methods for producing allulose.

[35] For example, the phosphate concentrations in the reaction may range from about 0 mM to about 300 mM, from about 0 mM to about 150 mM, from about 1 mM to about 50 mM, preferably from about 5 mM to about 50 mM, or more preferably from about 10 mM to about 50 mM. For example, the phosphate concentration in the reaction 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, approximately 8 mM, approximately 9 mM, approximately 10 mM, approximately 15 mM, approximately 20 mM, approximately 25 mM, approximately 30 mM, approximately 35 mM, approximately 40 mM, approximately 45 mM, approximately 50 mM or approximately 55 mM.

[36] Therefore, low phosphate concentration leads to reduced production cost due to low total phosphate and thus reduced cost of phosphate removal. It also prevents A6PP from being inhibited by high concentrations of free phosphate and reduces the potential for phosphate contamination.

[37] In addition, 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(H), ie without NAD(H). Other advantages also include the fact that at least one step of the disclosed processes for producing allulose involves an energetically favorable chemical reaction (FIG. 7).

[38] Examples of enzymes used to convert a saccharide into G1P include α-glucan phosphorylase (αGP, EC 2.4.1.1, which also includes maltodextrin phosphorylase, starch phosphorylase, glycogen phosphorylase and other α-1,4 glycosidic bond-breaking phosphorylases), 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 a combination thereof. The choice of enzyme or combination of enzymes depends on the saccharide used in the method.

[39] The saccharides used to produce G1P may be polysaccharides, oligosaccharides and/or disaccharides. For example, the saccharide may be starch, one or more starch derivatives, cellulose, one or more cellulose derivatives, sucrose, one or more sucrose derivatives, or a combination thereof.

[40] Starch is the most widely used energy storage compound in nature and is primarily 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.

[41] Starch derivatives can be produced by enzymatic hydrolysis of starch or by acid hydrolysis of starch. In particular, enzymatic hydrolysis of starch can be catalyzed or enhanced by isoamylase (IA, EC. 3.2.1.68), which hydrolyzes α-1,6-glucosidic bonds; pullulanase (PA, EC. 3.2.1.41), which hydrolyzes α-1,6-glucosidic bonds; 4-α-glucanotransferase (4GT, EC 2.4.1.25), which catalyzes the transglycosylation of short maltooligosaccharides, yielding longer maltooligosaccharides; or α-amylase (EC 3.2.1.1), which cleaves α-1,4-glucosidic bonds.

[42] In addition, cellulose derivatives can be produced by enzymatic hydrolysis of cellulose catalyzed by cellulase mixtures, by acids, or by pretreatment of biomass.

[43] In certain embodiments, the enzymes used to convert the saccharide to G1P contain αGP. At this stage, when saccharides include starch, G1P is created from starch by αGP; when the saccharides contain soluble starch, amylodextrin or maltodextrin, G1P is prepared from soluble starch, amylodextrin or maltodextrin using αGP.

[44] When the saccharides include maltose and the enzymes contain maltose phosphorylase, G1P is created from maltose via maltose phosphorylase. If the saccharides include sucrose and the enzymes contain sucrose phosphorylase, G1P is created from sucrose by sucrose phosphorylase.

[45] In yet another embodiment, when the saccharides contain cellobiose and the enzymes contain cellobiose phosphorylase, G1P is created from cellobiose using cellobiose phosphorylase.

[46] In a further embodiment, when the saccharides contain cellodextrins and the enzymes contain cellodextrin phosphorylase, G1P is created from the cellodextrins using cellodextrin phosphorylase.

[47] In an alternative embodiment of converting a saccharide to G1P, where the saccharides contain cellulose and the enzymes contain cellulose phosphorylase, G1P is created from cellulose by cellulose phosphorylase.

[48] According to the invention, allulose can also be obtained from fructose. An example of the method is shown in Fig. 4. For example, the method involves the creation of F6P from fructose and polyphosphate, catalyzed by polyphosphate fructokinase (PPFK); conversion of F6P to A6P catalyzed by A6PE; and the conversion of A6P to allulose catalyzed by A6PP. Fructose can be obtained, for example, through the enzymatic conversion of sucrose.

[49] In other embodiments, allulose can be produced from sucrose. An example of such a method is shown in Fig. 6. The method involves an in vitro synthesis route, which includes the following enzymatic steps: creation of G1P from sucrose and free phosphate, catalyzed by sucrose phosphorylase (SP); conversion of G1P to G6P catalyzed by PGM; conversion of G6P to F6P catalyzed by PGI; conversion of F6P to A6P catalyzed by A6PE; and the conversion of A6P to allulose catalyzed by A6PP.

[50] Phosphate ions produced during the conversion of A6P to allulose can then be reused in the step of converting sucrose to G1P. Additionally, as shown in FIG. 6, PPFK and polyphosphate can be used to increase the yield of allulose through the production of F6P from fructose produced through phosphorolytic cleavage of sucrose by SP.

[51] In some embodiments, a method for producing allulose includes the following steps: creating glucose from polysaccharides and oligosaccharides using enzymatic hydrolysis or acid hydrolysis, converting glucose to G6P catalyzed by at least one enzyme, creating fructose from polysaccharides and oligosaccharides using enzymatic hydrolysis or acid hydrolysis, and converting fructose to G6P catalyzed by at least one enzyme. Examples of polysaccharides and oligosaccharides are listed above.

[52] In other embodiments, G6P is produced from glucose and sodium polyphosphate using polyphosphate glucokinase.

[53] The present disclosure provides methods for converting saccharides, such as polysaccharides and oligosaccharides in starch, cellulose, sucrose and their derivatives, into allulose. In certain embodiments, artificial (non-natural) ATP-free enzymatic pathways are provided to convert starch, cellulose, sucrose, and their derivative products into allulose using cell-free enzymatic cocktails.

[54] As shown above, several enzymes can be used to hydrolyze starch to increase G1P yield. Such enzymes include isoamylase, pullulanase and α-amylase. Corn starch contains many branches that interfere with the action of αGP. Isoamylase can be used to debranch starch, yielding linear amylodextrin. Isoamylase pretreated starch can lead to higher concentrations of F6P in the final product. Isoamylase and pullulanase cleave α-1,6-glycosidic bonds, which allows for more complete destruction of starch by α-glucan phosphorylase. α-amylase cleaves α-1,4-glycosidic bonds, therefore α-amylase is used to break starch into fragments for faster conversion into allulose.

[55] As shown in FIG. 2, maltose phosphorylase (MP) can be used to increase the yield of allulose through phosphorolytic cleavage of the breakdown product of maltose into G1P and glucose. Alternatively, 4-glucan transferase (4GT) can be used to increase allulose yield by recycling the breakdown products of glucose, maltose and maltotriose into longer malto-oligosaccharides; which can be phosphorolytically cleaved by αGP to produce G1P.

[56] Additionally, cellulose is the most abundant bioresource and is a major component of plant cell walls. Non-edible 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, and so on, can be obtained through a number of treatments. Partially hydrolyzed cellulose substrates include water-insoluble cellodextrins with a degree of polymerization greater than 7, water-soluble cellodextrins with a degree of polymerization of 3-6, cellobiose, glucose and fructose.

[57] In certain embodiments, cellulose and its derivative products can be converted to allulose through a series of steps. An example of such a method is shown in Fig. 3. The method involves an in vitro synthesis route, which includes the following steps: creation of G1P from cellodextrin and cellobiose and without phosphate, catalyzed by cellodextrin phosphorylase (CDP) and cellobiose phosphorylase (CBP), respectively; conversion of G1P to G6P catalyzed by PGM; conversion of G6P to F6P catalyzed by PGI; conversion of F6P to A6P catalyzed by A6PE; and the conversion of A6P to allulose catalyzed by A6PP. In this method, phosphate ions can be reused in the step of converting cellodextrin and cellobiose to G1P.

[58] Several enzymes can be used to hydrolyze solid cellulose into water-soluble cellodextrins and cellobiose. Such enzymes include endoglucanase and cellobiohydrolase, but do not include β-glucosidase (cellobiase).

[59] Before cellulose is hydrolyzed to produce G1P, cellulose and biomass can be pretreated to increase their reactivity and reduce the degree of polymerization of cellulose chains. Cellulose and biomass pretreatment methods include dilute acid pretreatment, cellulose solvent-based lignocellulose fractionation, ammonia fiber expansion, ammonia water infiltration, ionic liquid treatment, and partial hydrolysis using concentrated acids, including hydrochloric acid, sulfuric acid, phosphoric acid, and combinations thereof. .

[60] In some embodiments, polyphosphate and polyphosphate glucokinase (PPGK) can be added to the process, thereby increasing allulose yield by phosphorylating the glucose degradation product to G6P, as shown in FIG. 3.

[61] In other embodiments, allulose can be created from glucose. An example of such a method is shown in Fig. 5. The method includes the steps of creating G6P from glucose and polyphosphate, catalyzed by polyphosphate glucokinase (PPGK); conversion of G6P to F6P catalyzed by PGI; enzyme-catalyzed conversion of F6P to A6P; and the conversion of A6P to allulose, catalyzed by A6PP.

[62] Any suitable biological buffer known in the art can be used in the method of the invention, such as HEPES, PBS, BIS-TRIS, MOPS, DIPSO, Trizma, etc. The reaction buffer for all embodiments may have a pH of range 5.0-8.0. 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.

[63] The reaction buffer may also contain key metal cations. Examples of metal ions include Mg ²⁺ , Co ²⁺ and Zn ²⁺ .

[64] The reaction temperature at which the process steps are carried out may be in the range of 37-85°C. More preferably, the steps can be carried out at a temperature in the range of from about 40°C to about 70°C. The temperature may be, for example, about 40°C, about 45°C, about 50°C, about 55°C, or about 60°C. Preferably, the reaction temperature is approximately 50°C.

[65] The reaction time in the disclosed methods can be adjusted as necessary and can range from 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, approximately 38 hours, approximately 40 hours, approximately 42 hours, approximately 44 hours, approximately 46 hours or approximately 48 hours. More preferably, the reaction time is approximately 24 hours.

[66] The processes according to the invention can achieve high yields due to a very favorable equilibrium constant for the overall reaction. Theoretically, yields of up to 99% can be achieved if the starting material is completely converted into the intermediate product.

[67] The methods of the invention use inexpensive starting materials and reduce the cost of production by reducing the costs associated with starting materials and product recovery. Starch, cellulose, sucrose and some of their derivatives are less expensive feedstocks than, for example, fructose. When allulose is produced from fructose, yields are lower than in the present invention, and allulose must be separated from fructose through chromatography, which leads to higher production costs.

[68] Also, the step of converting A6P to allulose according to the invention is an irreversible phosphatase reaction, regardless of the feedstock. Consequently, allulose is produced in very high yields, while effectively minimizing subsequent product recovery costs.

[69] Unlike cellular production methods, the invention involves cell-free production of allulose, has a relatively high reaction rate due to the elimination of the cell membrane, which often slows the transport of substrate/product into and out of the cell. It also has a final product free of nutrient rich fermentation media/cellular metabolites.

EXAMPLES

Materials and methods

[70] Chemical compounds

[71] All chemicals, including corn starch, soluble starch, maltodextrins, maltose, glucose, filter paper, were reagent grade or higher and were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Fisher Scientific (Pittsburgh, PA, USA) unless otherwise noted. Restriction enzymes, T4 ligase, and Phusion DNA polymerase were purchased from New England Biolabs (Ipswich, MA, USA). Oligonucleotides were synthesized using either Integrated DNA Technologies (Coralville, IA, USA) or Eurofins MWG Operon (Huntsville, AL, USA). The nucleotide sequence, SEQ ID No. 1, encodes thermophilic A6PE from Thermoanaerobacterium thermosaccharolyticum (UNIPROT ID D9TQJ4). SEQ ID No. 2 is a codon optimized version of this nucleotide sequence. SEQ ID No. 3 is the amino acid sequence for the enzyme. The nucleotide sequence of SEQ ID No. 4 encodes thermophilic A6PE from Bacillus thermoamylovorans (UNIPROT ID A0A090IXZ8). SEQ ID No. 5 is a codon optimized version of this nucleotide sequence. SEQ ID No. 6 is the amino acid sequence for the enzyme. The nucleotide sequence of SEQ ID No. 7 encodes thermophilic A6PP from Clostridium thermocellum (UNIPROT ID A3DC21). SEQ ID No. 8 is a codon optimized version of the nucleotide sequence. SEQ ID No. 9 is the amino acid sequence corresponding to the enzyme. Regenerated amorphous cellulose used in enzymatic purification was obtained from Avicel PH105 (FMC BioPolymer, Philadelphia, PA, USA), through its dissolution and regeneration 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 in as a host cell for expression of the recombinant protein. ZYM-5052 media containing 100 mg L ^-1 ampicillin or 50 mg L ^-1 kanamycin were used for E. coli cell growth and recombinant protein expression. Cellulase from Trichoderma reesei (cat. no.: C2730) and pullulanase (cat. no.: P1067) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and prepared using Novozymes (Franklinton, NC, USA). Maltose phosphorylase (cat. no.: M8284) was purchased from Sigma-Aldrich.

[72] Preparation and purification of recombinant enzymes

[73] E. coli strain BL21 (DE3), carrying a protein expression plasmid, was incubated in a 1 L Erlenmeyer flask with 100 ml of ZYM-5052 media containing 100 mg L ^-1 ampicillin or 50 mg L ^-1 kanamycin. Cells were grown at 30°C with rotational shaking at 220 rpm for 16-24 hours. Cells were collected by centrifugation at 12°C and washed once with 20 mM HEPES (pH 7.5) containing 50 mM NaCl and 5 mM MgCl ₂ (thermoprecipitation and cellulose binding module), or 20 mM HEPES (pH 7.5) containing 300 mM NaCl and 5 mM imidazole (Ni purification). Cell pellets were resuspended in the same buffer and lysed by sonication (Fisher Scientific Sonic Dismembrator Model 500; pulse on 5 s and off 10 s for a total of 21 min at 50% amplitude). After centrifugation, the target proteins in the supernatants were purified.

[74] Three approaches have been used to purify various recombinant proteins. Histidine-tagged proteins were purified using Profinity IMAC Ni-Charged Resin (Bio-Rad, Hercules, CA, USA). Fusion proteins containing a cellulose-binding module (CBM) and a self-excision intein were purified through high-affinity adsorption onto a large area of regenerated amorphous cellulose surface. Thermoprecipitation at 70-95°C for 5-30 min was used to purify hyperthermostable enzymes. The purity of the recombinant proteins was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A6PE was purified using 80 μM CoCl ₂ present in growth media, elution buffers, dialysis buffer and protein storage buffer.

[75] Enzymes used and analysis of their activity

[76] α-Glucan phosphorylase (αGP) from Thermotoga maritima (UNIPROT ID G4FEH8) was used. Activity was assayed in 50 mM sodium phosphate buffer (pH 7.2) containing 1 mM MgCl ₂ , 5 mM DTT and 30 mM maltodextrin at 50°C. The reaction was stopped by enzyme filtration using a Vivaspin 2 (10,000 MWCO) concentrator (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. The unit (U) is described as µmol/min.

[77] Phosphoglucomutase (PGM) from Thermococcus kodakaraensis (UNIPROT ID Q68BJ6) was used. Activity was measured in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl ₂ and 5 mM G1P at 50°C. The reaction was stopped by enzyme filtration using a Vivaspin 2 concentrator (10,000 MWCO). The product glucose-6-phosphate (G6P) was determined using a hexokinase/G6PDH assay kit (Sigma Aldrich, cat. no. GAHK20-1KT).

[78] Two different sources of phosphoglucoisomerase (PGI) from Clostridium thermocellum (UNIPROT ID A3DBX9) and Thermus thermophilus (UNIPROT ID Q5SLL6) were used. Activity was measured in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl ₂ and 10 mM G6P at 50°C. The reaction was stopped by enzyme filtration using a Vivaspin 2 concentrator (10,000 MWCO). The product, fructose 6-phosphate (F6P), was determined using a coupled fructose 6-phosphate kinase (F6PK)/pyruvate dehydrogenase (PK)/lactate dehydrogenase (LD) enzyme assay, where a decrease in absorbance at 340 nm indicates production of F6P. This 200 μl reaction contained 50 mM HEPES (pH 7.2), 5 mM MgCl ₂ , 10 mM G6P, 1.5 mM ATP, 1.5 mM phosphoenolpyruvate, 200 μM NADH, 0.1 U. PGI, 5 Units PK and 5 Units L.D.

[79] Allulose-6-phosphate-3-epimerase (A6PE) from Thermoanaerobacterium thermosaccharolyticum (UNIPROT ID D9TQJ4), SEQ ID No. 3 was used. Activity was measured in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl ₂ . 80 µM CoCl ₂ , 1 U/ml A6PP and 10 mM F6P, at 50°C. The reaction was stopped by enzyme filtration using a Vivaspin 2 concentrator (10,000 MWCO). The product, allulose-6-phosphate (A6P), was determined using allulose-6-phosphate phosphatase and detecting the release of free phosphate. In order 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 the 50 μl reaction. These were mixed, followed by 125 μl of 5% ascorbic acid (pH 5). This solution was mixed and then incubated at 30°C for 20 min. Absorbance at 850 nm was read to determine the release of free phosphate.

[80] Allulose-6-phosphate phosphatase (A6PP) from Clostridium thermocellum (UNIPROT ID A3DC21), SEQ ID No. 9 was used. Activity was measured in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl ₂ , 80 μM CoCl ₂ , 1 U/ml A6PE and 10 mM F6P, at 50°C. The reaction was stopped by enzyme filtration using a Vivaspin 2 concentrator (10,000 MWCO). The product, allulose, was determined via free phosphate release detection as described for A6PE.

[81] 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 analyzed as described.

[82] Recombinant polyphosphate glucokinase from Thermobifida fusca YX is 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 activities were analyzed as described.

[83] 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 activities were analyzed as described.

[84] Recombinant 4-α-glucanol transferase 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.

[85] Sucrose phosphorylase from Caldithrix abyssi (UNIPROT H1XT50) was used. Its activity was measured in 50 mM HEPES buffer (pH 7.5) containing 10 mM sucrose and 12 mM organic phosphate. Glucose-1-phosphate (G1P) was measured using a glucose hexokinase/G6PDH assay kit supplemented with 25 U/ml phosphoglucomutase, as for α-glucan phosphorylase.

[86] The units of enzyme used in each example below can be increased or decreased to adjust the reaction time as desired. For example, if one wants to carry out example 9 in 8 hours instead of 24 hours, the enzyme units should be increased by approximately 3 times. Conversely, if one wishes to perform Example 9 in 48 hours instead of 24 hours, the enzyme units should be reduced by approximately 2 times. These examples illustrate how the number of enzyme units can be used to increase or decrease reaction time while maintaining constant productivity.

[87] Example 1

[88] In order to validate the technical implementation of the enzymatic biosynthesis of fructose-6-phosphate made of starch, three enzymes were reconciled: α-gluanthosphorus from T. Maritima (Uniprot ID G4FEH8), phosphorus from Thermococcus Kodakaraansis Niprot ID Q68BJ6) and phosphoglucoisomerase from Clostridium thermocellum (UNIPROT ID A3DBX9). Recombinant proteins were overexpressed in E. coli BL21 (DE3) and purified as described above.

[89] 0.20 ml of a reaction mixture containing 10 g/l soluble starch, 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCl ₂ , 0.5 mM ZnCl ₂ , 0.01 Units. αGP, 0.01 Units PGM and 0.01 Units PGI, incubated at 50°C for 24 hours. The reaction was stopped by enzyme filtration using a Vivaspin 2 concentrator (10,000 MWCO). The product, fructose 6-phosphate (F6P), was determined using a coupled fructose 6-phosphate kinase (F6PK)/pyruvate dehydrogenase (PK)/lactate dehydrogenase (LD) enzyme assay, where a decrease in absorbance at 340 nm indicates production of F6P as described higher. The final F6P concentration after 24 hours was 3.6 g/L.

[90] Example 2

[91] The same tests as in example 1 (except for the reaction temperature) were 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-hour reactions. These results suggest that increasing the reaction temperature for this set of enzymes increased the yield of F6P, but too high a temperature may reduce the activity of some enzymes.

[92] Example 3

[93] It was found that at 80°C, a ratio of αGP:PGM:PGI enzyme units of approximately 1:1:1 led to rapid formation of F6P. It was noted that the enzyme ratio does not significantly affect the final F6P concentration if the reaction time is long enough. However, the ratio of enzymes affects reaction rates and the overall cost of enzymes used in the system.

[94] Example 4

[95] 0.20 ml reaction mixture containing 10 g/l maltodextrin, 50 mm phosphate-buffered saline pH 7.2, 5 mm MgCl ₂ , 0.5 mm ZnCl ₂ , 0.01 Units. αGP, 0.01 Units PGM and 0.01 Units PGI, incubated at 50°C for 24 hours. The reaction was stopped by enzyme filtration using a Vivaspin 2 concentrator (10,000 MWCO). The product, fructose 6-phosphate (F6P), was determined using a coupled fructose 6-phosphate kinase (F6PK)/pyruvate dehydrogenase (PK)/lactate dehydrogenase (LD) enzyme assay, where a decrease in absorbance at 340 nm indicates production of F6P as described higher. The final F6P concentration after 24 hours was 3.6 g/L.

[96] Example 5

[97] To test the production of F6P from Avicel, Sigma, cellulase was used to hydrolyze cellulose at 50°C. To remove β-glucosidase from commercial cellulase, 10 units of filter paper/ml cellulase was mixed with 10 g/L Avicel in an ice-water bath for 10 min. After centrifugation at 4°C, the supernatant containing β-glucosidase was decanted. Avicel, which was bound to cellulase-containing endoglucanase and cellobiohydrolase, was resuspended in citrate buffer (pH 4.8) for hydrolysis at 50°C for 3 days. Cellulose hydrolyzate 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 MgCl ₂ and 0.5 mM ZnCl ₂ . The reaction was carried out at 60°C for 72 hours and high concentrations of F6P were detected (small amounts of glucose and no cellobiose). F6P was detected using the coupled enzyme assay described above. Glucose was detected using a hexokinase/G6PDH assay kit as described above.

[98] Example 6

[99] To increase the yield of F6Pa from Avicel, Avicel was pretreated with concentrated phosphoric acid to produce amorphous cellulose (RAC) as described in 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 units of filter paper/mL cellulase was mixed with 10 g/L RAC in an ice-water bath for 5 min. After centrifugation at 4°C, the supernatant containing β-glucosidase was decanted. RAC, which was bound to cellulase-containing endoglucanase and cellobiohydrolase, was resuspended in citrate buffer (pH 4.8) for hydrolysis at 50°C for 12 hours. The RAC hydrolyzate 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 MgCl ₂ and 0.5 mM ZnCl ₂ . The reaction was carried out at 60°C for 72 hours. High concentrations of F6P and glucose were recovered because no enzymes were added to convert glucose to F6P. F6P was detected using the coupled enzyme assay described above. Glucose was detected using a hexokinase/G6PDH assay kit as described above.

[100] Example 7

[101] To further increase the yield of F6P from RAC, polyphosphate glucokinase and polyphosphate were added. To remove β-glucosidase from commercial cellulase, 10 units of filter paper/mL cellulase was mixed with 10 g/L RAC in an ice-water bath for 5 min. After centrifugation at 4°C, the supernatant containing β-glucosidase was decanted. RAC, which was bound to cellulase-containing endoglucanase and cellobiohydrolase, was resuspended in citrate buffer (pH 4.8) for hydrolysis at 50°C and incubated in citrate buffer (pH 4.8) for hydrolysis at 50°C for 12 hours. RAC hydrolyzate was mixed with 5 U/ml polyphosphate glucokinase, 5 U/ml cellodextrin phosphorylase, 5 U/ml 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 MgCl ₂ and 0.5 mM ZnCl ₂ . The reaction was carried out at 50°C for 72 hours. F6P was found in high concentrations with only small amounts of glucose now present. F6P was detected using the coupled enzyme assay described above. Glucose was detected using a hexokinase/G6PDH assay kit as described above.

[102] Example 8

[103] To validate the production of allulose from F6P, 2 g/L F6P was mixed with 1 U/ml A6PE and 1 U/ml A6PP in 50 mM HEPES buffer (pH 7.2) containing 5 mM MgCl ₂ and 80 µM CoCl ₂ . The reaction was incubated for 6 hours at 50°C. 99% conversion of F6P to allulose was observed via HPLC (Agilent 1100 series) using an Agilent Hi-Plex H column and refractive index detector. The sample was run in 5 mM H ₂ SO ₄ at 0.6 ml/min.

[104] Example 9

[105] In order to validate the production of allulose from maltodextrin, a 0.20 ml reaction mixture containing 20 g/l maltodextrin, 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCl ₂ , 80 μM CoCl ₂ , 0.05 Unit αGP, 0.05 Units PGM, 0.05 Units PGI, 0.05 Units A6PE and 0.05 U A6PP, incubate at 50°C for 24 hours. The reaction is stopped by filtering the enzyme using a Vivaspin 2 concentrator (10,000 MWCO). Allulose is detected and quantified using an Agilent 1100 series HPLC with a refractive index detector and an Agilent Hi-Plex H column. The mobile phase is 5 mM H ₂ SO ₄ flowing at 0.6 ml/min. Standards of various allulose concentrations are used to quantify the yield of the invention.

[106] Example 10

A reaction mixture containing 200 g/L maltodextrin, 10 mM acetate buffer (pH 5.5), 5 mM MgCl ₂ , 80 μM CoCl ₂ and 0.1 g/L isoamylase is incubated at 80°C for 24 hours. This is used to create another reaction mixture containing 20 g/L isoamylase-treated maltodextrin, 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCl ₂ , 0.05 U. αGP, 0.05 Units PGM, 0.05 Units PGI, 0.05 Units A6PE and 0.05 Units A6PP, incubate at 50°C for 24 hours. The production of allulose is quantified as in example 9.

[107] Example 11

A reaction mixture containing 200 g/L maltodextrin, 10 mM acetate buffer (pH 4.5), 5 mM MgCl ₂ and a 1:200 dilution of Novozymes D6 pullulanase is incubated at 50°C for 4 hours. This is used to create another reaction mixture containing 20 g/L pullulanase-treated maltodextrin, 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCl ₂ , 80 μM CoCl ₂ , 0.05 U. αGP, 0.05 Units PGM, 0.05 Units PGI, 0.05 Units A6PE and 0.05 U A6PP, incubate at 50°C for 24 hours. The production of allulose is quantified as in example 9.

[108] Example 12

[109] In order to further increase the yield of allulose from maltodextrin, 0.05 U. 4-glucan transferase (4GT) is added to the reaction described in example 9.

[110] 0.2 ml of a reaction mixture containing 20 g/l of isoamylase-treated maltodextrin (see example 9), 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCl ₂ , 80 μM CoCl ₂ , 0.05 U . αGP, 0.05 Units PGM, 0.05 Units PGI, 0.05 Units A6PE, 0.05 Units A6PP and 0.05 Units 4GT, incubated at 50°C for 24 hours. The production of allulose is quantified as in example 9.

[111] Example 13

[112] To determine the concentration range of phosphate-buffered saline (PBS), 0.20 ml of the reaction mixture 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 MgCl ₂ ; 0.1 Units aGP; 0.1 Units P.G.M.; and 0.1 Units PGI, incubate at 50°C for 6 hours. The short duration ensures that completion is not reached and hence differences in performance can be clearly seen. F6P production is quantified using a coupled fructose-6-phosphate kinase (F6PK)/pyruvate dehydrogenase (PK)/lactate dehydrogenase (LD) enzyme assay, where a decrease in absorbance at 340 nm indicates F6P production. Accordingly, yields of 4.5 g/L, 5.1 g/L, 5.6 g/L, 4.8 g/L, or 4.9 g/L F6P are obtained for reactions containing 6.25 mM, 12. 5 mM, 25 mM, 37.5 mM, or 50 mM phosphate-buffered saline pH 7.2 (Table 1). These results indicate that a concentration of 25 mM PBS pH 7.2 is ideal for these specific reaction conditions. It is important to note that even using 6.25 mM PBS at pH 7.2 results in significant turnover due to phosphate recycling. This shows that the disclosed phosphate recycling processes can maintain low phosphate levels even at industrial volumetric production levels (eg, 200-300 g/L maltodextrin).

Table 1

PBS concentration pH 7.2 (mM) F6P, g/l 6.25 4.5 12.5 5.1 25 5.6 37.5 4.8 50 4.9

[113] Example 14

[114] To determine the pH range of the cascade reaction, 0.20 ml of the 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 MgCl ₂ ; 0.02 Units αGP; 0.02 Units P.G.M.; and 0.02 Units PGI, incubate at 50°C for 16 hours. The units are reduced to ensure that no completions are achieved and therefore differences in efficiency can be clearly seen. The production of F6P is quantified as in example 12. Accordingly, the yield is 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 F6P was 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 (Table 2). These results indicate that pH 6.8 is ideal for these particular reaction conditions, despite the system operating over a wide pH range.

table 2

pH in PBS F6P, g/l 6.0 4.0 6.2 4.1 6.4 4.2 6.6 4.1 6.8 4.4 7.0 4.1 7.2 3.8 7.3 4.0

[115] Example 15

[116] To study scaling, 20 ml reaction mixture containing 50 g/l isoamylase-treated maltodextrin (see example 10), 50 mM phosphate-buffered saline pH 7.2, 5 mM MgCl ₂ , 80 μM CoCl ₂ , 10 U . αGP, 10 Units PGM, 10 Units PGI, 10 Units A6PE and 10 Units A6PP, incubate at 50°C for 24 hours. The production of allulose was quantified as in example 9.

[117] Example 16

[118] In order to further increase the yield of allulose from maltodextrin, 0.05 U. Maltose phosphorylase is added to the reaction described in example 9.

[119] Example 17

[120] In order to further increase the yield of allulose from maltodextrin, 0.05 U. Polyphosphate glucokinase and 75 mM polyphosphate are added to the reaction described in example 9.

[121] Example 18

[122] To obtain allulose from fructose, a reaction mixture containing 10 g/l fructose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl ₂ , 80 μM CoCl ₂ , 0.05 U. fructose polyphosphate kinase, 0.05 units. A6PE and 0.05 Units A6PP, incubate at 50°C for 24 hours. The production of allulose is quantified as in example 9.

[123] Example 19

[124] To obtain allulose from glucose, a reaction mixture containing 10 g/l glucose, 50 mM Tris buffer pH 7.0, 75 mM polyphosphate, 5 mM MgCl ₂ , 80 μM CoCl ₂ , 0.05 U. glucose polyphosphate kinase, 0.05 units. PGI, 0.05 Units A6PE and 0.05 Units A6PP, incubate at 50°C for 24 hours. The production of allulose is quantified as in example 9.

[125] Example 20

[126] To obtain allulose from sucrose, a reaction mixture containing 10 g/l sucrose, 50 mM phosphate-buffered saline pH 7.0, 5 mM MgCl ₂ , 80 μM CoCl ₂ , 0.05 U. sucrose phosphorylase, 0.05 PGM, 0.05 U. PGI, 0.05 Units A6PE and 0.05 Units A6PP, incubate at 50°C for 24 hours. The production of allulose is quantified as in example 9.

[127] Example 21

[128] In order to further increase the yield of allulose from sucrose, 75 mM polyphosphate and 0.05 polyphosphate fructokinase were added to the reaction mixture in Example 20. Allulose production was quantified as in Example 9.

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LIST OF SEQUENCES

<110> BONUMOSE LLC

<120> ENZYMATIVE PRODUCTION OF D-ALLULOSE

<130> 146.0003-WO00

<140> PCT/US17/66298

<141> 2017-12-14

<150> 62/434,033

<151> 2016-12-14

<160> 9

<170> PatentIn version 3.5

<210> 1

<211> 663

<212> DNA

<213> Thermoanaerobacterium thermosaccharolyticum

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gga 663

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<213> Artificial sequence

<220>

<223> A6PE from Thermoanaerobacterium thermosaccharolyticum

with codon optimization

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Ala Phe Arg Ile Ile Asp Arg Ile Lys Ser His Gly Leu Met Val Gly

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Asn Lys Ile Asp Lys Ile Thr Ile Met Thr Val Asp Pro Gly Phe Ala

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145 150 155 160

Arg Leu Lys Asp Asp Asn Asn Tyr Asn Tyr Leu Ile Glu Ala Asp Gly

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Val Phe Val Leu Gly Ser Ser Gly Leu Phe Asn Leu Ser Asp Asp Leu

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Gly Lys Ala Trp Glu Ile Met Ile Gly Asn Phe Asn Gly

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atgagcaaca aaattgaatt ttcaccgtct ttaatgacaa tggatttaga caagtttaaa 60

gaacagatta cttttttaaa taatcatgtc ggttcttacc atatcgatat tatggacgga 120

cattatgtac ctaatataac tctatcccct tggtttgtcc aagaggtacg gaaaattagt 180

gatgttccga tgtctgccca cttgatggtc acaaacccaa gtttttgggt acaacaactc 240

attgatatta agtgtgaatg gatttgcatg cacgtagaaa cccttgatgg gttagctttc 300

cgcttaattg atcaaatcca cgatgcggga ttaaaagcag gggtcgtatt aaatcctgaa 360

acaagtgttg atgcgattcg cccgtacatt gatttagtgg ataaagtcac cattatgact 420

gtcgacccag gttttgcagg tcaacgcttt attgatagta cattggagaa aatcgtggaa 480

ttaagaaaat tacgggaaga acacggttat aaatatgtga ttgaaatgga tggatcttcg 540

aatcggaaat ccttcaagaa aatttatgaa gccggtcctg acatttatat tataggtcgc 600

agcggtttgt ttggattaca cgaagatatc gaaaaagcat gggaaatcat gtgcaaagat 660

tttgaggaaa tgactggcga aaaagtatta 690

<210> 5

<211> 690

<212> DNA

<213> Artificial sequence

<220>

<223> Thermophilic A6PE from Bacillus thermoamylovorans

with codon optimization

<400> 5

atgagtaaca agatcgaatt cagcccgagc ctgatgacca tggatctgga taaatttaaa 60

gaacagatca cctttctgaa caatcatgtt ggcagttatc atattgatat catggatggt 120

cattacgtgc cgaatattac cctgagcccg tggtttgttc aggaagtgcg caaaattagc 180

gatgttccga tgagcgcaca tctgatggtt accaatccga gtttttgggt tcagcagctg 240

attgatatta aatgtgaatg gatttgcatg catgttgaaa ccctggatgg cctggccttt 300

cgtctgattg atcagattca tgatgccggc ctgaaagcag gtgtggttct gaatccggaa 360

accagtgtgg atgccattcg tccgtatatt gatctggttg ataaagttac catcatgacc 420

gtggatccgg gctttgccgg ccagcgcttt attgatagca ccctggaaaa aattgtggaa 480

ctgcgtaaac tgcgtgaaga acatggttat aaatatgtga ttgagatgga tggcagcagc 540

aatcgcaaaa gctttaaaaa aatttacgag gcaggtccgg atatttatat tattggccgt 600

agtggcctgt ttggcctgca tgaagatatt gaaaaagcat gggaaattat gtgtaaggat 660

tttgaagaaa tgaccggtga aaaagtgctg 690

<210> 6

<211> 230

<212> Protein

<213> Bacillus thermoamylovorans

<400> 6

Met Ser Asn Lys Ile Glu Phe Ser Pro Ser Leu Met Thr Met Asp Leu

1 5 10 15

Asp Lys Phe Lys Glu Gln Ile Thr Phe Leu Asn Asn His Val Gly Ser

20 25 30

Tyr His Ile Asp Ile Met Asp Gly His Tyr Val Pro Asn Ile Thr Leu

35 40 45

Ser Pro Trp Phe Val Gln Glu Val Arg Lys Ile Ser Asp Val Pro Met

50 55 60

Ser Ala His Leu Met Val Thr Asn Pro Ser Phe Trp Val Gln Gln Leu

65 70 75 80

Ile Asp Ile Lys Cys Glu Trp Ile Cys Met His Val Glu Thr Leu Asp

85 90 95

Gly Leu Ala Phe Arg Leu Ile Asp Gln Ile His Asp Ala Gly Leu Lys

100 105 110

Ala Gly Val Val Leu Asn Pro Glu Thr Ser Val Asp Ala Ile Arg Pro

115 120 125

Tyr Ile Asp Leu Val Asp Lys Val Thr Ile Met Thr Val Asp Pro Gly

130 135 140

Phe Ala Gly Gln Arg Phe Ile Asp Ser Thr Leu Glu Lys Ile Val Glu

145 150 155 160

Leu Arg Lys Leu Arg Glu Glu His Gly Tyr Lys Tyr Val Ile Glu Met

165 170 175

Asp Gly Ser Ser Asn Arg Lys Ser Phe Lys Lys Ile Tyr Glu Ala Gly

180 185 190

Pro Asp Ile Tyr Ile Ile Gly Arg Ser Gly Leu Phe Gly Leu His Glu

195 200 205

Asp Ile Glu Lys Ala Trp Glu Ile Met Cys Lys Asp Phe Glu Glu Met

210 215 220

Thr Gly Glu Lys Val Leu

225 230

<210> 7

<211> 651

<212> DNA

<213> Clostridium thermocellum

<400> 7

atgattaaat acaaagcggt attctttgat tttgactata cgctggcaga ttcatctaaa 60

gctgttatag aatgtattaa ctatgcactg caaaaaatgg gttatcccga atcttctccg 120

gaaagtattt gcagaacaat aggacttacg ttggccgagg cttttaaaat acttagcggg 180

gatacttctg attccaatgc ggaccttttc cgccaatact ttaaagaaag agcagatctg 240

gttatgtgtg accggactgt aatgtacagc actngttgaat gtgttttgaa gaagctgaaa 300

aaggcggatg taaaaacagg aattgtttca acgaagtaca gatacaggat agaggatata 360

ttaaaaaggg acaaactttt acagtatttt gatgtaattg tcggcgggga agatgttgcg 420

gcccataaac cggatccgga agggcttcta aaggccatat cgatggttgg ctgccaaaag 480

gaagaagtcc tttttgtcgg agacagtacg gtggatgcaa ggactgcaaa aaatgcggga 540

gtggattttg tggcggttct tacggggaca accggggcaa atgagttttc agagtataat 600

cctggtgctg tgattgaaga tttgagtggt ttattggata tgtttatgtt a 651

<210> 8

<211> 651

<212> DNA

<213> Artificial sequence

<220>

<223> A6PP from Clostridium thermocellum with codon optimization

<400> 8

atgatcaagt acaaggccgt tttttttgat tttgattaca ccctggcaga tagcagcaaa 60

gccgttattg aatgtattaa ttacgccctg cagaaaatgg gctatccgga aagcagtccg 120

gaaagcattt gtcgtaccat tggcctgacc ctggcagaag catttaaaat tctgagcggt 180

gataccagcg atagcaatgc cgatctgttt cgccagtatt ttaaagaacg cgcagatctg 240

gttatgtgtg atcgcaccgt gatgtatagc accgtggaat gcgtgctgaa aaaactgaaa 300

aaagcagatg ttaagaccgg tattgtgagc accaaatatc gctatcgtat tgaagatatt 360

ctgaaacgtg ataaactgct gcagtatttt gatgttattg ttggtggcga agatgttgcc 420

gcccataaac cggatccgga aggcctgctg aaagcaatta gcatggtggg ctgccagaaa 480

gaagaagttc tgtttgttgg tgatagcacc gttgatgcac gtaccgccaa aaatgcaggc 540

gtggattttg tggccgttct gaccggcacc accggcgcaa atgaatttag cgaatataat 600

ccgggcgccg tgattgaaga tctgagcggt ctgctggata tgtttatgct g 651

<210> 9

<211> 217

<212> Protein

<213> Clostridium thermocellum

<400> 9

Met Ile Lys Tyr Lys Ala Val Phe Phe Asp Phe Asp Tyr Thr Leu Ala

1 5 10 15

Asp Ser Ser Lys Ala Val Ile Glu Cys Ile Asn Tyr Ala Leu Gln Lys

20 25 30

Met Gly Tyr Pro Glu Ser Ser Pro Glu Ser Ile Cys Arg Thr Ile Gly

35 40 45

Leu Thr Leu Ala Glu Ala Phe Lys Ile Leu Ser Gly Asp Thr Ser Asp

50 55 60

Ser Asn Ala Asp Leu Phe Arg Gln Tyr Phe Lys Glu Arg Ala Asp Leu

65 70 75 80

Val Met Cys Asp Arg Thr Val Met Tyr Ser Thr Val Glu Cys Val Leu

85 90 95

Lys Lys Leu Lys Lys Ala Asp Val Lys Thr Gly Ile Val Ser Thr Lys

100 105 110

Tyr Arg Tyr Arg Ile Glu Asp Ile Leu Lys Arg Asp Lys Leu Leu Gln

115 120 125

Tyr Phe Asp Val Ile Val Gly Gly Glu Asp Val Ala Ala His Lys Pro

130 135 140

Asp Pro Glu Gly Leu Leu Lys Ala Ile Ser Met Val Gly Cys Gln Lys

145 150 155 160

Glu Glu Val Leu Phe Val Gly Asp Ser Thr Val Asp Ala Arg Thr Ala

165 170 175

Lys Asn Ala Gly Val Asp Phe Val Ala Val Leu Thr Gly Thr Thr Gly

180 185 190

Ala Asn Glu Phe Ser Glu Tyr Asn Pro Gly Ala Val Ile Glu Asp Leu

195 200 205

Ser Gly Leu Leu Asp Met Phe Met Leu

210 215

<---

Claims (21)

1. A method for producing allulose, where the method includes: conversion of fructose 6-phosphate (F6P) to allulose 6-phosphate (A6P), catalyzed by allulose 6-phosphate 3-epimerase (A6PE); And conversion of the resulting A6P to allulose, catalyzed by allulose-6-phosphate phosphatase (A6PP), wherein A6PE contains an amino acid sequence having at least 55% sequence identity to SEQ ID NO: 3 or 6, and A6PP contains an amino acid sequence having at least 55% sequence identity to SEQ ID NO: 9. 2. The method according to claim 1, which further includes the step of converting glucose-6-phosphate (G6P) to F6P, where the step is catalyzed by phosphoglucoisomerase (PGI). 3. The method according to claim 2, which further includes the step of converting glucose-1-phosphate (G1P) to G6P, where the step is catalyzed by phosphoglucomutase (PGM). 4. The method of claim 3, which further comprises the step of converting the saccharide to G1P, wherein the step is catalyzed by at least one enzyme selected from the group consisting of α-glucan phosphorylase (αGP), maltose phosphorylase, sucrose phosphorylase, cellodextrin phosphorylase, cellobiose phosphorylase and cellulose phosphorylase wherein the saccharide is selected from the group consisting of starch or a derivative thereof, cellulose or a derivative thereof and sucrose. 5. The method of claim 4, wherein the saccharide is starch or a derivative thereof selected from the group consisting of amylose, amylopectin, soluble starch, amylodextrin, maltodextrin, maltose and glucose. 6. The method according to claim 5, which further includes the step of converting starch into a starch derivative, wherein the starch derivative is obtained by enzymatic hydrolysis of starch or by acid hydrolysis of starch. 7. The method according to claim 5, which includes adding 4-glucan transferase (4GT). 8. The method of claim 6, wherein the starch derivative is produced by enzymatic hydrolysis of starch catalyzed by isoamylase, pullulanase, α-amylase or a combination thereof. 9. The method according to claim 1, which additionally includes: a step for converting fructose to F6P, where the step is catalyzed by polyphosphate fructokinase (PPFK); and optionally a step of converting sucrose to fructose, where the step is catalyzed by sucrose phosphorylase (SP). 10. The method according to claim 2, which additionally includes: a step of converting glucose to G6P, where the step is catalyzed by polyphosphate fructokinase (PPFK), and optionally a step of converting sucrose to glucose, where the step is catalyzed by sucrose phosphorylase (SP). 11. The method of claim 1, wherein A6PE contains an amino acid sequence having at least 90%, at least 95%, or at least 100% sequence identity to SEQ ID Nos. 3 or 6. 12. The method according to claim 1, in which A6PE contains an (α/β) _8- barrel domain for catalysis, Ser at the end of the 7th β-barrel strand, Ser at the end of the 8th β-barrel strand, Gly in the active loop center, His in the 2nd and 3rd barrel β-strands, Asp in the 2nd and 7th barrel β-strands, and the His-hydrophobic residue-Asp signature in the 2nd barrel β-strand. 13. The method of claim 1, wherein A6PP comprises an amino acid sequence having at least 90%, at least 95%, or at least 100% sequence identity to SEQ ID NO: 9. 14. The method of claim 1, wherein A6PP contains a Rossmann-like fold domain for catalysis, a C1 capping domain, a DxD signature in the 1st β-strand of the Rossmann-like fold, a Thr or Ser at the end of the 2nd β-strand Rossmann-like fold, Lys on the N-α-helix closer to the C-terminus relative to the 3rd β-strand of the Rossmann-like fold, and the GDxxxD signature at the end of the 4th β-strand of the Rossmann-like fold. 15. Method according to any one of paragraphs. 1-14, wherein the process steps are carried out at a temperature ranging from about 37°C to about 85°C, at a pH ranging from about 5.0 to about 8.0 and/or for about 8 hours to about 48 hours. 16. Method according to any one of paragraphs. 1-14, in which the process steps are carried out in a single bioreactor or in a plurality of bioreactors arranged in series. 17. Method according to any one of paragraphs. 1-14, in which the process steps are carried out without ATP, without NAD(H), at a phosphate concentration of from about 0 mM to about 150 mM, and/or the phosphate is recycled.