RU2776637C2 - Acellular sugar production - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, and is a method for the production of allulose, providing following stages: conversion of polymer glucose carbohydrate into glucose-1-phosphate (hereinafter – G1P), using α-glucan or cellodextrin phosphorylase; conversion of G1P into glucose-6-phosphate (hereinafter – G6P), using phosphoglucomutase; conversion of G6P into fructose-6-phosphate (hereinafter – F6P), using phosphoglucoisomerase; conversion of F6P into alluloso-6-phosphate (hereinafter – A6P), using alluloso-6-phosphatepimerase (hereinafter – A6PE); and conversion of A6P into allulose, using alluloso-6-phosphatephosphatase (hereinafter – A6PP); where A6PP is A6PP Ruminiclostridium thermocellum, or where A6PE is A6PE Brevibacillus thermoruber.

EFFECT: invention allows for the production of allulose with high degree of efficiency.

15 cl, 8 dwg, 3 tbl, 14 ex

Description

Link to related applications

This application claims priority under § 119(e) 35 U.S.C. as per U.S. Provisional Application No. 62/443,447, filed January 6, 2017, and U.S. Patent Application No. 62/538,181, filed July 28, 2017, each of which is incorporated herein by reference in its entirety .

Background of the Invention

Existing technologies for converting starch to simple sugars use multiple biotransformation reactions with deep purification methods after each biotransformation. Although biotransformation methods are relatively inexpensive due to the use of immobilized enzymes and continuous production systems, post-processing has a significant cost impact.

Brief summary of the present invention

The present invention relates to cell-free systems, methods, compositions and kits for the enzymatic conversion of polymeric glucose such as starch (e.g. amylose and/or amylopectin), glycogen or any partially hydrolysed derivative thereof such as maltodextrin or cellodextrin (which can be used interchangeably with cellulose), into pentose (eg ribose, arabinose or xylulose) or hexose (eg allulose, glucose or fructose) sugars. The methods of the present disclosure implement sugar production pathways in cell-free reactions (eg, in a one-pot (single) cell-free reaction) to convert starch and/or cellulose/cellodextrin to hexose and/or pentose sugars. In contrast to the methods that typically involve the phosphorylation of substrates such as glucose to glucose-6-phosphate and use high-energy phosphate sources such as ATP and phosphoenoylpyruvate, the methods described herein typically replace high-energy phosphate sources, such as inexpensive inorganic phosphate (P _i ). In some embodiments, α-glucan phosphorylase (also called starch phosphorylase) (EC 2.4.1.1) is used to convert starch to glucose-1-phosphate, which is then converted to glucose-6-phosphate by phosphoglucomutase (EC 5.4.2.2, 5.4 .2.5 or 5.4.2.6). According to other embodiments, cellodextrin phosphorylase (also called cellulose phosphorylase or β-(1-4) glucan phosphorylase) (EC 2.4.1.49) is used to convert cellulose/cellodextrin to glucose-1-phosphate, which is then converted to glucose-6-phosphate via phosphoglucomutase (EC 5.4.2.2, 5.4.2.5 or 5.4.2.6). The subsequent one or more enzymatic reactions of a particular sugar pathway presented herein are highly product specific. Sugar phosphatase (EC 3.1.3.-) is used to convert to the final product. Thus, such thermodynamics of the reaction, i.e., phosphorylation of the substrate - dephosphorylation of the product, contributes to the production of the product.

In addition, the enzymatic conversion reactions described herein are virtually irreversible, thus maintaining high yields of the desired hexose and pentose sugars. In contrast, conventional biotransformation methods to convert starch or cellulose/cellodextrin to allulose, for example, use three separate processes, two of which are reversible, with the final product concentration determined by the thermodynamics of the enzymes used. For example, starch is converted to glucose, glucose is isomerized to fructose, and fructose is epimerized to allulose. The isomerization of glucose to fructose is characterized by a yield of approximately 45%, therefore, significant post-treatment is necessary to obtain a pure product and recycle the non-catalytically reacted substrate. Similarly, the epimerization of fructose to allulose has a ~20% yield, which again requires significant post-treatment to obtain a purified product and recycle the uncatalyzed substrate. The ability to directly convert starch to the product of interest in the cell-free systems described herein reduces costs by reducing post-processing and substrate wastage.

Preferably, many of the enzymes used in the methods provided herein are thermostable, which (1) provides thermal inactivation of molecules with undesirable activity contained in cell lysates in which the transformation process is carried out, and (2) reduces the likelihood of contamination by microorganisms, adversely affecting production cycles. The enzymes in these pathways can be isolated from thermophilic, mesophilic, or psychrophilic organisms and/or, in some embodiments, engineered to increase (or decrease) the thermal stability of such enzymes. The thermophilic organism (thermophile) actively reproduces at high temperatures in the range of 41°C to 122°C (106°F to 252°F). The mesophilic organism (mesophile) actively reproduces at moderate temperatures in the range of 20°C to 45°C (68°F to 113°F). The psychrophilic organism (psychrophile) actively reproduces at low temperatures ranging from -20°C to 10°C (from -4°F to 50°F).

Thus, certain aspects of the present disclosure relate to methods for producing sugar (e.g., allulose, glucose, fructose, sorbitol, ribulose, ribose, and/or arabinose), wherein the method comprises (a) culturing at least two cell populations, cells from each populations are engineered to express at least one thermostable enzyme of the sugar production pathway described herein to produce at least two cultured cell populations expressing different enzymes, (b) lysing the cells of at least two cultured populations to obtain at least two cell lysates, (c) combining at least two cell lysates to form a mixture of cell lysates that contains thermostable enzymes of the sugar pathway, (d) heating the mixture of cell lysates to a temperature that inactivates undesirable natural molecules with enzymatic activity, but does not inactivate heat-stable fermen you from step (c), to obtain a heat-inactivated lysate; and (e) incubating the reaction mixture in the presence of a substrate (for example, starch, glycogen, or any partially hydrolyzed derivative thereof) and a source of phosphate (for example, inorganic phosphate) to obtain sugar.

In some embodiments, a cell-free method for producing sugar (e.g., allulose, glucose, fructose, sorbitol, ribulose, ribose, and/or arabinose) involves (a) culturing at least two cell populations, each population being engineered to express a at least one sugar pathway enzyme described herein to produce at least two cultured cell populations expressing different enzymes, wherein at least one of the sugar pathway enzymes is thermostable, (b) lysing the cells of at least two cultured populations to obtain at least two cell lysates, (c) optionally, heating one or more cell lysates from step (b) to a temperature that inactivates undesirable natural molecules with enzymatic activity, but does not inactivate one or more thermostable enzymes from step (a ), resulting in inactivation heat-treated lysate, (d) combining the cell lysates from steps (b) and (c) to obtain a cell lysate mixture that contains sugar pathway enzymes, wherein at least one of the above enzymes is thermostable, and (e) incubating the reaction mixture in the presence of a substrate (eg, starch, glycogen, or any partially hydrolyzed derivative thereof) and a source of phosphate (eg, inorganic phosphate) to form a sugar.

In some embodiments, a cell-free method for producing sugar (e.g., allulose, glucose, fructose, sorbitol, ribulose, ribose, and/or arabinose) involves (a) culturing at least two cell populations, each population being engineered to express a at least one thermostable sugar pathway enzyme described herein to produce at least two cultured cell populations expressing different enzymes, (b) lysing the cells of at least two cultured populations to obtain at least two cell lysates, (c ) combining at least two cell lysates to obtain a mixture of cell lysates, (d) heating the mixture of cell lysates to a temperature that inactivates undesirable natural molecules with enzymatic activity, but does not inactivate thermostable enzymes from step (a), to obtain a heat-inactivated lysate, (f) adding to a heat-inactivated lysate of at least one purified sugar pathway enzyme; and (e) incubating the reaction mixture in the presence of a substrate (e.g., starch, glycogen, or any partially hydrolyzed derivative thereof) and a phosphate source (e.g., inorganic phosphate) to produce sugar.

In some embodiments, a cell-free method for producing sugar (e.g., allulose, glucose, fructose, sorbitol, ribulose, ribose, and/or arabinose) involves (a) culturing at least two cell populations, each population being engineered to express a at least one sugar pathway enzyme described herein to produce at least two cultured cell populations expressing different enzymes, wherein at least one of the aforementioned enzymes is thermostable, (b) lysing the cells of at least two cultured populations with obtaining at least two cell lysates, (c) optionally heating one or more cell lysates from step (b) to a temperature that inactivates undesirable natural molecules with enzymatic activity, but does not inactivate thermostable enzymes from step (a), to obtain a heat-inactivated lysate , (d) combining the cell lysates from steps (b) and (c) to obtain a mixture of cell lysates, (e) adding at least one purified sugar pathway enzyme to the mixture of cell lysates, and (f) incubating the reaction mixture in the presence of a substrate ( eg starch, glycogen or any partially hydrolyzed derivative thereof) and a source of phosphate (eg inorganic phosphate) to form a sugar.

Some aspects of the present disclosure relate to cell-free methods for producing allulose, comprising (a) culturing cells engineered to express α-glucan phosphorylase (also called starch phosphorylase), phosphoglucomutase, phosphoglucoisomerase, allulose-6-phosphate epimerase, and allulose-6-phosphate phosphatase to obtain cultured cells, which express enzymes, wherein at least one of the above enzymes is thermostable, (b) lysing the cultured cells to obtain a cell lysate, (c) heating the cell lysate to a temperature that inactivates the natural molecule with enzymatic activity, but does not inactivate one or more thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and (d) incubating the heat-inactivated lysate in the presence of starch, glycogen, or any partially hydrolysed derivative thereof, and a source of phosphate (e.g., inorganic phosphate) with the production of allulose. According to some embodiments, at least one purified enzyme is added to the cell lysate before or after step (c). It should be understood that cells can be lysed by any means, including mechanical, chemical, enzymatic, osmotic, or thermal lysis. Thus, the lysing step and the heating (heat inactivation) step can be combined into one step of heating the cells to a temperature that lyses the cells and inactivates the natural molecule with enzymatic activity.

In some embodiments, the cell-free methods comprise (a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of α-glucan phosphorylases, phosphoglucomutases, phosphoglucoisomerases, allulose- 6-phosphate epimerases and allulose-6-phosphate phosphatases, to obtain at least two cultured populations of cells expressing different enzymes, and at least one of the above enzymes is thermostable, (b) lysing the cells of at least two cultured populations to obtain at least at least two cell lysates, (c) combining at least two cell lysates to obtain a mixture of cell lysates, which contains α-glucan phosphorylase, phosphoglucomutase, phosphoglucoisomerase, allulose-6-phosphate epimerase and allulose-6-phosphate phosphatase, (d) heating the mixture of cell lysates up to a temperature that inactivates t a naturally occurring molecule with enzymatic activity, but does not inactivate one or more of the thermostable enzymes from step (c), to produce a heat-inactivated lysate; and (e) incubating the reaction mixture in the presence of starch, glycogen, or any of its partially hydrolysed derivatives, and a source of phosphate (e.g., inorganic phosphate) to produce allulose.

According to other embodiments, the cell-free methods comprise (a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of α-glucan phosphorylases, phosphoglucomutases, phosphoglucoisomerases, allulose- 6-phosphate epimerases and allulose-6-phosphate phosphatases, to obtain at least two cultured populations of cells expressing different enzymes, and at least one of the above enzymes is thermostable, (b) lysing the cells of at least two cultured populations to obtain at least at least two cell lysates, (c) combining at least two cell lysates to obtain a mixture of cell lysates, (d) heating the mixture of cell lysates to a temperature that inactivates the natural molecule with enzymatic activity, but does not inactivate one or more thermostable enzymes from step ( a), with by reducing the heat-inactivated lysate, (f) adding to the heat-inactivated lysate at least one purified enzyme selected from the group consisting of α-glucan phosphorylases, phosphoglucomutases, phosphoglucoisomerases, allulose-6-phosphate epimerases, and allulose-6-phosphate phosphatases, to obtain a reaction mixture containing α-glucan phosphorylase, phosphoglucomutase, phosphoglucoisomerase, allulose-6-phosphate epimerase, and allulose-6-phosphate phosphatase, and (f) incubating the reaction mixture in the presence of starch, glycogen, or any of its partially hydrolysed derivatives, and a phosphate source (e.g., inorganic phosphate) with obtaining allulose.

According to some aspects of the present disclosure, it may be preferable to use cellulose/cellodextrin as the starting substrate. Thus, certain aspects of the present disclosure pertain to cell-free methods for producing allulose, comprising (a) culturing cells engineered to express cellodextrin phosphorylase, phosphoglucomutase, phosphoglucoisomerase, allulose-6-phosphate epimerase, and allulose-6-phosphate phosphatase to produce cultured cells that express the enzymes wherein at least one of the above enzymes is thermostable, (b) lysing the cultured cells to obtain a cell lysate, (c) heating the cell lysate to a temperature that inactivates the natural molecule with enzymatic activity, but does not inactivate one or more thermostable enzymes from step (a) to obtain a heat-inactivated lysate; and (d) incubating the heat-inactivated lysate in the presence of cellodextrin and a source of phosphate (eg, inorganic phosphate) to obtain allulose.

In some embodiments, the cell-free methods comprise (a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of cellodextrin phosphorylases, phosphoglucomutases, phosphoglucoisomerases, allulose-6- phosphate epimerases and allulose-6-phosphate phosphatases, to obtain at least two cultured populations of cells expressing different enzymes, and at least one of the above enzymes is thermostable, (b) lysing the cells of at least two cultured populations to obtain at least two cell lysates, (c) combining at least two cell lysates to obtain a mixture of cell lysates that contains cellodextrin phosphorylase, phosphoglucomutase, phosphoglucoisomerase, allulose-6-phosphate epimerase and allulose-6-phosphate phosphatase, (d) heating the mixture of cell lysates to a temperature that and inactivates the natural molecule with enzymatic activity, but does not inactivate one or more thermostable enzymes from step (c), to obtain a heat-inactivated lysate; and (e) incubation of the reaction mixture in the presence of cellodextrin and a source of phosphate (for example, inorganic phosphate) to obtain allulose.

According to other embodiments, the cell-free methods comprise (a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of cellodextrin phosphorylases, phosphoglucomutases, phosphoglucoisomerases, allulose-6- phosphate epimerases and allulose-6-phosphate phosphatases, to obtain at least two cultured populations of cells expressing different enzymes, and at least one of the above enzymes is thermostable, (b) lysing the cells of at least two cultured populations to obtain at least two cell lysates, (c) combining at least two cell lysates to obtain a mixture of cell lysates, (d) heating the mixture of cell lysates to a temperature that inactivates the natural molecule with enzymatic activity, but does not inactivate one or more thermostable enzymes from step (a) , With obtaining a heat-inactivated lysate, (f) adding to the heat-inactivated lysate at least one purified enzyme selected from the group consisting of cellodextrinphosphorylases, phosphoglucomutases, phosphoglucoisomerases, allulose-6-phosphate epimerases and allulose-6-phosphate phosphatases, to obtain a reaction mixture containing cellodextrinphosphorylase, phosphoglucomutase, phosphoglucoisomerase, allulose-6-phosphate epimerase and allulose-6-phosphate phosphatase, and (f) incubating the reaction mixture in the presence of cellodextrin and a source of phosphate (eg, inorganic phosphate) to obtain allulose.

The present disclosure also covers the cell-free production of other sugars such as glucose, fructose, mannose, sorbitol, ribulose, ribose, and arabinose.

According to some embodiments, the sugar pathways presented require balancing of energy cofactors such as NADH, NADPH, NAD ⁺ or NADP ⁺ . This can be done using cofactor regeneration systems. In these cases, NADH and NADPH are called "reduced cofactors" or "reducing agents" and NAD ⁺ and NADP ⁺ are called "oxidized cofactors" or "oxidizing agents". In cases with excess reducing agents, NAD(P)H oxidase (EC No. 1.6.3.1, 1.6.3.2, 1.6.2.3 or 1.6.3.4) can be used to burn excess reduced cofactors to form either H ₂ O ₂ or O ^- ₂ , or H ₂ O, depending on the type of oxidase. When H ₂ O ₂ and O ₂ ^{- are} formed, which can harm the lysate, superoxide dismutase (EC No. 1.15.1.1) and/or catalase (EC No. 1.11.1.6) can be used in tandem to convert harmful compounds into H ₂ O and O ₂ . In cases with an excess of oxidizing agents, a cofactor regeneration system can be used to restore oxidized cofactors back to their reduced forms. Some examples include the use of formate dehydrogenase (EC No. 1.2.1.2) to oxidize formate to CO ₂ while reducing NAD(P) ⁺ to NAD(P)H, or the use of phosphonate dehydrogenase (EC No. 1.20.1.1) or sulfite oxidoreductase (EC No. 1.8.1.2 ) to oxidize the corresponding inorganic salts to phosphate and sulfate, resulting in reduced NAD(P)H.

Also provided herein are engineered cells, cell lysates, and reaction mixtures containing enzymes, such as thermostable enzymes, used to produce a particular sugar of interest (eg, allulose, glucose, fructose, sorbitol, ribulose, ribose, and/or arabinose).

Brief description of the drawings

Fig. 1 is a schematic representation of the enzymatic pathway for converting starch to allulose. The abbreviations have the following meanings: G1P = glucose-1-phosphate, G6P = glucose-6-phosphate, F6P = fructose-6-phosphate, A6P = allulose-6-phosphate and PO ₄ = inorganic phosphate.

Fig. 2 is a schematic representation of two enzymatic pathways for converting starch to glucose. The abbreviations have the following meanings: G1P = glucose-1-phosphate, G6P = glucose-6-phosphate and PO ₄ = inorganic phosphate.

Fig. 3 is a schematic representation of two enzymatic pathways for converting starch to fructose. The abbreviations have the following meanings: G1P = glucose-1-phosphate, G6P = glucose-6-phosphate, F6P = fructose-6-phosphate and PO ₄ = inorganic phosphate.

Fig. 4 is a schematic representation of the enzymatic pathway for converting starch to sorbitol. The abbreviations have the following meanings: G1P = glucose-1-phosphate, G6P = glucose-6-phosphate, S6P = sorbitol-6-phosphate, F6P = fructose-6-phosphate, NADPH = nicotinamide adenine dinucleotide phosphate (reduced form), NADP ⁺ = nicotinamide adenine dinucleotide phosphate, and PO ₄ = inorganic phosphate.

Fig. 5 is a schematic representation of the enzymatic pathway for converting starch to ribulose. The abbreviations have the following meanings: G1P = glucose-1-phosphate, G6P = glucose-6-phosphate, 6PGL = 6-phosphogluconolactone, 6PG = 6-phosphogluconate, Ru5P = ribulose-5-phosphate, NADPH = nicotinamide adenine dinucleotide phosphate (reduced form), NADP ⁺ = nicotinamide adenine dinucleotide phosphate, CO ₂ = carbon dioxide and PO ₄ = inorganic phosphate.

Fig. 6 is a schematic representation of the enzymatic pathway for converting starch to ribose. The abbreviations have the following meanings: G1P = glucose-1-phosphate, G6P = glucose-6-phosphate, 6PGL = 6-phosphogluconolactone, 6PG = 6-phosphogluconate, Ru5P = ribulose-5-phosphate, R5P = ribose-5-phosphate, NADPH = nicotinamide adenine dinucleotide phosphate (reduced dinucleotide), NADP ⁺ = nicotinamide adenine dinucleotide phosphate, CO ₂ = carbon dioxide and PO ₄ = inorganic phosphate.

Fig. 7 is a schematic representation of the enzymatic pathway for converting starch to arabinose. The abbreviations have the following meanings: G1P = glucose-1-phosphate, G6P = glucose-6-phosphate, 6PGL = 6-phosphogluconolactone, 6PG = 6-phosphogluconate, Ru5P = arabinose-5-phosphate, Ar5P = ribose-5-phosphate, NADPH = nicotinamide adenine dinucleotide phosphate (reduced dinucleotide), NADP ⁺ = nicotinamide adenine dinucleotide phosphate, CO ₂ = carbon dioxide and PO ₄ = inorganic phosphate.

Fig. 8 is a schematic representation of the enzymatic pathway for converting starch to mannose. The abbreviations have the following meanings: G1P = glucose-1-phosphate, G6P = glucose-6-phosphate, F6P = fructose-6-phosphate, M6P = mannose-6-phosphate and PO4 = inorganic phosphate.

Detailed disclosure of the present invention

This document describes enzymatic pathways used to convert starch (eg, amylose or amylopectin) or cellulose/cellodextrin to pentose (eg, ribose, arabinose, or xylulose) and/or hexose (eg, allulose, glucose, or fructose) sugars. Enzymatic pathways use at least one α-glucan phosphorylase (also called starch phosphorylase) (EC 2.4.1.1) or at least one cellodextrin phosphorylase (also called cellulose phosphorylase) (EC 2.4.1.49), at least one phosphoglucomutase (EC 5.4.2.2 , 5.4.2.5 or 5.4.2.6) and any number of isomerases, epimerases and/or sugar phosphatases, depending on the final product. In accordance with some embodiments, the enzymes or a portion of the enzymes are thermostable. Such thermostable enzymes can withstand the heating step in the sugar production process, which inactivates molecules with undesirable activity contained in cell lysates in which conversion processes are carried out. This heat inactivation step reduces the likelihood of microbial contamination adversely affecting production cycles.

Thus, the present disclosure relates, in accordance with certain embodiments, to highly efficient and cost effective methods, compositions, and systems for producing sugars such as hexose and pentose sugars. Non-limiting examples of sugar pathways and enzymes of these pathways are shown in Table 1 below.

Table 1. Brief description of enzymes of illustrative pathways

Path substrate Enzymes getting glucose α(1-4)- or β(1-4)-glucans α- or β-(1-4)-glucan phosphorylase (EC 2.4.1.1, 2.4.1.49),
phosphoglucomutase (EC 5.4.2.2, 5.4.2.5, 5.4.2.6),
glucose-6-phosphate phosphatase (EC 3.1.3.9, 3.1.3.58) getting glucose α(1-4)- or β(1-4)-glucans α- or β-(1-4)-glucan phosphorylase (EC 2.4.1.1, 2.4.1.49),
glucose-1-phosphate phosphatase (EC 3.1.3.10) getting fructose α(1-4)- or β(1-4)-glucans α- or β-(1-4)-glucan phosphorylase (EC 2.4.1.1, 2.4.1.49),
phosphoglucomutase (EC 5.4.2.2, 5.4.2.5, 5.4.2.6),
phosphoglucoisomerase (EC 5.3.1.9) and
fructose-6-phosphate phosphatase (EC 3.1.3.-, 3.1.3.58) allulose production α(1-4)- or β(1-4)-glucans α- or β-(1-4)-glucan phosphorylase (EC 2.4.1.1, 2.4.1.49),
phosphoglucomutase (EC 5.4.2.2, 5.4.2.5, 5.4.2.6),
phosphoglucoisomerase (EC 5.3.1.9),
allulose-6-phosphate epimerase (EC 5.1.3.-) and
allulose-6-phosphate phosphatase (EC 3.1.3.-, 3.1.3.58) getting sorbitol α(1-4)- or β(1-4)-glucans α- or β-(1-4)-glucan phosphorylase (EC 2.4.1.1, 2.4.1.49),
phosphoglucomutase (EC 5.4.2.2, 5.4.2.5, 5.4.2.6),
aldose dehydrogenase (EC 1.1.1.200) and
sorbitol-6-phosphate phosphatase (EC 3.1.3.50, 3.1.3.58) getting sorbitol α(1-4)- or β(1-4)-glucans α- or β-(1-4)-glucan phosphorylase (EC 2.4.1.1, 2.4.1.49),
phosphoglucomutase (EC 5.4.2.2, 5.4.2.5, 5.4.2.6),
phosphoglucoisomerase (EC 5.3.1.9)
sorbitol-6-phosphate-2-dehydrogenase (EC 1.1.1.140)
sorbitol-6-phosphate phosphatase (EC 3.1.3.50, 3.1.3.58) obtaining ribulose α(1-4)- or β(1-4)-glucans α- or β-(1-4)-glucan phosphorylase (EC 2.4.1.1, 2.4.1.49),
phosphoglucomutase (EC 5.4.2.2, 5.4.2.5, 5.4.2.6),
glucose-6-phosphate dehydrogenase (EC 1.1.1.49, 1.1.1.388, 1.1.1.363),
6-phosphogluconolactonase (EC 3.1.1.31),
6-phosphogluconate dehydrogenase (EC 1.1.1.44, 1.1.1.343, 1.1.1.351) and
ribulose-5-phosphate phosphatase (EC 3.1.3.-, 3.1.3.58) getting ribose α(1-4)- or β(1-4)-glucans α- or β-(1-4)-glucan phosphorylase (EC 2.4.1.1, 2.4.1.49),
phosphoglucomutase (EC 5.4.2.2, 5.4.2.5, 5.4.2.6),
glucose-6-phosphate dehydrogenase (EC 1.1.1.49, 1.1.1.388, 1.1.1.363),
6-phosphogluconolactonase (EC 3.1.1.31),
6-phosphogluconate dehydrogenase (EC 1.1.1.44, 1.1.1.343, 1.1.1.351) and
ribose-5-phosphate isomerase (EC 5.3.1.6) and
ribose-5-phosphate phosphatase (EC 3.1.3.-, 3.1.3.58) getting arabinose α(1-4)- or β(1-4)-glucans α- or β-(1-4)-glucan phosphorylase (EC 2.4.1.1, 2.4.1.49),
phosphoglucomutase (EC 5.4.2.2, 5.4.2.5, 5.4.2.6),
glucose-6-phosphate dehydrogenase (EC 1.1.1.49, 1.1.1.388, 1.1.1.363),
6-phosphogluconolactonase (EC 3.1.1.31),
6-phosphogluconate dehydrogenase (EC 1.1.1.44, 1.1.1.343, 1.1.1.351) and
arabinose-5-phosphate isomerase (EC 5.3.1.13) and
arabinose-5-phosphate phosphatase (EC 3.1.3.-, 3.1.3.58) getting mannose α(1-4)- or β(1-4)-glucans α- or β-(1-4)-glucan phosphorylase (EC 2.4.1.1, 2.4.1.49),
phosphoglucomutase (EC 5.4.2.2, 5.4.2.5, 5.4.2.6),
phosphoglucoisomerase (EC 5.3.1.9),
mannose-6-phosphate isomerase (EC 5.3.1.8) and
mannose-6-phosphate phosphatase (EC 3.1.3.-, 3.1.3.58)

Allulose production

Some aspects of the present disclosure relate to methods, compositions and systems for producing allulose. Such methods, in some embodiments, involve culturing cells engineered to express at least one α-glucan phosphorylase and/or at least one cellodextrin phosphorylase, at least one phosphoglucomutase, at least one phosphoglucoisomerase, at least one alluloso -6-phosphate epimerase, at least one allulose-6-phosphate phosphatase, or a combination of at least two (eg, at least three or at least four) of the above enzymes. In some embodiments, α-glucan phosphorylase (and/or cellodextrin phosphorylase) and phosphoglucomutase are expressed as a single fusion (chimeric) protein or bifunctional protein.

A fusion protein can be created by joining two or more genes or gene segments that code for separate proteins. Translation of such a hybrid gene results in one or more polypeptides with functional properties derived from each of the original proteins. A polyfunctional protein is a single protein that has at least two distinct activities, where such functional property is a natural biological function or is the result of hybridization of engineered enzymes. Other enzymes may also be expressed as a single fusion protein or as a polyfunctional protein. Thus, the fusion protein may have a variety of functional properties of any of the enzyme pathways described herein.

Allulose pathway enzymes provided herein are typically heterologous to the host cell (originally cloned or derived from a different cell type), although some of the enzymes may be endogenous (native) to the host cell. Thus, in some embodiments, at least one enzyme (eg, a thermostable enzyme) used to convert starch and/or cellodextrin to allulose is heterologous to the host cell. In some embodiments, at least two, at least three, or at least four enzymes are heterologous with respect to the host cell. In accordance with some embodiments, at least one enzyme is endogenous (natural) in relation to the host cell. In accordance with some embodiments, at least two, at least three, or at least four enzymes are endogenous to the host cell.

The host cells can be prokaryotic cells such as bacterial cells (eg Escherichia coli cells ) or eukaryotic cells such as yeast or plant cells. Other types of cells are described below.

According to some embodiments, at least one of the enzymes used to convert starch and/or cellulose/cellodextrin to allulose is a thermostable enzyme. In accordance with some embodiments, at least two (eg, at least three or at least four) enzymes are thermostable enzymes. According to some embodiments, all enzymes are thermostable enzymes. Thus, according to some embodiments, the methods involve culturing cells engineered to express at least one thermostable α-glucan phosphorylase, at least one thermostable phosphoglucomutase, at least one thermostable phosphoglucoisomerase, at least one thermostable allulose-6- phosphate epimerase, at least one thermostable allulose-6-phosphate phosphatase, or a combination of at least two or more of the above thermostable enzymes. According to some embodiments, the methods include culturing cells engineered to express at least one thermostable cellodextrin phosphorylase, at least one thermostable phosphoglucomutase, at least one thermostable phosphoglucoisomerase, at least one thermostable allulose-6-phosphate epimerase, at least one thermostable allulose-6-phosphate phosphatase; or a combination of at least two or more of the above thermostable enzymes.

In some embodiments, methods for producing allulose involve lysing (e.g., thermal, mechanical, chemical, or enzymatic lysis) cultured cells to produce at least one (e.g., at least two, at least three, or at least four) cell lysate. It should be understood that multiple cell lysates (and hence multiple cell populations, e.g. from the same organism (e.g. bacteria) or from different organisms (e.g. bacteria, yeast and/or plant cells)). For example, one cell population may be engineered to express one or more of the allulose pathway enzymes, while another cell population (or several other cell populations) may be engineered to express another (at least one other) allulose pathway enzyme. Thus, according to some embodiments, the methods include culturing at least one population of cells engineered to express at least one α-glucan phosphorylase, culturing at least one population of cells engineered to express at least one phosphoglucomutase, culturing at least one population of cells engineered to express at least one phosphoglucoisomerase, culturing at least one population of cells engineered to express at least one allulose-6-phosphate epimerase, and/or culturing at least one population of cells engineered to express at least one allulose-6-phosphate phosphatase. According to some embodiments, the methods include culturing at least one cell population engineered to express at least one cellodextrin phosphorylase, culturing at least one cell population engineered to express at least one phosphoglucomutase, culturing at least one cell population engineered to express at least one phosphoglucoisomerase, culturing at least one population of cells engineered to express at least one allulose-6-phosphate epimerase, and/or culturing at least one population of cells engineered to express at least one alluloso -6-phosphate phosphatase. After cell lysis, the cell lysates are combined so that the enzymes are present in a single cell lysate/single reaction mixture.

It should be understood that in any of the methods described herein, cells can be lysed by any method, including mechanical, chemical, enzymatic, osmotic and/or thermal lysis. Thus, the lysing step and the heating (heat inactivation) step can be combined into one step of heating the cells to a temperature that lyses the cells and inactivates naturally occurring molecules with undesirable enzymatic activity.

In accordance with some embodiments, the methods further comprise heating one or more cell lysates (or a mixture of cell lysates) to a temperature that inactivates natural molecules with undesired enzymatic activity, but does not inactivate any thermostable enzymes of the production route, to obtain a heat-inactivated lysate. . One or more cell lysates, in accordance with some embodiments, are heated to a temperature of at least 50°C. For example, one or more cell lysates can be heated to a temperature of at least 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C or 90°C. A native enzyme (or other non-thermostable enzyme) is considered inactivated, according to some embodiments, when its activity level is reduced by at least 50%. According to some embodiments, a naturally occurring enzyme (or other non-thermostable enzyme) is considered inactivated when its activity level is reduced by at least 55%, 60%, 65%, 70%, 75%, 80%, 85% , 90%, 95%, 98%, 99% or 100%.

One or more cell lysates can be heated for a period of time sufficient to inactivate native enzymes (or other than thermostable enzymes) of the cell. For example, one or more cell lysates can be heated for at least 2, 3, 4, or 5 minutes. In accordance with some embodiments, one or more cell lysates are heated for more than 5 minutes. In some embodiments, one or more cell lysates are heated for a period of time sufficient to reduce the activity of at least some naturally occurring enzymes (or other non-thermostable enzymes) by at least 50% (e.g., at least 60%, 70%, 80% or 90%).

After heat inactivation, according to some embodiments, at least one (eg, at least two or at least three) purified enzyme (or partially purified enzyme) is added to the cell lysate/reaction mixture. Thus, the reaction mixture, in accordance with some embodiments, includes a combination of enzymes present in the cell lysate (expressed by one or more engineered host cells) and at least one purified enzyme. The at least one purified enzyme may be selected from the group consisting of α-glucan phosphorylases or cellodextrin phosphorylases, phosphoglucomutases, phosphoglucoisomerases, allulose-6-phosphate epimerases, and allulose-6-phosphate phosphatases.

In some embodiments, the methods also include incubating one or more heat-inactivated lysates in the presence of starch and inorganic phosphate to produce allulose. In some embodiments, the methods also include incubating one or more heat-inactivated lysates in the presence of cellodextrin and inorganic phosphate to produce allulose. According to some embodiments, the heat-inactivated lysates are incubated at a temperature of at least 50°C. According to some embodiments, the heat-inactivated lysates are incubated for at least 2 minutes (eg, at least 3, 4, or 5 minutes). For example, heat-inactivated lysates can be incubated for 2-5 minutes or 2-10 minutes. The starch may be, for example, amylose, amylopectin or a mixture of amylose and amylopectin. According to some embodiments, biomass is used instead of starch. For example, the reaction may involve the participation of one or more cellodextrinphosphorylase. In accordance with some embodiments, the starch or cellodextrin is present as a component of the mixture (eg, part of the biomass). For example, in some embodiments, one or more heat-inactivated lysates (eg, lysates of microorganism cells) are incubated in the presence of cornmeal and inorganic phosphate to produce allulose (or any other sugar described herein).

The present invention also relates to cells and cell lysates used to obtain allulose. Thus, the engineered cell (e.g., bacterial cell, yeast cell, and/or plant cell) or one or more cell lysates of the present disclosure may include at least one (e.g., at least two, at least three, or at least four) an enzyme selected from the group consisting of α-glucan phosphorylases, phosphoglucomutases, phosphoglucoisomerases, allulose-6-phosphate epimerases, and allulose-6-phosphate phosphatases. In accordance with some embodiments, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure may include at least one (e.g., at least two, at least three, or at least at least four) an enzyme selected from the group consisting of cellodextrin phosphorylases, phosphoglucomutases, phosphoglucoisomerases, allulose-6-phosphate epimerases, and allulose-6-phosphate phosphatases. In accordance with some embodiments, the engineered cell (e.g., bacterial cell, yeast cell, and/or plant cell) or one or more cell lysates of the present disclosure comprises at least one (e.g., at least two, at least three, or at least four) an enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable phosphoglucoisomerases, thermostable allulose-6-phosphate epimerases, and thermostable allulose-6-phosphate phosphatases. In accordance with some embodiments, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure comprises at least one (e.g., at least two, at least three, or at least four) an enzyme selected from the group consisting of thermostable cellodextrinphosphorylases, thermostable phosphoglucomutases, thermostable phosphoglucoisomerases, thermostable allulose-6-phosphate epimerases, and thermostable allulose-6-phosphate phosphatases.

Table 2. Exemplary enzymes of the allulose pathway

Path stage Enzyme name EC No. natural organism NCBI No. one α-glucan phosphorylase 2.4.1.1 Aquifex aeolicus WP_010880430 Thermocrinis minervae WP_079654205 Thermosulfidibacter takaii WP_068550435 Thermosulfurimonas dismutans WP_068671361 Thermococcus litoralis WP_004066514 Palaeococcus pacificus WP_048164525 Thermotoga neapolitana WP_015919877 Ruminiclostridium thermocellum WP_003512623 Pyrococcus abyssi WP_048146597 Thermococcus thioreducens WP_055429034 Deinococcus radiodurans NP_295917 Sulfolobus acidocaldarius WP_011277212 Thermus caldophilus AAV68178 Meiothermus silvanus ADH63988 Oceanithermus profundus WP_013457575 Ardenticatena maritima WP_054491617 Thermococcus barophilus WP_013466486 Pseudothermotoga thermarum WP_013931760 Hydrogenobacter thermophilus WP_012962737 Thermus oshimai AFV76231 Meiothermus ruber WP_013013285 Marinitoga piezophila WP_014295659 Cellodextrin phosphorylase (also called cellodextrin phosphorylase) 2.4.2.49 Clostridium thermocellum BAB71818 Clostridium straminisolvens GAE90338 Thermotoga RQ2 WP_011943512 Ignisphaera aggregans ADM28607 Thermotoga maritima WP_004082399 Spirochaeta thermophila WP_013314871 Caldicellulosiruptor bescii WP_015907054 Dictyoglomus thermophilum WP_012548338 Thermoanaerobacterium thermosaccharolyticum WP_013297089 Thermosipho africanus ACJ76363 Caldisalinibacter kiritimatiensis WP_006313788 Defluviitalea phaphyphila WP_058486419 Caldicellulosiruptor kronotskyensis WP_013429146 Thermococcus sibiricus WP_015848606 Thermosphaera aggregans WP_013129904 2 Phosphoglucomutase 5.4.2.6 Thermococcus kodakaraensis BAD42440 Pyrococcus kukulkanii WP_068320630 Ammonifex degensii WP_015738524 Methanothermobacter wolfei WP_074359679 Methanothermus fervidus WP_013413625 Sulfolobus acidocaldarius WP_011277678 Archaeoglobus fulgidus WP_010877965 Ferroglobus placidus WP_012964640 Geoglobus ahangari WP_048096365 Archaeoglobus veneficus WP_013683858 Archaeoglobus sulfaticallidus WP_015589873 Aciduliprofundum boonie WP_012997480 Clostridium thermocellum WP_003517493 Defluviitalea phaphyphila WP_058485855 Caminicella sporogenes WP_072968430 Caloranaerobacter ferrireducens WP_069650396 Thermosipho malanesiensis WP_012056981 Fervidobacterium pennivorans WP_014451812 Symbiobacterium thermophilum WP_011196853 Spirochaeta thermophila ADN02136 Thermoanaerobacter wiegelii AEM79998 3 Phosphoglucoisomerase 5.3.1.9 Thermus thermophilus WP_041443619 Meiothermus timidus WP_018467230 Thermus filiformis WP_038061840 marinithermus hydrothermalis WP_013704730 Thermosipho africanus WP_004103575 Sulfurihydrogenibium azorense WP_012674892 Persephonella marina WP_012675923 Marinitoga piezophila WP_014295589 Kosmotoga olearia WP_012744692 Thermotoga maritima WP_004081585 Geobacillus stearothermophilus KZE97846 Anoxybacillus flavithermus WP_041638934 Thermosulfidibacter takaii BAT72177 Fervidobacterium nodosum WP_011994042 Clostridium thermocellum WP_003512317 Thermoanaerobacterium thermosaccharolyticum WP_013297353 Methanococcus jannaschii WP_010871130 Methanotorris igneus WP_013799854 Methanocaldococcus villosus WP_004589908 Methanothermococcus okinawensis WP_013867340 Pseudothermotoga thermarum WP_013931655 Deferribacter desulfuricans WP_013007743 Thermovibrio ammonificans WP_013537727 four Allulose-6-phosphate epimerase 5.3.1.- Thermobacterium thermosaccharolyticum WP_013298194 Thermoanaerobacter brockii WP_003868217 Caldanaerobacter subterraneus WP_011025758 Deferribacter desulfuricans WP_013008817 Thermocrinis ruber WP_025305325 Hydrogenivirga sp. 128-5-R1-1 WP_008287078 Brevibacillus thermoruber WP_029098887 Thermosipho atlanticus WP_073071389 Thermosulfidibacter takaii WP_068550718 5 Allulose-6-phosphate phosphatase 3.1.3.- Thermoanaerobacter wiegelii WP_014063120 Thermoanaerobacter ethanolicus WP_003870772 Thermus islandicus WP_022799086 Deinococcus geothermalis DSM 11300 ABF44399 Thermosphaera aggregans WP_013129214 Crenarchaeota archaeon 13_1_40CM_3_53_5 WP_013335457 Pyrococcus horikoshii Ot3 WP_010884566 Aquifex aeolicus WP_010880861 Ruminiclostridium thermocellum WP_003512401 Desulfotomaculum kuznetsovii AEG14852 Caldanaerobacter subterraneus WP_009610632 Acidothermus cellulolyticus WP_011718939 Methanothermobacter thermautotrophicus WP_010877362 Thermobifida fusca AAZ54262 Thermotoga neapolitana ACM23496 Petrotoga mobilis WP_012207996 Thermodesulfatator indicus WP_013908370 Thermus thermophilus AAS81813 Bacteroides vulgatus ABR41712 Bacteroides fragilus CAH06673

It should be understood that the allulose pathway may include any combination of enzymes selected from each step 1-5 of the pathway from Table 2. For example, the step 1 α-glucan phosphorylase of the pathway can be selected from any of the α-glucan phosphorylases from Aquifex aeolicus , Thermocrinis minervae, Thermosulfidibacter takaii , Thermosulfurimonas dismutans, Thermococcus litoralis, Palaeococcus pacificus, Thermotoga neapolitana, Ruminiclostridium thermocellum, Pyrococcus abyssi, Thermococcus thioreducens, Deinococcus radiodurans, Sulfolobus acidocaldarius, Thermus caldophilus, Meiothermus silvanus, Oceanithermus profundus, Ardenticatena maritima, Thermococcus barophilus, Pseudothermotoga thermarum, Hydrogenobacter thermophilus, Thermus oshimai, Meiothermus ruber and Marinitoga piezophila and combined with a phosphoglucomutase from stage 2 of the pathway selected from any of the phosphoglucomutase from Thermococcus kodakaraensis, Pyrococcus kukulkanii, Ammonifex degensii, Methanothermobacter wolfeii, Methanothermus fervidus, Sulfolobus acid ocaldarius, Archaeoglobus fulgidus, Ferroglobus placidus, Geoglobus ahangari, Archaeoglobus veneficus, Archaeoglobus sulfaticallidus, Aciduliprofundum boonie, Clostridium thermocellum, Defluviitalea phaphyphila, Caminicella sporogenes, Caloranaerobacter ferrireducens, Thermosipho malanesiensis, Fervidobacterium pennivorans, Symbiobacterium thermophilum, Spirochaeta thermophila и Thermoanaerobacter wiegelii.

Getting glucose

Other aspects of the present disclosure relate to methods, compositions and systems for producing glucose. Such methods, in some embodiments, involve culturing cells engineered to express at least one α-glucan phosphorylase and/or at least one cellodextrin phosphorylase, at least one phosphoglucomutase, at least one glucose-6-phosphate phosphatase, or a combination at least two of the above enzymes. In some embodiments, α-glucan phosphorylase (and/or cellodextrin phosphorylase) and phosphoglucomutase are expressed as a single fusion (chimeric) protein or bifunctional protein. In some embodiments, such methods involve culturing cells engineered to express at least one α-glucan phosphorylase and/or at least one cellodextrin phosphorylase, at least one glucose-1-phosphate phosphatase, or a combination of at least two of the above enzymes. . In some embodiments, α-glucan phosphorylase (and/or cellodextrin phosphorylase) and phosphoglucomutase are expressed as a single fusion (chimeric) protein or bifunctional protein.

The glucose production pathway enzymes presented herein are typically heterologous to the host cell (originally cloned or derived from a different cell type), although some of these enzymes may be endogenous (native) to the host cell. Thus, in some embodiments, at least one enzyme (eg, a thermostable enzyme) used to convert starch and/or cellodextrin to glucose is heterologous to the host cell. In accordance with some embodiments, at least two enzymes are heterologous with respect to the host cell. In accordance with some embodiments, at least one enzyme (eg, a thermostable enzyme) is endogenous (natural) with respect to the host cell. According to some embodiments, at least two of the enzymes are endogenous to the host cell.

The host cells can be prokaryotic cells such as bacterial cells (eg Escherichia coli cells ) or eukaryotic cells such as yeast or plant cells. Other types of cells are described below.

According to some embodiments, at least one of the enzymes used to convert starch and/or cellodextrin to glucose is a thermostable enzyme. In accordance with some embodiments, at least two of the enzymes are thermostable enzymes. According to some embodiments, all enzymes are thermostable enzymes. Thus, according to some embodiments, the methods involve culturing cells engineered to express at least one thermostable α-glucan phosphorylase, at least one thermostable phosphoglucomutase, at least one thermostable glucose-6-phosphate phosphatase, or a combination of at least two or more of the above thermostable enzymes. In some embodiments, the methods involve culturing cells engineered to express at least one thermostable cellodextrin phosphorylase, at least one thermostable phosphoglucomutase, at least one thermostable glucose-6-phosphate phosphatase, or a combination of at least two or more of the above. thermostable enzymes. In some embodiments, the methods involve culturing cells engineered to express at least one thermostable α-glucan phosphorylase, at least one thermostable glucose-1-phosphate phosphatase, or a combination of at least two or more of the above thermostable enzymes. In some embodiments, the methods involve culturing cells engineered to express at least one thermostable cellodextrin phosphorylase, at least one thermostable glucose-1-phosphate phosphatase, or a combination of at least two or more of the above thermostable enzymes.

In some embodiments, methods for producing glucose involve lysing (e.g., thermal, mechanical, chemical, or enzymatic lysis) cultured cells to produce at least one (e.g., at least two, at least three, or at least four) cell lysate. It should be understood that multiple cell lysates (and hence multiple cell populations, e.g. from the same organism (e.g. bacteria) or from different organisms (e.g. bacteria, yeast and/or plant cells)). For example, one cell population may be engineered to express one or more glucose pathway enzymes, while another cell population (or several other cell populations) may be engineered to express another (at least one other) glucose pathway enzyme. Thus, according to some embodiments, the methods include culturing at least one population of cells engineered to express at least one α-glucan phosphorylase, culturing at least one population of cells engineered to express at least one phosphoglucomutase, and/ or culturing at least one population of cells engineered to express at least one glucose-6-phosphate phosphatase. According to some embodiments, the methods include culturing at least one cell population engineered to express at least one cellodextrin phosphorylase, culturing at least one cell population engineered to express at least one phosphoglucomutase, and/or culturing at least one population of cells engineered to express at least one glucose-6-phosphate phosphatase. According to some embodiments, the methods include culturing at least one population of cells engineered to express at least one α-glucan phosphorylase and/or culturing at least one population of cells engineered to express at least one glucose-1- phosphate phosphatase. According to some embodiments, the methods include culturing at least one population of cells engineered to express at least one cellodextrin phosphorylase and/or culturing at least one population of cells engineered to express at least one glucose-1-phosphate phosphatase. After cell lysis, the cell lysates are combined so that the enzymes are present in a single cell lysate/single reaction mixture.

In accordance with some embodiments, the methods further comprise heating one or more cell lysates (or a mixture of cell lysates) to a temperature that inactivates natural molecules with undesired enzymatic activity, but does not inactivate any thermostable enzymes of the production route, to obtain a heat-inactivated lysate. . One or more cell lysates, in accordance with some embodiments, are heated to a temperature of at least 50°C. For example, one or more cell lysates can be heated to a temperature of at least 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C or 90°C.

One or more cell lysates can be heated for a period of time sufficient to inactivate native enzymes (or other than thermostable enzymes) of the cell. For example, one or more cell lysates can be heated for at least 2, 3, 4, or at least 5 minutes. In accordance with some embodiments, one or more cell lysates are heated for more than 5 minutes. In some embodiments, one or more cell lysates are heated for a period of time sufficient to reduce the activity of natural enzymes (or other non-thermostable enzymes) by at least 50% (e.g., at least 60%, 70 %, 80% or 90%).

After heat inactivation, in accordance with some embodiments, at least one (eg, at least two or at least three) purified enzyme is added to the cell lysate/reaction mixture. Thus, the reaction mixture, in accordance with some embodiments, includes a combination of enzymes present in the cell lysate (expressed by one or more engineered host cells) and at least one purified enzyme. The at least one purified enzyme may be selected from the group consisting of α-glucan phosphorylases or cellodextrin phosphorylases, phosphoglucomutases, glucose-6-phosphate phosphatases. Alternatively, at least one purified enzyme may be selected from the group consisting of α-glucan phosphorylases or cellodextrin phosphorylases and glucose-1-phosphate phosphatases.

In some embodiments, the methods also include incubating one or more heat-inactivated lysates in the presence of starch and inorganic phosphate to produce glucose. According to some embodiments, the methods also include incubating one or more heat-inactivated lysates in the presence of cellodextrin and inorganic phosphate to produce glucose. According to some embodiments, the heat-inactivated lysates are incubated at a temperature of at least 50°C. According to some embodiments, the heat-inactivated lysates are incubated for at least 2 minutes (eg, at least 3, 4, or 5 minutes). For example, heat-inactivated lysates can be incubated for 2-5 minutes or 2-10 minutes.

The starch may be, for example, amylose, amylopectin or a mixture of amylose and amylopectin. According to some embodiments, biomass is used instead of starch. According to such embodiments, the reaction involves one or more cellodextrin phosphorylases. In accordance with some embodiments, starch or cellodextrin is present as a component of the mixture.

The present invention also relates to cells and cell lysates used to obtain glucose. Thus, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure may include at least one (e.g., at least two) enzyme selected from the group consisting of α-glucan phosphorylases. , phosphoglucomutase and glucose-6-phosphate phosphatase. In accordance with some embodiments, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure may include at least one (e.g., at least two) enzyme selected from the group consisting of from cellodextrinphosphorylases, phosphoglucomutases and glucose-6-phosphate phosphatases. The engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure may include at least one (e.g., at least two) enzyme selected from the group consisting of α-glucan phosphorylases and glucose- 1-phosphate phosphatase. In accordance with some embodiments, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure may include at least one (e.g., at least two) enzyme selected from the group consisting of from cellodextrinphosphorylases and glucose-1-phosphate phosphatases. In accordance with some embodiments, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure comprises at least one (e.g., at least two) enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases and thermostable glucose-6-phosphate phosphatases. In accordance with some embodiments, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure comprises at least one (e.g., at least two) enzyme selected from the group consisting of thermostable cellodextrinphosphorylases, thermostable phosphoglucomutases and thermostable glucose-6-phosphate phosphatases. In accordance with some embodiments, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure comprises at least one (e.g., at least two) enzyme selected from the group consisting of thermostable α-glucan phosphorylases and thermostable glucose-1-phosphate phosphatases. In accordance with some embodiments, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure comprises at least one (e.g., at least two) enzyme selected from the group consisting of thermostable cellodextrinphosphorylases and thermostable glucose-1-phosphate phosphatases.

Getting fructose

Still other aspects of the present disclosure relate to methods, compositions and systems for producing fructose. Such methods, in some embodiments, involve culturing cells engineered to express at least one α-glucan phosphorylase and/or cellodextrin phosphorylase, at least one phosphoglucomutase, at least one phosphoglucoisomerase, at least one fructose-6-phosphate phosphatase or combinations of at least two of the above enzymes. In some embodiments, α-glucan phosphorylase (and/or cellodextrin phosphorylase) and phosphoglucomutase are expressed as a single fusion (chimeric) protein or bifunctional protein.

The enzymes of the fructose pathways presented herein are typically heterologous to the host cell (originally cloned or derived from a different cell type), although some of the enzymes may be endogenous (native) to the host cell. Thus, in some embodiments, at least one enzyme (eg, a thermostable enzyme) used to convert starch and/or cellodextrin to fructose is heterologous to the host cell. In accordance with some embodiments, at least two or at least three enzymes are heterologous with respect to the host cell. In accordance with some embodiments, at least one enzyme (eg, a thermostable enzyme) is endogenous (natural) with respect to the host cell. In accordance with some embodiments, at least two or at least three enzymes are endogenous to the host cell.

The host cells can be prokaryotic cells such as bacterial cells (eg Escherichia coli cells ) or eukaryotic cells such as yeast or plant cells. Other types of cells are described below.

According to some embodiments, at least one of the enzymes used to convert starch and/or cellodextrin to fructose is a thermostable enzyme. In accordance with some embodiments, at least two or at least three of the enzymes are thermostable enzymes. According to some embodiments, all enzymes are thermostable enzymes. Thus, according to some embodiments, the methods involve culturing cells engineered to express at least one thermostable α-glucan phosphorylase, at least one thermostable phosphoglucomutase, at least one thermostable phosphoglucoisomerase, at least one thermostable fructose-6- phosphate phosphatase; or a combination of at least two or more of the above thermostable enzymes. In some embodiments, the methods involve culturing cells engineered to express at least one thermostable cellodextrin phosphorylase, at least one thermostable phosphoglucomutase, at least one thermostable phosphoglucoisomerase, at least one thermostable fructose-6-phosphate phosphatase, or a combination of at least at least two or more of the above thermostable enzymes.

In some embodiments, methods for producing fructose involve lysing (e.g., thermal, mechanical, chemical, or enzymatic lysis) cultured cells to produce at least one (e.g., at least two, at least three, or at least four) cell lysate. It should be understood that multiple cell lysates (and hence multiple cell populations, e.g. from the same organism (e.g. bacteria) or from different organisms (e.g. bacteria, yeast and/or plant cells)). For example, one cell population may be engineered to express one or more fructose pathway enzymes, while another cell population (or several other cell populations) may be engineered to express another (at least one other) fructose pathway enzyme. Thus, according to some embodiments, the methods include culturing at least one population of cells engineered to express at least one α-glucan phosphorylase, culturing at least one population of cells engineered to express at least one phosphoglucomutase, culturing at least one population of cells engineered to express at least one phosphoglucoisomerase and/or culturing at least one population of cells engineered to express at least one fructose-6-phosphate phosphatase. According to some embodiments, the methods include culturing at least one cell population engineered to express at least one cellodextrin phosphorylase, culturing at least one cell population engineered to express at least one phosphoglucomutase, culturing at least one cell population engineered to express at least one phosphoglucoisomerase, and/or culturing at least one population of cells engineered to express at least one fructose-6-phosphate phosphatase. After cell lysis, the cell lysates are combined so that the enzymes are present in a single cell lysate/single reaction mixture.

In accordance with some embodiments, the methods further comprise heating one or more cell lysates (or a mixture of cell lysates) to a temperature that inactivates natural molecules with undesired enzymatic activity, but does not inactivate any thermostable enzymes of the production route, to obtain a heat-inactivated lysate. . One or more cell lysates, in accordance with some embodiments, are heated to a temperature of at least 50°C. For example, one or more cell lysates can be heated to a temperature of at least 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C or 90°C.

One or more cell lysates can be heated for a period of time sufficient to inactivate native enzymes (or other than thermostable enzymes) of the cell. For example, one or more cell lysates can be heated for at least 2, 3, 4, or at least 5 minutes. In accordance with some embodiments, one or more cell lysates are heated for more than 5 minutes. In some embodiments, one or more cell lysates are heated for a period of time sufficient to reduce the activity of native enzymes (or other non-thermostable enzymes) by at least 50% (e.g., at least 60%, 70 %, 80% or 90%).

After heat inactivation, in accordance with some embodiments, at least one (eg, at least two or at least three) purified enzyme is added to the cell lysate/reaction mixture. Thus, the reaction mixture, in accordance with some embodiments, includes a combination of enzymes present in the cell lysate (expressed by one or more engineered host cells) and at least one purified enzyme. The at least one purified enzyme may be selected from the group consisting of α-glucan phosphorylases or cellodextrin phosphorylases, phosphoglucomutases, phosphoglucoisomerases, and fructose-6-phosphate phosphatases.

In some embodiments, the methods also include incubating one or more heat-inactivated lysates in the presence of starch and inorganic phosphate to produce fructose. According to some embodiments, the methods also include incubating one or more heat-inactivated lysates in the presence of cellodextrin and inorganic phosphate to produce fructose. According to some embodiments, the heat-inactivated lysates are incubated at a temperature of at least 50°C. According to some embodiments, the heat-inactivated lysates are incubated for at least 2 minutes (eg, at least 3, 4, or 5 minutes). For example, heat-inactivated lysates can be incubated for 2-5 minutes or 2-10 minutes. The starch may be, for example, amylose, amylopectin or a mixture of amylose and amylopectin. According to some embodiments, biomass is used instead of starch. According to such embodiments, the reaction involves one or more cellodextrin phosphorylases. In accordance with some embodiments, starch or cellodextrin is present as a component of the mixture.

The present invention also relates to cells and cell lysates used to obtain fructose. Thus, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure may include at least one (e.g., at least two or at least three) enzymes selected from the group, consisting of α-glucan phosphorylases, phosphoglucomutases and fructose-6-phosphate phosphatases. In accordance with some embodiments, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure comprises at least one (e.g., at least two or at least three) enzymes selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases and thermostable fructose-6-phosphate phosphatase. In accordance with some embodiments, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure comprises at least one (e.g., at least two or at least three) enzymes selected from the group consisting of thermostable cellodextrinphosphorylases, thermostable phosphoglucomutases and thermostable fructose-6-phosphate phosphatase.

Obtaining mannose

Some other aspects of the present disclosure relate to methods, compositions and systems for producing mannose. Such methods, in some embodiments, involve culturing cells engineered to express at least one α-glucan phosphorylase and/or at least one cellodextrin phosphorylase, at least one phosphoglucomutase, at least one phosphoglucoisomerase, at least one mannose -6-phosphate isomerase, at least one mannose-6-phosphate phosphatase, or a combination of at least two of the above enzymes. In some embodiments, α-glucan phosphorylase (and/or cellodextrin phosphorylase) and phosphoglucomutase are expressed as a single fusion (chimeric) protein or bifunctional protein.

The mannose pathway enzymes provided herein are typically heterologous to the host cell (originally cloned or derived from a different cell type), although some of the enzymes may be endogenous (native) to the host cell. Thus, in some embodiments, at least one enzyme (eg, a thermostable enzyme) used to convert starch and/or cellodextrin to mannose is heterologous to the host cell. In some embodiments, at least two, at least three, at least four, at least five, or at least six enzymes are heterologous with respect to the host cell. In accordance with some embodiments, at least one enzyme (eg, a thermostable enzyme) is endogenous (natural) with respect to the host cell. In some embodiments, at least two, at least three, at least four, at least five, or at least six enzymes are endogenous to the host cell.

The host cells can be prokaryotic cells such as bacterial cells (eg Escherichia coli cells ) or eukaryotic cells such as yeast or plant cells. Other types of cells are described below.

According to some embodiments, at least one of the enzymes used to convert starch and/or cellodextrin to mannose is a thermostable enzyme. In accordance with some embodiments, at least two, at least three, at least four, at least five, or at least six enzymes are thermostable enzymes. According to some embodiments, all enzymes are thermostable enzymes. Thus, according to some embodiments, the methods involve culturing cells engineered to express at least one thermostable α-glucan phosphorylase, at least one thermostable phosphoglucomutase, at least one thermostable phosphoglucoisomerase, at least one thermostable mannose-6- phosphate isomerase, at least one thermostable mannose-6-phosphate phosphatase, or a combination of at least two or more of the above thermostable enzymes. According to some embodiments, the methods include culturing cells engineered to express at least one thermostable cellodextrin phosphorylase, at least one thermostable phosphoglucomutase, at least one thermostable phosphoglucoisomerase, at least one thermostable mannose-6-phosphate isomerase, at least one thermostable mannose-6-phosphate phosphatase; or a combination of at least two or more of the above thermostable enzymes.

In some embodiments, methods for producing mannose involve lysing (e.g., thermal, mechanical, chemical, or enzymatic lysis) cultured cells to produce at least one (e.g., at least two, at least three, or at least four) cell lysate. It should be understood that multiple cell lysates (and hence multiple cell populations, e.g. from the same organism (e.g. bacteria) or from different organisms (e.g. bacteria, yeast and/or plant cells)). For example, one cell population may be engineered to express one or more mannose pathway enzymes, while another cell population (or several other cell populations) may be engineered to express another (at least one other) mannose pathway enzyme. Thus, according to some embodiments, the methods include culturing at least one population of cells engineered to express at least one α-glucan phosphorylase, culturing at least one population of cells engineered to express at least one phosphoglucomutase, culturing at least one population of cells engineered to express at least one phosphoglucoisomerase, culturing at least one population of cells engineered to express at least one mannose-6-phosphate isomerase, and/or culturing at least one population of cells engineered to express at least one mannose-6-phosphate phosphatase. According to some embodiments, the methods include culturing at least one cell population engineered to express at least one cellodextrin phosphorylase, culturing at least one cell population engineered to express at least one phosphoglucomutase, culturing at least one cell population engineered to express at least one phosphoglucoisomerase, culturing at least one population of cells engineered to express at least one mannose-6-phosphate isomerase, and/or culturing at least one population of cells engineered to express at least one mannose -6-phosphate phosphatase. After cell lysis, the cell lysates are combined so that the enzymes are present in a single cell lysate/single reaction mixture.

In accordance with some embodiments, the methods further comprise heating one or more cell lysates (or a mixture of cell lysates) to a temperature that inactivates natural molecules with undesired enzymatic activity, but does not inactivate any thermostable enzymes of the production route, to obtain a heat-inactivated lysate. . One or more cell lysates, in accordance with some embodiments, are heated to a temperature of at least 50°C. For example, one or more cell lysates can be heated to a temperature of at least 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C or 90°C.

One or more cell lysates can be heated for a period of time sufficient to inactivate native enzymes (or other than thermostable enzymes) of the cell. For example, one or more cell lysates can be heated for at least 2, 3, 4, or at least 5 minutes. In accordance with some embodiments, one or more cell lysates are heated for more than 5 minutes. In some embodiments, one or more cell lysates are heated for a period of time sufficient to reduce the activity of native enzymes (or other non-thermostable enzymes) by at least 50% (e.g., at least 60%, 70 %, 80% or 90%).

After heat inactivation, in accordance with some embodiments, at least one (eg, at least two or at least three) purified enzyme is added to the cell lysate/reaction mixture. Thus, the reaction mixture, in accordance with some embodiments, includes a combination of enzymes present in the cell lysate (expressed by one or more engineered host cells) and at least one purified enzyme. The at least one purified enzyme can be selected from the group consisting of α-glucan phosphorylases or cellodextrin phosphorylases, phosphoglucomutases, phosphoglucoisomerases, mannose-6-phosphate isomerases, and mannose-6-phosphate phosphatases.

In some embodiments, the methods also include incubating one or more heat-inactivated lysates in the presence of starch and inorganic phosphate to produce mannose. In some embodiments, the methods also include incubating one or more heat-inactivated lysates in the presence of cellodextrin and inorganic phosphate to produce mannose. According to some embodiments, the heat-inactivated lysates are incubated at a temperature of at least 50°C. According to some embodiments, the heat-inactivated lysates are incubated for at least 2 minutes (eg, at least 3, 4, or 5 minutes). For example, heat-inactivated lysates can be incubated for 2-5 minutes or 2-10 minutes. The starch may be, for example, amylose, amylopectin or a mixture of amylose and amylopectin. According to some embodiments, biomass is used instead of starch. According to such embodiments, the reaction involves one or more cellodextrin phosphorylases. In accordance with some embodiments, starch or cellodextrin is present as a component of the mixture.

The present invention also relates to cells and cell lysates used for the production of mannose. Thus, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure may include at least one (e.g., at least two, at least three, at least four, at least five or at least six) an enzyme selected from the group consisting of α-glucan phosphorylases, phosphoglucomutases, phosphoglucoisomerases, mannose-6-phosphate isomerases, and mannose-6-phosphate phosphatases. The engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure may include at least one (e.g., at least two, at least three, at least four, at least five or at least six) an enzyme selected from the group consisting of cellodextrin phosphorylases, phosphoglucomutases, phosphoglucoisomerases, mannose-6-phosphate isomerases, and mannose-6-phosphate phosphatases. In accordance with some embodiments, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure comprises at least one (e.g., at least two, at least three, at least four, at least five, or at least six) an enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable phosphoglucoisomerases, thermostable mannose-6-phosphate isomers, and thermostable mannose-6-phosphate phosphatases. In accordance with some embodiments, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure comprises at least one (e.g., at least two, at least three, at least four, at least five, or at least six) an enzyme selected from the group consisting of thermostable cellodextrin phosphorylases, thermostable phosphoglucomutases, thermostable phosphoglucoisomerases, thermostable mannose-6-phosphate isomerases, and thermostable mannose-6-phosphate phosphatases.

Obtaining sorbitol

Still other aspects of the present disclosure relate to methods, compositions and systems for producing sorbitol. Such methods, in some embodiments, involve culturing cells engineered to express at least one α-glucan phosphorylase and/or at least one cellodextrin phosphorylase, at least one phosphoglucomutase, at least one aldose dehydrogenase, at least one sorbitol -6-phosphate phosphatase; or a combination of at least two of the above enzymes. In some embodiments, the methods involve culturing cells engineered to express at least one α-glucan phosphorylase and/or at least one cellodextrin phosphorylase, at least one phosphoglucomutase, at least one phosphoglucoisomerase, at least one sorbitol-6 -phosphate-2-dehydrogenase, at least one sorbitol-6-phosphate phosphatase, or a combination of at least two of the enzymes mentioned above. In some embodiments, α-glucan phosphorylase (and/or cellodextrin phosphorylase) and phosphoglucomutase are expressed as a single fusion (chimeric) protein or bifunctional protein.

The sorbitol pathway enzymes provided herein are typically heterologous to the host cell (originally cloned or derived from a different cell type), although some of the enzymes may be endogenous (native) to the host cell. Thus, in some embodiments, at least one enzyme (eg, a thermostable enzyme) used to convert starch and/or cellodextrin to sorbitol is heterologous to the host cell. In accordance with some embodiments, at least two or at least three enzymes are heterologous with respect to the host cell. In accordance with some embodiments, at least one enzyme (eg, a thermostable enzyme) is endogenous (natural) with respect to the host cell. In accordance with some embodiments, at least two or at least three enzymes are endogenous to the host cell.

The host cells can be prokaryotic cells such as bacterial cells (eg Escherichia coli cells ) or eukaryotic cells such as yeast or plant cells. Other types of cells are described below.

According to some embodiments, at least one of the enzymes used to convert starch and/or cellodextrin to sorbitol is a thermostable enzyme. In accordance with some embodiments, at least two, at least three, or at least four of the enzymes are thermostable enzymes. According to some embodiments, all enzymes are thermostable enzymes. Thus, according to some embodiments, the methods involve culturing cells engineered to express at least one thermostable α-glucan phosphorylase, at least one thermostable phosphoglucomutase, at least one thermostable aldose dehydrogenase, at least one thermostable sorbitol-6- phosphate phosphatase; or a combination of at least two or more of the above thermostable enzymes. In some embodiments, the methods involve culturing cells engineered to express at least one thermostable cellodextrin phosphorylase, at least one thermostable phosphoglucomutase, at least one thermostable aldose dehydrogenase, at least one thermostable sorbitol-6-phosphate phosphatase, or a combination of at least at least two or more of the above thermostable enzymes. According to some embodiments, the methods involve culturing cells engineered to express at least one thermostable α-glucan phosphorylase, at least one thermostable phosphoglucomutase, at least one thermostable phosphoglucoisomerase, at least one thermostable sorbitol-6-phosphate-2 -dehydrogenase, at least one thermostable sorbitol-6-phosphate phosphatase, or a combination of at least two or more of the above thermostable enzymes. According to some embodiments, the methods involve culturing cells engineered to express at least one thermostable cellodextran phosphorylase, at least one thermostable phosphoglucomutase, at least one thermostable phosphoglucoisomerase, at least one thermostable sorbitol-6-phosphate-2-dehydrogenase , at least one thermostable sorbitol-6-phosphate phosphatase, or a combination of at least two or more of the above thermostable enzymes.

In some embodiments, methods for producing sorbitol involve lysing (e.g., thermal, mechanical, chemical, or enzymatic lysis) cultured cells to produce at least one (e.g., at least two, at least three, or at least four) cell lysate. It should be understood that multiple cell lysates (and hence multiple cell populations, e.g. from the same organism (e.g. bacteria) or from different organisms (e.g. bacteria, yeast and/or plant cells)). For example, one cell population may be engineered to express one or more sorbitol pathway enzymes, while another cell population (or several other cell populations) may be engineered to express another (at least one other) sorbitol pathway enzyme. Thus, according to some embodiments, the methods include culturing at least one population of cells engineered to express at least one α-glucan phosphorylase, culturing at least one population of cells engineered to express at least one phosphoglucomutase, culturing at least one cell population engineered to express at least one aldose dehydrogenase and/or culturing at least one cell population engineered to express at least one sorbitol-6-phosphate phosphatase. According to some embodiments, the methods include culturing at least one cell population engineered to express at least one cellodextrin phosphorylase, culturing at least one cell population engineered to express at least one phosphoglucomutase, culturing at least one cell population engineered to express at least one aldose dehydrogenase and/or culturing at least one population of cells engineered to express at least one sorbitol-6-phosphate phosphatase. After cell lysis, the cell lysates are combined so that the enzymes are present in a single cell lysate/single reaction mixture.

In accordance with some embodiments, the methods further comprise heating one or more cell lysates (or a mixture of cell lysates) to a temperature that inactivates natural molecules with undesired enzymatic activity, but does not inactivate any thermostable enzymes of the production route, to obtain a heat-inactivated lysate. . One or more cell lysates, in accordance with some embodiments, are heated to a temperature of at least 50°C. For example, one or more cell lysates can be heated to a temperature of at least 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C or 90°C.

One or more cell lysates can be heated for a period of time sufficient to inactivate native enzymes (or other than thermostable enzymes) of the cell. For example, one or more cell lysates can be heated for at least 2, 3, 4, or at least 5 minutes. In accordance with some embodiments, one or more cell lysates are heated for more than 5 minutes. In some embodiments, one or more cell lysates are heated for a period of time sufficient to reduce the activity of natural enzymes (or other non-thermostable enzymes) by at least 50% (e.g., at least 60%, 70 %, 80% or 90%).

After heat inactivation, in accordance with some embodiments, at least one (eg, at least two or at least three) purified enzyme is added to the cell lysate/reaction mixture. Thus, the reaction mixture, in accordance with some embodiments, includes a combination of enzymes present in the cell lysate (expressed by one or more engineered host cells) and at least one purified enzyme. The at least one purified enzyme can be selected from the group consisting of α-glucan phosphorylases or cellodextrin phosphorylases, phosphoglucomutases, phosphoglucoisomerases, sorbitol-6-phosphate-2-dehydrogenases, aldose dehydrogenases, and sorbitol-6-phosphate phosphatases.

According to some embodiments, the methods also include incubating one or more heat-inactivated lysates in the presence of starch and inorganic phosphate to produce sorbitol. In some embodiments, the methods also include incubating one or more heat-inactivated lysates in the presence of cellodextrin and inorganic phosphate to produce sorbitol. According to some embodiments, the heat-inactivated lysates are incubated at a temperature of at least 50°C. According to some embodiments, the heat-inactivated lysates are incubated for at least 2 minutes (eg, at least 3, 4, or 5 minutes). For example, heat-inactivated lysates can be incubated for 2-5 minutes or 2-10 minutes. The starch may be, for example, amylose, amylopectin or a mixture of amylose and amylopectin. According to some embodiments, biomass is used instead of starch. According to such embodiments, the reaction involves one or more cellodextrin phosphorylases. In accordance with some embodiments, starch or cellodextrin is present as a component of the mixture.

The present invention also relates to cells and cell lysates used to obtain sorbitol. Thus, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure may include at least one (e.g., at least two or at least three) enzymes selected from the group, consisting of α-glucan phosphorylases, phosphoglucomutases, aldose dehydrogenases and sorbitol-6-phosphate phosphatases. The engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure may include at least one (e.g., at least two or at least three) enzymes selected from the group consisting of cellodextrin phosphorylases , phosphoglucomutase, aldose dehydrogenase and sorbitol-6-phosphate phosphatase. In accordance with some embodiments, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure comprises at least one (e.g., at least two or at least three) enzymes selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable aldose dehydrogenases and thermostable sorbitol-6-phosphate phosphatases. In accordance with some embodiments, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure comprises at least one (e.g., at least two or at least three) enzymes selected from the group consisting of thermostable cellodextrinphosphorylases, thermostable phosphoglucomutases, thermostable aldose dehydrogenases and thermostable sorbitol-6-phosphate phosphatases. Thus, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure may include at least one (e.g., at least two or at least three) enzymes selected from the group, consisting of α-glucan phosphorylases, phosphoglucomutases, phosphoglucoisomerases, sorbitol-6-phosphate-2-dehydrogenases and sorbitol-6-phosphate phosphatases. The engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure may include at least one (e.g., at least two or at least three) enzymes selected from the group consisting of cellodextrin phosphorylases , phosphoglucomutases, phosphoglucoisomerases, sorbitol-6-phosphate-2-dehydrogenases and sorbitol-6-phosphate phosphatases. In accordance with some embodiments, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure comprises at least one (e.g., at least two or at least three) enzymes selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable phosphoglucoisomerases, thermostable sorbitol-6-phosphate aldose dehydrogenases and thermostable sorbitol-6-phosphate phosphatases. In accordance with some embodiments, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure comprises at least one (e.g., at least two or at least three) enzymes selected from the group consisting of thermostable cellodextrinphosphorylases, thermostable phosphoglucomutases, thermostable phosphoglucoisomerases, thermostable sorbitol-6-phosphate-2-dehydrogenases and thermostable sorbitol-6-phosphate phosphatases.

Getting ribulose

Additional aspects of the present disclosure relate to methods, compositions and systems for producing ribulose. Such methods, in some embodiments, involve culturing cells engineered to express at least one α-glucan phosphorylase and/or cellodextrin phosphorylase, at least one phosphoglucomutase, at least one glucose-6-phosphate dehydrogenase, at least one 6 -phosphogluconolactonase, at least one 6-phosphogluconate dehydrogenase, at least one ribulose-5-phosphate phosphatase, or a combination of at least two of the above enzymes. In some embodiments, α-glucan phosphorylase (and/or cellodextrin phosphorylase) and phosphoglucomutase are expressed as a single fusion (chimeric) protein or bifunctional protein.

The ribulose pathway enzymes provided herein are typically heterologous to the host cell (originally cloned or derived from a different cell type), although some of the enzymes may be endogenous (native) to the host cell. Thus, in some embodiments, at least one enzyme (eg, a thermostable enzyme) used to convert starch and/or cellodextrin to ribulose is heterologous to the host cell. In some embodiments, at least two, at least three, at least four, or at least five enzymes are heterologous with respect to the host cell. In accordance with some embodiments, at least one enzyme (eg, a thermostable enzyme) is endogenous (natural) with respect to the host cell. In some embodiments, at least two, at least three, at least four, or at least five enzymes are endogenous to the host cell.

The host cells can be prokaryotic cells such as bacterial cells (eg Escherichia coli cells ) or eukaryotic cells such as yeast or plant cells. Other types of cells are described below.

According to some embodiments, at least one of the enzymes used to convert starch and/or cellodextrin to ribulose is a thermostable enzyme. In accordance with some embodiments, at least two, at least three, at least four, or at least five enzymes are thermostable enzymes. According to some embodiments, all enzymes are thermostable enzymes. Thus, according to some embodiments, the methods involve culturing cells engineered to express at least one thermostable α-glucan phosphorylase, at least one thermostable phosphoglucomutase, at least one thermostable glucose-6-phosphate dehydrogenase, at least one thermostable 6-phosphogluconolactonase, at least one thermostable 6-phosphogluconate dehydrogenase, at least one thermostable ribulose-5-phosphate phosphatase, or a combination of at least two or more of the above thermostable enzymes. In some embodiments, the methods involve culturing cells engineered to express at least one thermostable cellodextrin phosphorylase, at least one thermostable phosphoglucomutase, at least one thermostable glucose-6-phosphate dehydrogenase, at least one thermostable 6-phosphogluconolactonase, at least one thermostable 6-phosphogluconate dehydrogenase, at least one thermostable ribulose-5-phosphate phosphatase, or a combination of at least two or more of the above thermostable enzymes.

In some embodiments, methods for producing ribulose involve lysing (e.g., thermal, mechanical, chemical, or enzymatic lysis) cultured cells to produce at least one (e.g., at least two, at least three, or at least four) cell lysate. It should be understood that multiple cell lysates (and hence multiple cell populations, e.g. from the same organism (e.g. bacteria) or from different organisms (e.g. bacteria, yeast and/or plant cells)). For example, one cell population may be engineered to express one or more ribulose pathway enzymes, while another cell population (or more than one cell population) may be engineered to express another (at least one other) ribulose pathway enzyme. Thus, according to some embodiments, the methods include culturing at least one population of cells engineered to express at least one α-glucan phosphorylase, culturing at least one population of cells engineered to express at least one phosphoglucomutase, culturing at least one population of cells engineered to express at least one glucose-6-phosphate dehydrogenase, culturing at least one population of cells engineered to express at least one 6-phosphogluconolactonase, culturing at least one population of cells engineered to express at least one 6-phosphogluconate dehydrogenase, and/or culturing at least one population of cells engineered to express at least one ribulose-5-phosphate phosphatase. According to some embodiments, the methods include culturing at least one cell population engineered to express at least one cellodextrin phosphorylase, culturing at least one cell population engineered to express at least one phosphoglucomutase, culturing at least one cell population engineered to express at least one glucose-6-phosphate dehydrogenase, culturing at least one population of cells engineered to express at least one 6-phosphogluconolactonase, culturing at least one population of cells engineered to express at least one 6- phosphogluconate dehydrogenase, and/or culturing at least one population of cells engineered to express at least one ribulose-5-phosphate phosphatase. After cell lysis, the cell lysates are combined so that the enzymes are present in a single cell lysate/single reaction mixture.

In accordance with some embodiments, the methods further comprise heating one or more cell lysates (or a mixture of cell lysates) to a temperature that inactivates natural molecules with undesired enzymatic activity, but does not inactivate any thermostable enzymes of the production route, to obtain a heat-inactivated lysate. . One or more cell lysates, in accordance with some embodiments, are heated to a temperature of at least 50°C. For example, one or more cell lysates can be heated to a temperature of at least 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C or 90°C.

One or more cell lysates can be heated for a period of time sufficient to inactivate native enzymes (or other than thermostable enzymes) of the cell. For example, one or more cell lysates can be heated for at least 2, 3, 4, or at least 5 minutes. In accordance with some embodiments, one or more cell lysates are heated for more than 5 minutes. In some embodiments, one or more cell lysates are heated for a period of time sufficient to reduce the activity of natural enzymes (or other non-thermostable enzymes) by at least 50% (e.g., at least 60%, 70 %, 80% or 90%).

After heat inactivation, in accordance with some embodiments, at least one (eg, at least two or at least three) purified enzyme is added to the cell lysate/reaction mixture. Thus, the reaction mixture, in accordance with some embodiments, includes a combination of enzymes present in the cell lysate (expressed by one or more engineered host cells) and at least one purified enzyme. The at least one purified enzyme can be selected from the group consisting of α-glucan phosphorylases or cellodextrin phosphorylases, phosphoglucomutases, glucose-6-phosphate dehydrogenases, 6-phosphogluconolactonases, 6-phosphogluconate dehydrogenases, and ribulose-5-phosphate phosphatases.

In some embodiments, the methods also include incubating one or more heat-inactivated lysates in the presence of starch and inorganic phosphate to produce ribulose. In some embodiments, the methods also include incubating one or more heat-inactivated lysates in the presence of cellodextrin and inorganic phosphate to produce ribulose. According to some embodiments, the heat-inactivated lysates are incubated at a temperature of at least 50°C. According to some embodiments, the heat-inactivated lysates are incubated for at least 2 minutes (eg, at least 3, 4, or 5 minutes). For example, heat-inactivated lysates can be incubated for 2-5 minutes or 2-10 minutes. The starch may be, for example, amylose, amylopectin or a mixture of amylose and amylopectin. According to some embodiments, biomass is used instead of starch. According to such embodiments, the reaction involves one or more cellodextrin phosphorylases. In accordance with some embodiments, starch or cellodextrin is present as a component of the mixture.

The present invention also relates to cells and cell lysates used to obtain ribulose. Thus, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure may include at least one (e.g., at least two or at least three) enzymes selected from the group, consisting of α-glucan phosphorylases, phosphoglucomutases, glucose-6-phosphate dehydrogenases, 6-phosphogluconolactonases, 6-phosphogluconate dehydrogenases, and ribulose-5-phosphate phosphatases. The engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure may include at least one (e.g., at least two or at least three) enzymes selected from the group consisting of cellodextrin phosphorylases , phosphoglucomutase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase and ribulose-5-phosphate phosphatase. In accordance with some embodiments, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure comprises at least one (e.g., at least two or at least three) enzymes selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable glucose-6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases, thermostable 6-phosphogluconate dehydrogenases, and thermostable ribulose-5-phosphate phosphatases. In accordance with some embodiments, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure comprises at least one (e.g., at least two or at least three) enzymes selected from the group consisting of thermostable cellodextrinphosphorylases, thermostable phosphoglucomutases, thermostable glucose-6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases, thermostable 6-phosphogluconate dehydrogenases and thermostable ribulose-5-phosphate phosphatases.

Obtaining ribose

Some other aspects of the present disclosure relate to methods, compositions and systems for producing ribose. Such methods, in some embodiments, involve culturing cells engineered to express at least one α-glucan phosphorylase and/or at least one cellodextrin phosphorylase, at least one phosphoglucomutase, at least one glucose-6-phosphate dehydrogenase, at least one 6-phosphogluconolactonase, at least one 6-phosphogluconate dehydrogenase, at least one ribose-5-phosphate isomerase, at least one ribose-5-phosphate phosphatase, or a combination of at least two of the above enzymes. In some embodiments, α-glucan phosphorylase (and/or cellodextrin phosphorylase) and phosphoglucomutase are expressed as a single fusion (chimeric) protein or bifunctional protein.

The ribose pathway enzymes provided herein are typically heterologous to the host cell (originally cloned or derived from a different cell type), although some of the enzymes may be endogenous (native) to the host cell. Thus, in some embodiments, at least one enzyme (eg, a thermostable enzyme) used to convert starch and/or cellodextrin to ribose is heterologous to the host cell. In some embodiments, at least two, at least three, at least four, at least five, or at least six enzymes are heterologous with respect to the host cell. In accordance with some embodiments, at least one enzyme (eg, a thermostable enzyme) is endogenous (natural) with respect to the host cell. In some embodiments, at least two, at least three, at least four, at least five, or at least six enzymes are endogenous to the host cell.

The host cells can be prokaryotic cells such as bacterial cells (eg Escherichia coli cells ) or eukaryotic cells such as yeast or plant cells. Other types of cells are described below.

According to some embodiments, at least one of the enzymes used to convert starch and/or cellodextrin to ribose is a thermostable enzyme. In accordance with some embodiments, at least two, at least three, at least four, at least five, or at least six enzymes are thermostable enzymes. According to some embodiments, all enzymes are thermostable enzymes. Thus, according to some embodiments, the methods involve culturing cells engineered to express at least one thermostable α-glucan phosphorylase, at least one thermostable phosphoglucomutase, at least one thermostable glucose-6-phosphate dehydrogenase, at least one thermostable 6-phosphogluconolactonase, at least one thermostable 6-phosphogluconate dehydrogenase, at least one thermostable ribose-5-phosphate isomerase, at least one thermostable ribose-5-phosphate phosphatase, or a combination of at least two or more of the above thermostable enzymes. In some embodiments, the methods involve culturing cells engineered to express at least one thermostable cellodextrin phosphorylase, at least one thermostable phosphoglucomutase, at least one thermostable glucose-6-phosphate dehydrogenase, at least one thermostable 6-phosphogluconolactonase, at least one thermostable 6-phosphogluconate dehydrogenase, at least one thermostable ribose-5-phosphate isomerase, at least one thermostable ribose-5-phosphate phosphatase, or a combination of at least two or more of the above thermostable enzymes.

In some embodiments, methods for producing ribose involve lysing (e.g., thermal, mechanical, chemical, or enzymatic lysis) cultured cells to produce at least one (e.g., at least two, at least three, or at least four) cell lysate. It should be understood that multiple cell lysates (and hence multiple cell populations, e.g. from the same organism (e.g. bacteria) or from different organisms (e.g. bacteria, yeast and/or plant cells)). For example, one cell population may be engineered to express one or more ribose pathway enzymes, while another cell population (or several other cell populations) may be engineered to express another (at least one other) ribose pathway enzyme. Thus, according to some embodiments, the methods include culturing at least one population of cells engineered to express at least one α-glucan phosphorylase, culturing at least one population of cells engineered to express at least one phosphoglucomutase, culturing at least one population of cells engineered to express at least one glucose-6-phosphate dehydrogenase, culturing at least one population of cells engineered to express at least one 6-phosphogluconolactonase, culturing at least one population of cells engineered to express at least one 6-phosphogluconate dehydrogenase, culturing at least one population of cells engineered to express at least one ribose-5-phosphate isomerase, and/or culturing at least one population of cells engineered to express sessions of at least one ribose-5-phosphate phosphatase. According to some embodiments, the methods include culturing at least one cell population engineered to express at least one cellodextrin phosphorylase, culturing at least one cell population engineered to express at least one phosphoglucomutase, culturing at least one cell population engineered to express at least one glucose-6-phosphate dehydrogenase, culturing at least one population of cells engineered to express at least one 6-phosphogluconolactonase, culturing at least one population of cells engineered to express at least one 6- phosphogluconate dehydrogenase, culturing at least one population of cells engineered to express at least one ribose-5-phosphate isomerase, and/or culturing at least one population of cells engineered to express the at least one ribose-5-phosphate phosphatase. After cell lysis, the cell lysates are combined so that the enzymes are present in a single cell lysate/single reaction mixture.

In accordance with some embodiments, the methods further comprise heating one or more cell lysates (or a mixture of cell lysates) to a temperature that inactivates natural molecules with undesired enzymatic activity, but does not inactivate any thermostable enzymes of the production route, to obtain a heat-inactivated lysate. . One or more cell lysates, in accordance with some embodiments, are heated to a temperature of at least 50°C. For example, one or more cell lysates can be heated to a temperature of at least 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C or 90°C.

One or more cell lysates can be heated for a period of time sufficient to inactivate native enzymes (or other than thermostable enzymes) of the cell. For example, one or more cell lysates can be heated for at least 2, 3, 4, or at least 5 minutes. In accordance with some embodiments, one or more cell lysates are heated for more than 5 minutes. In some embodiments, one or more cell lysates are heated for a period of time sufficient to reduce the activity of natural enzymes (or other non-thermostable enzymes) by at least 50% (e.g., at least 60%, 70 %, 80% or 90%).

After heat inactivation, in accordance with some embodiments, at least one (eg, at least two or at least three) purified enzyme is added to the cell lysate/reaction mixture. Thus, the reaction mixture, in accordance with some embodiments, includes a combination of enzymes present in the cell lysate (expressed by one or more engineered host cells) and at least one purified enzyme. The at least one purified enzyme can be selected from the group consisting of α-glucan phosphorylases or cellodextrin phosphorylases, phosphoglucomutases, glucose-6-phosphate dehydrogenases, 6-phosphogluconolactonases, 6-phosphogluconate dehydrogenases, ribose-5-phosphate isomerase and ribose-5-phosphate phosphatases.

In some embodiments, the methods also include incubating one or more heat-inactivated lysates in the presence of starch and inorganic phosphate to produce ribose. In some embodiments, the methods also include incubating one or more heat-inactivated lysates in the presence of cellodextrin and inorganic phosphate to produce ribose. According to some embodiments, the heat-inactivated lysates are incubated at a temperature of at least 50°C. According to some embodiments, the heat-inactivated lysates are incubated for at least 2 minutes (eg, at least 3, 4, or 5 minutes). For example, heat-inactivated lysates can be incubated for 2-5 minutes or 2-10 minutes. The starch may be, for example, amylose, amylopectin or a mixture of amylose and amylopectin. According to some embodiments, biomass is used instead of starch. According to such embodiments, the reaction involves one or more cellodextrin phosphorylases. In accordance with some embodiments, starch or cellodextrin is present as a component of the mixture.

The present invention also relates to cells and cell lysates used to obtain ribose. Thus, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure may include at least one (e.g., at least two, at least three, at least four, at least five or at least six) an enzyme selected from the group consisting of α-glucan phosphorylases, phosphoglucomutases, glucose-6-phosphate dehydrogenases, 6-phosphogluconolactonases, 6-phosphogluconate dehydrogenases, ribose-5-phosphate isomerases, and ribose-5-phosphate phosphatases. The engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure may include at least one (e.g., at least two, at least three, at least four, at least five or at least six) an enzyme selected from the group consisting of cellodextrinphosphorylases, phosphoglucomutases, glucose-6-phosphate dehydrogenases, 6-phosphogluconolactonases, 6-phosphogluconate dehydrogenases, ribose-5-phosphate isomerases, and ribose-5-phosphate phosphatases. In accordance with some embodiments, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure comprises at least one (e.g., at least two, at least three, at least four, at least five or at least six) an enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable glucose-6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases, thermostable 6-phosphogluconate dehydrogenases, thermostable ribose-5- phosphisomerases and thermostable ribose-5-phosphate phosphatases. In accordance with some embodiments, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure comprises at least one (e.g., at least two, at least three, at least four, at least five, or at least six) an enzyme selected from the group consisting of thermostable cellodextrin phosphorylases, thermostable phosphoglucomutases, thermostable glucose-6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases, thermostable 6-phosphogluconate dehydrogenases, thermostable ribose-5-phosphate isomerases, and thermostable ribose-5-phosphate phosphatases.

Obtaining arabinose

Some other aspects of the present disclosure relate to methods, compositions and systems for producing arabinose. Such methods, in some embodiments, involve culturing cells engineered to express at least one α-glucan phosphorylase and/or at least one cellodextrin phosphorylase, at least one phosphoglucomutase, at least one glucose-6-phosphate dehydrogenase, at least one 6-phosphogluconolactonase, at least one 6-phosphogluconate dehydrogenase, at least one arabinose-5-phosphate isomerase, at least one ribose-5-phosphate phosphatase, or a combination of at least two of the above enzymes. In some embodiments, α-glucan phosphorylase (and/or cellodextrin phosphorylase) and phosphoglucomutase are expressed as a single fusion (chimeric) protein or bifunctional protein.

The arabinose pathway enzymes provided herein are typically heterologous to the host cell (originally cloned or derived from a different cell type), although some of the enzymes may be endogenous (native) to the host cell. Thus, in some embodiments, at least one enzyme (eg, a thermostable enzyme) used to convert starch and/or cellodextrin to arabinose is heterologous to the host cell. In some embodiments, at least two, at least three, at least four, at least five, or at least six enzymes are heterologous with respect to the host cell. In accordance with some embodiments, at least one enzyme (eg, a thermostable enzyme) is endogenous (natural) with respect to the host cell. In some embodiments, at least two, at least three, at least four, at least five, or at least six enzymes are endogenous to the host cell.

The host cells can be prokaryotic cells such as bacterial cells (eg Escherichia coli cells ) or eukaryotic cells such as yeast or plant cells. Other types of cells are described below.

According to some embodiments, at least one of the enzymes used to convert starch and/or cellodextrin to arabinose is a thermostable enzyme. In accordance with some embodiments, at least two, at least three, at least four, at least five, or at least six enzymes are thermostable enzymes. According to some embodiments, all enzymes are thermostable enzymes. Thus, according to some embodiments, the methods involve culturing cells engineered to express at least one thermostable α-glucan phosphorylase, at least one thermostable phosphoglucomutase, at least one thermostable glucose-6-phosphate dehydrogenase, at least one thermostable 6-phosphogluconolactonase, at least one thermostable 6-phosphogluconate dehydrogenase, at least one thermostable arabinose-5-phosphate isomerase, at least one thermostable arabinose-5-phosphate phosphatase, or a combination of at least two or more of the above thermostable enzymes. In some embodiments, the methods involve culturing cells engineered to express at least one thermostable cellodextrin phosphorylase, at least one thermostable phosphoglucomutase, at least one thermostable glucose-6-phosphate dehydrogenase, at least one thermostable 6-phosphogluconolactonase, at least one thermostable 6-phosphogluconate dehydrogenase, at least one thermostable arabinose-5-phosphate isomerase, at least one thermostable arabinose-5-phosphate phosphatase, or a combination of at least two or more of the above thermostable enzymes.

According to some embodiments, methods for producing arabinose involve lysing (e.g., thermal, mechanical, chemical, or enzymatic lysis) of cultured cells to produce at least one (e.g., at least two, at least three, or at least four) cell lysate. It should be understood that multiple cell lysates (and hence multiple cell populations, e.g. from the same organism (e.g. bacteria) or from different organisms (e.g. bacteria, yeast and/or plant cells)). For example, one cell population may be engineered to express one or more arabinose pathway enzymes, while another cell population (or more than one cell population) may be engineered to express another (at least one other) arabinose pathway enzyme. Thus, according to some embodiments, the methods include culturing at least one population of cells engineered to express at least one α-glucan phosphorylase, culturing at least one population of cells engineered to express at least one phosphoglucomutase, culturing at least one population of cells engineered to express at least one glucose-6-phosphate dehydrogenase, culturing at least one population of cells engineered to express at least one 6-phosphogluconolactonase, culturing at least one population of cells engineered to express at least one 6-phosphogluconate dehydrogenase, culturing at least one population of cells engineered to express at least one arabinose-5-phosphate isomerase, and/or culturing at least one population of cells engineered to express depression of at least one arabinose-5-phosphate phosphatase. According to some embodiments, the methods include culturing at least one cell population engineered to express at least one cellodextrin phosphorylase, culturing at least one cell population engineered to express at least one phosphoglucomutase, culturing at least one cell population engineered to express at least one glucose-6-phosphate dehydrogenase, culturing at least one population of cells engineered to express at least one 6-phosphogluconolactonase, culturing at least one population of cells engineered to express at least one 6- phosphogluconate dehydrogenase, culturing at least one population of cells engineered to express at least one arabinose-5-phosphate isomerase, and/or culturing at least one population of cells engineered to express at least one arabinose-5-phosphate phosphatase. After cell lysis, the cell lysates are combined so that the enzymes are present in a single cell lysate/single reaction mixture.

In accordance with some embodiments, the methods further comprise heating one or more cell lysates (or a mixture of cell lysates) to a temperature that inactivates natural molecules with undesired enzymatic activity, but does not inactivate any thermostable enzymes of the production route, to obtain a heat-inactivated lysate. . One or more cell lysates, in accordance with some embodiments, are heated to a temperature of at least 50°C. For example, one or more cell lysates can be heated to a temperature of at least 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C or 90°C.

One or more cell lysates can be heated for a period of time sufficient to inactivate native enzymes (or other than thermostable enzymes) of the cell. For example, one or more cell lysates can be heated for at least 2, 3, 4, or at least 5 minutes. In accordance with some embodiments, one or more cell lysates are heated for more than 5 minutes. In some embodiments, one or more cell lysates are heated for a period of time sufficient to reduce the activity of natural enzymes (or other non-thermostable enzymes) by at least 50% (e.g., at least 60%, 70 %, 80% or 90%).

After heat inactivation, in accordance with some embodiments, at least one (eg, at least two or at least three) purified enzyme is added to the cell lysate/reaction mixture. Thus, the reaction mixture, in accordance with some embodiments, includes a combination of enzymes present in the cell lysate (expressed by one or more engineered host cells) and at least one purified enzyme. The at least one purified enzyme may be selected from the group consisting of α-glucan phosphorylases or cellodextrin phosphorylases, phosphoglucomutases, glucose-6-phosphate dehydrogenases, 6-phosphogluconolactonases, 6-phosphogluconate dehydrogenases, arabinose-5-phosphate isomerase and arabinose-5-phosphate phosphatases.

In some embodiments, the methods also include incubating one or more heat-inactivated lysates in the presence of starch and inorganic phosphate to produce arabinose. According to some embodiments, the methods also include incubating one or more heat-inactivated lysates in the presence of cellodextrin and inorganic phosphate to produce arabinose. According to some embodiments, the heat-inactivated lysates are incubated at a temperature of at least 50°C. According to some embodiments, the heat-inactivated lysates are incubated for at least 2 minutes (eg, at least 3, 4, or 5 minutes). For example, heat-inactivated lysates can be incubated for 2-5 minutes or 2-10 minutes. The starch may be, for example, amylose, amylopectin or a mixture of amylose and amylopectin. According to some embodiments, biomass is used instead of starch. According to such embodiments, the reaction involves one or more cellodextrin phosphorylases. In accordance with some embodiments, starch or cellodextrin is present as a component of the mixture.

The present invention also relates to cells and cell lysates used to obtain arabinose. Thus, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure may include at least one (e.g., at least two, at least three, at least four, at least five or at least six) an enzyme selected from the group consisting of α-glucan phosphorylases, phosphoglucomutases, glucose-6-phosphate dehydrogenases, 6-phosphogluconolactonases, 6-phosphogluconate dehydrogenases, arabinose-5-phosphate isomerases, and arabinose-5-phosphate phosphatases. The engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure may include at least one (e.g., at least two, at least three, at least four, at least five or at least six) an enzyme selected from the group consisting of cellodextrinphosphorylases, phosphoglucomutases, glucose-6-phosphate dehydrogenases, 6-phosphogluconolactonases, 6-phosphogluconate dehydrogenases, arabinose-5-phosphate isomerases, and arabinose-5-phosphate phosphatases. In accordance with some embodiments, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure comprises at least one (e.g., at least two, at least three, at least four, at least five, or at least six) an enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable glucose-6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases, thermostable 6-phosphogluconate dehydrogenases, thermostable arabinose-5- phosphisomerases and thermostable arabinose-5-phosphate phosphatases. In accordance with some embodiments, the engineered cell (e.g., bacterial cell and/or yeast cell) or one or more cell lysates of the present disclosure comprises at least one (e.g., at least two, at least three, at least four, at least five, or at least six) an enzyme selected from the group consisting of thermostable cellodextrin phosphorylases, thermostable phosphoglucomutases, thermostable glucose-6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases, thermostable 6-phosphogluconate dehydrogenases, thermostable arabinose-5-phosphate isomerases, and thermostable arabinose-5-phosphate phosphatases.

Substrate plasticity and debranching enzymes

In all of the pathways described herein, a variety of polymeric glucose substrates can be used. Non-limiting examples of polymeric glucose substrates include starch, glycogen, and cellodextrin. In accordance with some embodiments, the substrate is a starch. According to other embodiments, the substrate is glycogen. According to still other embodiments, the substrate is cellodextrin. In some embodiments, a partially hydrolysed variant of the polymeric glucose substrate (eg, starch, glycogen, or cellulose/cellodextrin) is used. Starch and glycogen include many glucose monomers linked predominantly by α(1-4) bonds, while cellodextrin includes the same glucose monomers linked by β(1-4) bonds. Another important difference between cellodextrin and the other two substrates is the presence of α(1-6) branches from α(1-4) chains. Both starch and glycogen contain these branch points, although glycogen is substantially more branched than starch. In the case of α(1-4) polymers, α-glucan phosphorylases, also called α-glucan phosphorylases or glycogen phosphorylases, depending on substrate preference, consume the polymers one glucose at a time, releasing glucose-1-phosphate. In the case of cellodextrin, cellodextrinphosphorylase carries out the same reaction, also releasing glucose-1-phosphate.

Long polymers of starch and cellulose/cellodextrin are often insoluble in aqueous solutions and, in addition to precipitating out of solution, can cause gelation and retrogradation of the solution. When starch and cellulose/cellodextrin are partially hydrolyzed to shorter chain length polymers, either by chemical (e.g. acid hydrolysis) or enzymatic (e.g. α-amylase) methods, the resulting products are maltodextrins and cellodextrins in the case of starch and cellulose, respectively. These hydrolyzed derivatives often dissolve and mix better than their parent molecules and therefore, in some embodiments, they are used in the routes presented herein.

In the case of glycogen, starch, or hydrolyzed maltodextrins, α(1-6)-arms will significantly reduce the yield of either sugar pathway, as glucan phosphorylase processes the polymers to the end of their arms, leaving the large central core of available glucose unchanged. For such substrates/pathways, debranching enzymes can be used to increase the availability of the substrate for glucan phosphorylase. There are two typical classes of debranching enzymes that can be used are isoamylases and pullulanases (see, for example, Table 3). Enzymatically, both classes perform the same function, but differ in substrate specificity. Although the use of a debranching enzyme increases yield, the time of application will depend on the process and the substrates used. In some embodiments, α-glucan is pre-treated with α-amylase and a debranching enzyme, and then the resulting one or more straight-chain maltodextrins are fed into a reactor with other pathway enzymes. According to other embodiments, debranching occurs concurrently with the implementation of the pathway and the branched α-glucans are fed into a reaction mixture containing all of the enzymes of the pathway as well as the debranching enzyme.

Table 3. Exemplary debranching enzymes

Enzyme name EC No. natural organism NCBI No. Pullulanase 3.2.1.41 Fervidobacterium pennavorans AAD30387 Thermotoga sp. RQ5 WP_012310857 Bacillus flavocaldarius BAB18516 Thermosipho africanus WP_004100450 Kosmotoga olearia WP_015868997 Isoamylase 3.2.1.68 Sulfolobus tokodaii BAB65940 Metallosphaera hakonensis AAS00512 Sphaerobacter thermophilus WP_012873143 Bacillus lentus AGL34022

Cell-free preparation

"Cell-free preparation" is the use of biological processes to synthesize a biomolecule or chemical compound without the use of living cells. Rather, the cells are lysed and the crude (unprocessed) portions containing the enzymes are used to obtain the desired product. As a non-limiting example, cells are cultured, harvested and lysed by high pressure homogenization. The cell-free reaction can be carried out in batch or fed-batch mode. In some cases, networks of biological reactions fill the working volume of the reactor and may be more dilute than the intracellular environment. However, essentially all cellular catalysts are represented, including membrane-bound catalysts. The inner membrane is fragmented during cell lysis, and fragments of these membranes form functional membrane vesicles. Thus, complex biotransformations occur as a result of catalysis. See, for example, Swartz, AIChE Journal , 2012, 58(1), 5-13, incorporated herein by reference.

Cell-free methods and systems of the present invention, in accordance with some embodiments, use cell lysates (eg, crude or partially purified cell lysates), which are discussed in more detail herein. Cell lysates can be obtained, for example, by mechanical means (eg, grinding or crushing). In accordance with some embodiments, cell lysates are different from chemically permeabilized cells. As discussed herein, in accordance with some embodiments, during cell lysis (eg, mechanical cell lysis), the inner cell membrane is fragmented such that inverted membrane vesicles are formed in cell lysates. Cells that undergo lysis (eg, at least 75%, 80%, 85%, 90%, or 95%) are no longer intact.

According to some embodiments, permeabilized cells are used. Permeabilized cells are intact cells containing perforations (small holes). According to some embodiments, cells can be permeabilized to release the contents of the cell for use in the reaction, as presented herein.

In accordance with some embodiments, partially purified cell fractions are used. A partially purified cell fraction is a cell lysate from which one or more cellular components (eg, cell membranes) have been partially or completely removed.

Thermostable enzymes

An enzyme is considered thermostable if the enzyme (a) retains a significant portion of its activity after exposure to high temperatures, which denature other natural enzymes, or (b) performs its functions at a relatively high rate after exposure to medium or high temperature, after which natural enzymes perform their functions. at low speed.

In accordance with some embodiments, a thermostable enzyme retains more than 50% activity after exposure to a relatively high temperature that would otherwise denature a similar (non-thermostable) natural enzyme. According to some embodiments, a thermostable enzyme retains 50-100% activity after exposure to a relatively high temperature that would otherwise denature a similar (non-thermostable) native enzyme. For example, a thermostable enzyme may retain 50-90%, 50-85%, 50-80%, 50-75%, 50-70%, 50-65%, 50-60%, or 50-55% of its activity after exposure to relatively high temperature, which would otherwise denature a similar (non-thermostable) natural enzyme. According to some embodiments, the thermostable enzyme retains 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90 %, 95%, 98%, 99% or 100% of its activity after exposure to a relatively high temperature, which would otherwise denature a similar (non-thermostable) natural enzyme.

According to some embodiments, the activity of the thermostable enzyme after exposure to a high temperature environment exceeds (e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% , 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% exceeds) the activity of a similar (non-thermostable) natural enzyme.

Thermostable enzymes (eg, phosphatases or phosphorylases) may remain active (able to catalyze the reaction), for example, at a temperature of 45°C to 80°C or higher. According to some embodiments, thermostable enzymes remain active at 45-80°C, 45-70°C, 45-60°C, 45-50°C, 50-80°C, 50-70°C, 50 - 60°C, 60-80°C, 60-70°C or 70-80°C. For example, thermostable enzymes can remain active at 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C. C, 55°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78° C, 79°C or 80°C. Thermostable enzymes can remain active at relatively high temperatures for a period of 15 minutes to 48 hours or longer after exposure to relatively high temperatures. For example, thermostable enzymes can remain active at relatively high temperatures for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 24, 36, 42 or 48 hours.

Engineered cells

The engineered cells of the present invention, in accordance with some embodiments, have at least one or all of the enzymatic activities necessary to convert starch and/or cellulose/cellodextrin to sugar. "Engineered cells" are cells that contain at least one engineered (eg, recombinant or synthetic) nucleic acid or are otherwise modified to be structurally and/or functionally different from their naturally occurring counterparts. Thus, a cell that contains an engineered nucleic acid is considered an "engineered cell".

The engineered cells of the present invention, in accordance with some embodiments, contain α-glucan phosphorylase (e.g., thermostable α-glucan phosphorylase) and/or cellodextrin phosphorylase (e.g., thermostable cellodextrin phosphorylase), phosphoglucomutase (e.g., thermostable phosphoglucomutase), and at least one enzyme ( eg, thermostable enzyme) selected from the group consisting of isomerases, epimerases, dehydrogenases, and sugar phosphatases.

The engineered cells, in accordance with some embodiments, express selectable markers. Selectable markers are typically used to select engineered cells that take up and express the engineered nucleic acid after transfection of the cell (or other procedure used to introduce a foreign nucleic acid into a cell). Thus, a nucleic acid that encodes a product may also encode a selectable marker. Examples of selectable markers include, without limitation, genes encoding proteins that increase or decrease antibiotic resistance or sensitivity (e.g., ampicillin resistance genes, kanamycin resistance genes, neomycin resistance genes, tetracycline resistance genes, and chloramphenicol resistance genes) or others. connections. Other selectable markers may be used in accordance with the present disclosure.

An engineered cell "expresses" a product if the product encoded by the nucleic acid (eg, the engineered nucleic acid) is produced in the cell. It is known in the art that gene expression refers to the process by which genetic instructions in the form of a nucleic acid are used to synthesize a product such as a protein (eg, an enzyme).

The engineered cells may be prokaryotic cells or eukaryotic cells. In some embodiments, the engineered cells are bacterial cells, yeast cells, insect cells, mammalian cells, or other cell types.

Engineered bacterial cells useful in the context of the present disclosure include, without limitation, engineered Escherichia spp., Streptomyces spp., Zymonas spp., Acetobacter spp., Citrobacter spp., Synechocystis spp., Rhizobium spp., Clostridium spp., Corynebacterium spp. , Streptococcus spp., Xanthomonas spp., Lactobacillus spp., Lactococcus spp., Bacillus spp., Alcaligenes spp., Pseudomonas spp., Aeromonas spp., Azotobacter spp., Comamonas spp., Mycobacterium spp., Rhodococcus spp., Gluconobacter spp., Ralstonia spp., Acidithiobacillus spp., Microlunatus spp., Geobacter spp., Geobacillus spp., Arthrobacter spp., Flavobacterium spp., Serratia spp., Saccharopolyspora spp., Thermus spp., Stenotrophomonas spp., Chromobacterium spp. , Sinorhizobium spp., Saccharopolyspora spp., Agrobacterium spp., Vibrio spp. and Pantoea spp.

Engineered yeast cells useful in the context of the present disclosure include, without limitation, engineered Saccharomyces spp., Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia, and Pichia.

In some embodiments, engineered cells useful in the context of the present disclosure are Escherichia coli , Bacillus subtilis , Pseudomonas putida , Saccharomyces cerevisiae , and/or Lactobacillus brevis engineered cells. In some embodiments, engineered cells useful in the context of the present disclosure are Escherichia coli engineered cells.

Engineered nucleic acids

A "nucleic acid" is at least two nucleotides covalently linked together, and in some cases may contain phosphodiester linkages (eg, a phosphodiester "backbone"). Nucleic acids (eg, components or parts of nucleic acids) may be naturally occurring or engineered. "Naturally occurring" nucleic acids are present in a cell that exists in nature without human intervention. "Designed nucleic acids" include recombinant nucleic acids and synthetic nucleic acids. The term "recombinant nucleic acid" means a molecule that is assembled by combining nucleic acid molecules (for example, from the same species or from different species) and, as a rule, can be replicated in a living cell. The term "synthetic nucleic acid" means a molecule that is synthesized by biological methods, synthesized by chemical methods, or synthesized or amplified by other methods. Synthetic nucleic acids include nucleic acids that have been modified by chemical means or modified by other means, but can form base pairs with naturally occurring nucleic acid molecules. Recombinant and synthetic nucleic acids also include those molecules that result from the replication of any of the above. The engineered nucleic acids may contain parts of the nucleic acids that occur naturally, but as a whole, the engineered nucleic acids do not occur naturally and require human intervention. According to some embodiments, the nucleic acid encoding a product of the present disclosure is a recombinant nucleic acid or a synthetic nucleic acid. According to other embodiments, the nucleic acid encoding the product is naturally occurring.

The engineered nucleic acid encoding the enzymes provided herein can be operably linked to a "promoter", which is a regulatory region of the nucleic acid that regulates the initiation and rate of transcription of the remainder of the nucleic acid. A promoter directs the expression or directs the transcription of the nucleic acid it regulates.

A promoter may be a promoter that is naturally associated with a gene or sequence, which can be obtained by isolating 5' non-coding sequences upstream of the coding segment of that gene or sequence. Such a promoter can be called "endogenous".

According to some embodiments, the nucleic acid coding sequence may be located under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the encoded sequence in its natural environment. Such promoters may include promoters of other genes; promoters isolated from any other cell; and synthetic promoters or enhancers that are "not naturally occurring" such as, for example, promoters that contain different elements of different transcriptional regulatory regions and/or mutations that alter expression introduced by genetic engineering methods that are known in the art . In addition to obtaining nucleic acid sequences from promoters and enhancers by synthetic methods, sequences can be obtained using recombinant cloning and/or nucleic acid amplification techniques, including polymerase chain reaction (PCR).

A promoter is considered "operably linked" when it is in the correct functional location and orientation with respect to the nucleic acid that it regulates to control ("direct") the initiation of transcription and/or the expression of such nucleic acid.

The engineered nucleic acids of the present disclosure may contain a constitutive promoter or an inducible promoter. The term "constitutive promoter" means a promoter that is permanently active in a cell. The term "inducible promoter" means a promoter that initiates or enhances transcriptional activity in the presence, exposure to, or contact with an inducer or inducing agent, or is activated in the absence of a repressive factor. Inducible promoters for use in accordance with the present disclosure include any inducible promoter described herein or known to those skilled in the art. Examples of inducible promoters include, without limitation, chemically/biochemically regulated and physically regulated promoters such as alcohol regulated promoters, tetracycline regulated promoters, steroid regulated promoters, metal regulated promoters, pathogenesis regulated, temperature/heat regulated, phosphate regulated promoters (e.g., PhoA), and light-regulated promoters.

The inducer or inducing agent may be endogenous or normally an exogenous state (eg, light), be a compound (eg, a chemical or non-chemical compound), or a protein that binds to an inducible promoter in such a way as to be active in regulating transcriptional activity. from the inducible promoter. Thus, a "signal that regulates transcription" of a nucleic acid means an inducer signal that acts on an inducible promoter. The signal that regulates transcription can activate or inactivate transcription, depending on the regulatory system used. Transcriptional activation may involve direct action on a promoter to direct transcription, or indirect action on a promoter by inactivating a repressor that prevents the promoter from directing transcription. Conversely, transcriptional deactivation may involve direct action on the promoter to prevent transcription, or indirect action on the promoter by activating a repressor which then acts on the promoter.

The engineered nucleic acids can be introduced into host cells by any means known in the art, including, without limitation, transformation, transfection (e.g., chemical (e.g., with calcium phosphate, cationic polymers, or liposomes), or non-chemical (e.g., electroporation, sonoporation). , impalefection, optical transfection, hydrodynamics) and transduction (eg, viral transduction).

Enzymes or other proteins encoded by natural intracellular nucleic acid may be referred to as "endogenous enzymes" or "endogenous proteins".

Protease targeting

The engineered cells of the present invention may express (eg, endogenously express) enzymes necessary for cell health, which may adversely affect the production of a sugar of interest (eg, allulose). Such enzymes are referred to herein as "target enzymes". For example, target enzymes expressed by engineered cells may compete for substrates or cofactors with an enzyme that increases the proportion of precursor supplied to the sugar pathway. As another example, target enzymes expressed by engineered cells may compete for substrates or cofactors with an enzyme that is a key enzyme to enter the sugar pathway. As another example, target enzymes expressed by engineered cells may compete for substrates or cofactors with an enzyme that supplies a substrate or cofactor in the sugar pathway.

To eliminate or reduce this negative effect, target enzymes can be modified to include a site-specific protease recognition sequence in their protein sequence so that the target enzyme can become a "target" and can be cleaved for inactivation during sugar production (see, for example, U.S. Patent Publication No. 2012/0052547 A1, published March 1, 2012, and International Publication No. WO 2015/021058 A2, published February 12, 2015, each of which is incorporated herein by reference).

Cleavage of a target enzyme containing a site-specific protease recognition sequence occurs as a result of contact with a cognate site-specific protease that is isolated in the periplasmic space of the cell (separate from the target enzyme) during the cell growth phase (e.g., as engineered cells are cultured ) and is brought into contact with the target enzyme during the conversion phase (eg, after cell lysis to obtain a cell lysate). Thus, the engineered cells of the present disclosure comprise, in accordance with some embodiments, (i) an engineered nucleic acid encoding a target enzyme that adversely affects the rate of conversion and includes a site-specific protease recognition sequence in the protein sequence of the target enzyme, and ( ii) an engineered nucleic acid encoding a site-specific protease that cleaves the site-specific protease recognition sequence of the target enzyme and includes a sequence directing it to the periplasmic space. This periplasmic space targeting sequence is responsible for isolating the site-specific protease in the periplasmic space of the cell until the cell is lysed. Examples of sequences directing to the periplasmic space are presented below.

Examples of proteases that can be used in accordance with the present disclosure include, but are not limited to, alanine carboxypeptidase, astacin, bacterial leucylaminopeptidase, cancer procoagulant, cathepsin B, clostripain, cytosolanylaminopeptidase, elastase, Brg-C endoproteinase, enterokinase, gastrixin, gelatinase, Gly-X carboxypeptidase , glycyl endopeptidase, human rhinovirus 3C protease, hypodermin C, Iga-specific serine endopeptidase, leucyl aminopeptidase, leucyl endopeptidase, lysC, lysosomal carboxypeptidase pro-X, lysyl aminopeptidase, methionyl aminopeptidase, myxobacter, nardylysin, pancreatic endopeptidase E, picorna peptidase 2, picorna peptidase 3 , prolylaminopeptidase, proprotein convertase I, proprotein convertase II, ruscellisin, saccharopepsin, semenogelase, T-plasminogen activator, thrombin, tissue kallikrein, tobacco etch virus (TEV) protease, togavirin, tryptophanylaminopeptidase, plasminogen activator U, V8, venombin B , venombin BB and aminopeptidase Xaa-pro.

Direction to the periplasmic space

The sugar pathway enzymes may include at least one enzyme that adversely affects the health (eg, viability) of the cell. To eliminate or reduce this negative effect, the enzyme can be modified to include a relocation sequence such that the enzyme is relocated to a cellular or extracellular compartment where it is not normally found and where the enzyme does not adversely affect cell health (see, e.g., , Publication No. US-2011-0275116-A1, published November 10, 2011, incorporated herein by reference). For example, a sugar pathway enzyme can be relocated to the periplasmic space of the cell.

Thus, in some embodiments, engineered cells of the present invention comprise at least one sugar pathway enzyme that is linked to a periplasmic space targeting sequence. A "periplasmic space targeting sequence" is an amino acid sequence that directs a protein to which it is associated to the periplasmic space of a cell. A protein that is linked to a periplasmic space targeting sequence will be sequestered in the periplasmic space of the cell in which the protein is expressed.

Periplasmic space targeting sequences can be obtained, for example, from the N-terminus of a bacterial secretory protein. The sequences vary in length from about 15 to about 70 amino acids. The primary amino acid sequences of the periplasmic space targeting sequences vary, but generally have a common structure including the following components: (i) the N-terminal portion is of variable length and usually carries a net positive charge; (ii) followed by a central hydrophobic core about 6 to about 15 amino acids in length; and (iii) the final component comprises four to six amino acids that define the cleavage site for signal peptidases.

The periplasmic space targeting sequences of the present invention, in some embodiments, can be derived from a protein that is secreted from a Gram-negative bacterium. The secreted protein may be encoded by a bacterium or by a bacteriophage that infects the bacterium. Examples of Gram-negative bacterial sources of secreted proteins include, but are not limited to, members of the genera Escherichia, Pseudomonas, Klebsiella, Salmonella, Caulobacter, Methylomonas, Acetobacter, Achromobacter, Acinetobacter, Aeromonas, Agrobacterium, Alcaligenes, Azotobacter, Burkholderia, Citrobacter, Comamonas, Enterobacter, Erwinia, Rhizobium, Vibrio and Xanthomonas.

Examples of periplasmic targeting sequences for use in accordance with the present disclosure include, without limitation, sequences selected from the group consisting of:

MKIKTGARILALSALTTMMFSASALA (SEQ ID NO: 1);

MKQSTIALALLPLLFTPVTKA (SEQ ID NO: 2);

MMITLRKLPLAVAVAAGVMSAQAMA (SEQ ID NO: 3);

MNKKVLTLSAVMASMLFGAAAHA (SEQ ID NO: 4);

MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 5);

MKKIWLALAGLVLAFSASA (SEQ ID NO: 6);

MMTKIKLLMLIIFYLIISASAHA (SEQ ID NO: 7);

MKQALRVAFGFLILWASVLHA (SEQ ID NO: 8);

MRVLLFLLLSLFMLPAFS (SEQ ID NO: 9); and

MANNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA (SEQ ID NO: 10).

Cell cultures and cell lysates

As a rule, engineered cells are cultured. "Cultivation" refers to the method by which cells are grown under controlled conditions, usually outside their natural environment. For example, engineered cells, such as engineered bacterial cells, can be grown as a cell suspension in a liquid nutrient broth, also referred to as a liquid "culture medium".

In some embodiments, unmodified starch is used as the cell growth substrate.

Examples of commonly used media for growing Escherichia coli bacteria include, but are not limited to, LB (Luria-Bertani) Miller Broth (1% NaCl): 1% peptone, 0.5% yeast extract and 1% NaCl; Lennox LB Broth (Luria-Bertani) (0.5% NaCl): 1% peptone, 0.5% yeast extract and 0.5% NaCl; SOB medium (Super Optimal Broth): 2% peptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4; SOC medium (Super Optimal Broth with catabolic repressor): SOB + 20 mM glucose; 2x YT broth (2x yeast extract and tryptone): 1.6% peptone, 1% yeast extract and 0.5% NaCl; TB medium (Terrific Broth): 1.2% peptone, 2.4% yeast extract, 72 mM K2HPO4, 17 mM KH2PO4 and 0.4% glycerol; and SB medium (Super Broth): 3.2% peptone, 2% yeast extract and 0.5% NaCl and/or Korz's medium (Korz, DJ et al. 1995).

Examples of high density Escherichia coli growth media include, but are not limited to, DNAGro™ medium, ProGro™ medium, AutoX™ medium, DetoX™ medium, InduX™ medium, and SecPro™ medium.

In accordance with some embodiments, the engineered cells are cultured under conditions that result in the expression of enzymes or nucleic acids. Such culture conditions may depend on the particular product being expressed and the amount of product required.

According to some embodiments, engineered cells are cultured at 3°C to 40°C. For example, engineered cells can be cultured at 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C. C. Typically engineered cells, such as engineered bacterial cells, are cultured at 37°C.

According to some embodiments, the engineered cells are cultured for a period of time from 12 hours to 72 hours or more. For example, the engineered cells can be cultured for a period of 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, or 72 hours. Typically, engineered cells, such as engineered bacterial cells, are cultured for a period of 12 to 24 hours. According to some embodiments, engineered cells are cultured for 12-24 hours at 37°C.

According to some embodiments, engineered cells are cultured (eg, in liquid cell culture medium) to an optical density measured at 600 nm (OD600) of 5-25. In some embodiments, the engineered cells are cultured to an OD600 of 5, 10, 15, 20, or 25.

According to some embodiments, the engineered cells are cultured to a density of 1×10 ⁴ to 1×10 ⁸ viable cells/mL of cell culture medium. According to some embodiments, the engineered cells are cultured to a density of 1×10 ⁴ , 2×10 ⁴ , 3×10 ⁴ , 4×10 ⁴ , 5×10 ⁴ , 6×10 ⁴ , 7×10 ⁴ , 8×10 ⁴ , 9×10 ⁴ , 1×10 ⁵ , 2×10 ⁵ , 3×10 ⁵ , 4×10 ⁵ , 5×10 ⁵ , 6×10 ⁵ , 7×10 ⁵ , 8×10 ⁵ , 9×10 ⁵ , 1×10 ⁶ , 2×10 ⁶ , 3×10 ⁶ , 4×10 ⁶ , 5×10 ⁶ , 6×10 ⁶ , 7×10 ⁶ , 8×10 ⁶ , 9×10 ⁶ , 1×10 ⁷ , 2×10 ⁷ , 3×10 ⁷ , 4×10 ⁷ , 5×10 ⁷ , 6×10 ⁷ , 7×10 ⁷ , 8×10 ⁷ , 9×10 ⁷ , 1×10 ⁸ , 1×10 ⁹ or 1×10 ¹⁰ viable cells/ml. In some embodiments, the engineered cells are cultured to a density of 1×10 ⁸ to 1×10 ¹⁰ viable cells/mL. In some embodiments, the engineered cells are cultured to a density of 2×10 ⁵ to 3×10 ⁷ viable cells/mL.

According to some embodiments, engineered cells are cultured in a bioreactor. A bioreactor is generally understood to mean a container in which cells are cultured, such as a culture flask, cup, or bag, which can be disposable (disposable), autoclavable, or sterilizable. The bioreactor may be made of glass, or it may be polymer, or it may be made of other materials.

Examples of bioreactors include, but are not limited to, mechanically agitated (e.g., vigorously agitated) and tubular (e.g., plug flow) bioreactors, bubbling bioreactors, agitated membrane tanks, centrifugal agitated tanks, vibratory mixers, fluidized bed reactors, and membrane bioreactors. The mode of operation of the bioreactor may be a batch or continuous process and will depend on the cultured engineered cells. The bioreactor is a continuous reactor, when the streams of raw materials and products are continuously supplied to and removed from the system. A batch bioreactor may have a continuous recycle stream but no continuous nutrient supply or continuous product collection. In the case of fed-batch cultures (or batch cultures), cells are inoculated at a lower density of viable cells in a medium that is similar in composition to batch medium. The cells are allowed to grow exponentially without virtually any external manipulation until nutrient depletion and the cells reach the stationary phase of growth. At this point, in the case of a batch feeding and collection process, a portion of the cells and product can be harvested and the culture medium removed can be replenished with fresh medium. This process can be repeated several times. To obtain recombinant proteins and antibodies, you can use the method with portioned feeding. As cells grow exponentially and nutrients are depleted, a concentrated nutrient medium (e.g., 10-15 times concentrated basal medium) is added either continuously or intermittently to provide additional nutrients, which allows further increase in cell concentration and the duration of the transition phase. Fresh medium can be added in proportion to the cell concentration without removing the culture medium (broth). In order to accommodate the addition of medium, inoculate the fed culture at a volume much less than the full capacity of the bioreactor (eg, about 40% - 50% of the maximum volume).

Some of the processes of the present disclosure relate to large scale sugar production. In the case of large-scale production methods, the engineered cells can be grown in liquid culture medium in a volume of 5 liters (l) to 50 liters or more. In some embodiments, engineered cells can be grown in liquid culture medium in a volume greater than (or equal to) 10 L. In some embodiments, the engineered cells are grown in liquid culture medium in a volume of 5 L, 10 L, 15 L, 20 L, 25 L, 30 L, 35 L, 40 L, 45 L, 50 L or more. In some embodiments, engineered cells can be grown in liquid culture medium in volumes of 5 L to 10 L, 5 L to 15 L, 5 L to 20 L, 5 L to 25 L, 5 L to 30 l, 5 l to 35 l, 5 l to 40 l, 5 l to 45 l, 10 l to 15 l, 10 l to 20 l, 10 l to 25 l, 20 l to 30 l , from 10 l to 35 l, from 10 l to 40 l, from 10 l to 45 l, from 10 l to 50 l, from 15 l to 20 l, from 15 l to 25 l, from 15 l to 30 l, 15 l to 35 l, 15 l to 40 l, 15 l to 45 l, or 15 to 50 l.

Typically, culturing engineered cells is followed by cell lysis. "Lysis" refers to the process by which cells are destroyed, for example, by viral, enzymatic, mechanical, chemical, thermal or osmotic mechanisms. "Cell lysate" means the fluid contained in the contents of lysed cells (eg, lysed engineered cells), including, for example, organelles, membrane lipids, proteins, nucleic acids, and inverted membrane vesicles. Cell lysates of the present disclosure can be obtained by lysing any of the engineered cell populations provided herein. "Cell lysate" may exclude permeabilized/perforated cells.

Methods for cell lysis, referred to as "lysis", are known in the art, any of which can be used in accordance with the present disclosure. Such cell lysis methods include, without limitation, physical/mechanical lysis, such as homogenization, as well as chemical, thermal, and/or enzymatic lysis.

Cell lysis can disrupt carefully controlled cellular environments, resulting in protein degradation and modification by unregulated endogenous proteases and phosphatases. Therefore, in some embodiments, protease inhibitors and/or phosphatase inhibitors can be added to the cell lysate or pre-lysis cells, or these active molecules can be eliminated by gene inactivation or protease targeting.

Cell lysates, in accordance with some embodiments, can be combined with at least one nutrient. For example, cell lysates can be combined with Na ₂ HPO ₄ , KH ₂ PO ₄ , NH ₄ Cl, NaCl, MgSO ₄ , CaCl ₂ . Examples of other nutrients include, without limitation, magnesium sulfate, magnesium chloride, magnesium orotate, magnesium citrate, potassium phosphate monobasic, potassium phosphate dibasic, potassium phosphate tribasic, sodium phosphate monobasic, sodium phosphate dibasic, sodium phosphate tribasic, ammonium phosphate monobasic, ammonium phosphate dibasic , ammonium sulfate, ammonium chloride, ammonium hydroxide.

In some embodiments, cell lysates may consist of disrupted cell suspensions that are further modified by chemical, thermal, enzymatic, or mechanical means to increase concentration, or purify, or decrease concentration, or eliminate specific components. For example, after being broken down by a mechanical, thermal, chemical, or enzymatic process as described above, the resulting material can be subjected to mechanical separation, such as membrane filtration, centrifugation, or otherwise, to partially increase the content of a selected molecule with enzymatic activity, or to eliminate an unwanted molecule. with enzymatic activity or a lysate component. Additional examples may include adding salts or solvents to the disrupted cell suspension or changing the pH or temperature of the disrupted cell suspension resulting in the precipitation of the desired active molecules followed by mechanical separation of these precipitated components as described above. Conversely, the addition of salts or solvents, or a change in pH or temperature, can be used to eliminate undesired active molecules by either inactivating those enzymes or by precipitation and subsequent mechanical separation of molecules with undesired enzymatic activity or undesired enzymatic activities.

Cell lysates, in accordance with some embodiments, can be combined with at least one cofactor. For example, cell lysates can be combined with adenosine diphosphate (ADP), adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NAD+), or other non-protein chemicals required for enzyme activity (eg, inorganic ions and coenzymes).

In some embodiments, the cell lysates are incubated under conditions that convert starch or cellulose/cellodextrin to sugar.

The volume of cell lysate used for one reaction may vary. In accordance with some variants of implementation, the volume of the cell lysate is 1-150 m ³ . For example, the volume of a cell lysate can be 1 m ³ , 5 m ³ , 10 m ³ , 15 m ³ , 20 m ³ , 25 m ³ , 30 m ³ , 35 m ³ , 40 m ³ , 45 m ³ , 50 m ³ , 55 m ³ , 60 m ³ , 65 m ³ , 70 m ³ , 75 m ³ , 80 m ³ , 85 m ³ , 90 m ³ , 95 m ³ , 100 m ³ , 105 m ³ , 110 m ³ , 115 m ³ , 120 m ³ , 125 m ³ , 130 m ³ , 135 m ³ , 140 m ³ , 145 m ³ or 150 m ³ . According to some embodiments, the cell lysate volume is 25 m ³ to 150 m ³ , 50 m ³ to 150 m ³ , or 100 m ³ to 150 m ³ .

Purified Enzymes

In accordance with some embodiments, the enzymes of the present invention may be purified before being added to a commercial reaction. Enzyme purification is to be understood as any increase in concentration or any extraction of a particular enzyme or molecule with enzymatic activity, or groups of enzymes or molecules with enzymatic activity, from a complex mixture of materials, for example, including, but not limited to, cell suspensions of broken cells or culture media left over from culture. Thus, a purified enzyme or protein should be understood to mean an enzyme or protein that has been isolated or increased in concentration from a complex matrix, thereby increasing its relative concentration compared to other components of the matrix. Enzyme purification methods include, without limitation, mechanical, chromatographic, chemical, pH dependent, or temperature methods. For example, adding salt to a disrupted cell suspension results in precipitation of the target enzyme or protein, followed by mechanical separation of the precipitated enzyme or protein, such as by membrane filtration or centrifugation. Additional examples may include separating the enzyme from the complex matrix using affinity chromatography techniques (eg, hexahistidine or streptavidin purification).

Enzymatic specificity

Enzymatic specificity is to be understood as a trait inherent in an enzyme, where it exhibits improved enzymatic kinetics, thermodynamics, or reaction rate with respect to one substrate as compared to another substrate. High specificity enzymes are best illustrated as having a high ratio of catalytic constant (defined as turnover number or Kcat) to Michaelis constant (Km) or Kcat/Km. It is advantageous to have an enzyme with high substrate specificity as this increases the rate of the reaction and improves the yield by reducing the production of non-target products. For example, in the allulose production route described herein, there are several intermediates that are similar in chemical structure, namely glucose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate, and allulose-6-phosphate. The final enzymatic step in this method is the dephosphorylation of allulose-6-phosphate to the final allulose with the participation of allulose-6-phosphate phosphatase. It is advantageous to use an enzyme with very high specificity for allulose-6-phosphate and relatively low specificity for other intermediates in the pathway, namely glucose-1-phosphate, glucose-6-phosphate and fructose-6-phosphate. Catalytic dephosphorylation of these intermediates will result in either glucose or fructose resulting in reduced yield and increased product complexity.

Additional Embodiments

1. A cell-free method for producing allulose, the method comprising:

(a) culturing cells engineered to express thermostable α-glucan phosphorylase (also called starch phosphorylase), thermostable phosphoglucomutase, thermostable phosphoglucoisomerase, thermostable allulose-6-phosphate epimerase, and thermostable allulose-6-phosphate phosphatase to obtain cultured cells that express thermostable enzymes;

(b) lysing the cultured cells to obtain a cell lysate;

(c) heating the cell lysate to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(d) incubating the heat inactivated lysate in the presence of starch and inorganic phosphate to produce allulose.

2. A cell-free method for producing allulose, the method comprising:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable phosphoglucoisomerases, thermostable allulose-6-phosphate epimerases, and thermostable alluloses -6-phosphate phosphatase, with obtaining at least two cultured populations of cells expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to obtain a mixture of cell lysates that contains thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, thermostable phosphoglucoisomerase, thermostable allulose-6-phosphate epimerase and thermostable allulose-6-phosphate phosphatase;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(e) incubating the reaction mixture in the presence of starch and inorganic phosphate to obtain allulose.

3. A cell-free method for producing allulose, the method comprising:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable phosphoglucoisomerases, thermostable allulose-6-phosphate epimerases, and thermostable alluloses -6-phosphate phosphatase, with obtaining at least two cultured populations of cells expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to obtain a mixture of cell lysates;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate;

(e) adding to the heat-inactivated lysate at least one purified enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable phosphoglucoisomerases, thermostable allulose-6-phosphate epimerases, and thermostable allulose-6-phosphate phosphatases to form a reaction mixture containing α-glucan phosphorylase, phosphoglucomutase, phosphoglucoisomerase, allulose-6-phosphate epimerase and allulose-6-phosphate phosphatase; and

(f) incubating the reaction mixture in the presence of starch and inorganic phosphate to obtain allulose.

4. A cell-free method according to any one of embodiments 1-3, wherein one or more thermostable α-glucan phosphorylases are selected from the group consisting of α-glucan phosphorylases Aquifex aeolicus, Thermocrinis minervae, Thermosulfidibacter takaii, Thermosulfurimonas dismutans, Thermococcus litoralis, Palaeococcus pacificus, Thermotoga neapolitana, Ruminiclostridium thermocellum, Pyrococcus abyssi, Thermococcus thioreducens, Deinococcus radiodurans, Sulfolobus acidocaldarius, Thermus caldophilus, Meiothermus silvanus, Oceanithermus profundus, Ardenticatena maritima, Thermococcus barophilus, Pseudothermotoga thermarum, Hydrogenobacter thermophilus, Therniothermusi rubietophili .

5. Cell-free method according to any one of embodiments 1-4, wherein one or more thermostable phosphoglucomutases are selected from the group consisting of phosphoglucomutases Thermococcus kodakaraensis, Pyrococcus kukulkanii, Ammonifex degensii, Methanothermobacter wolfeii, Methanothermus fervidus, Sulfolobus acidocaldarius, Archaeoglobus fulgidus, Ferroglobus placidus , Geoglobus ahangari, Archaeoglobus veneficus, Archaeoglobus sulfaticallidus, Aciduliprofundum boonie, Clostridium thermocellum, Defluviitalea phaphyphila, Caminicella sporogenes, Caloranaerobacter ferrireducens, Thermosipho malanesiensis, Fervidobacterium pennivorans, Symbiobacterium thermophilum, Spirochaeta thermophila and Thermoanagelobacter w .

6. Cell-free method according to any one of embodiments 1-5, wherein one or more thermostable phosphoglucomutases are selected from the group consisting of Thermus thermophilus, Meiothermus timidus, Thermus filiformis, Marinithermus hydrothermalis, Thermosipho africanus, Sulfurihydrogenibium azorense, Persephonella marina, Marinitoga pezophila phosphoglucomutases , Kosmotoga olearia, Thermotoga maritima, Geobacillus stearothermophilus, Anoxybacillus flavithermus, Thermosulfidibacter takaii, Fervidobacterium nodosum, Clostridium thermocellum, Thermoanaerobacterium thermosaccharolyticum, Methanococcus jannaschii, Methanotorris igneus, Methanocaldococcus villosus, Methanothermococcus okinawensis, Pseudothermotoga thermarum, Deferribacter desulfuricans и Thermovibrio ammonificans .

7. A cell-free method according to any one of embodiments 1-6, wherein one or more thermostable allulose-6-phosphate epimerases are selected from the group consisting of allulose-6-phosphate epimerases from Thermobacterium thermosaccharolyticum, Thermoanaerobacter brockii, Caldanaerobacter subterraneus, Deferribacter desulfuricans, Thermocrinis ruber, Hydrogenivirga sp. 128-5-R1-1, Brevibacillus thermoruber, Thermosipho atlanticus and Thermosulfidibacter takaii .

8. Cell-free method according to any one of embodiments 1-7, wherein one or more thermostable allulose-6-phosphate phosphatases are selected from the group consisting of allulose-6-phosphate phosphatases Thermoanaerobacter wiegelii , Thermoanaerobacter ethanolicus, Thermus islandicus, Deinococcus geothermalis DSM 11300, Thermosphaera aggregans, Crenarchaeota archaeon, Pyrococcus horikoshii Ot3, Aquifex aeolicus, Ruminiclostridium thermocellum, Desulfotomaculum kuznetsovii, Caldanaerobacter subterraneus, Acidothermus cellulolyticus, Methanothermobacter thermautotrophicus, Thermobifida fusca, Thermotoga neapolitana, Petrotoga mobilus, and Ther- modesulfat

9. A cell-free method for producing glucose, the method comprising:

(a) culturing cells engineered to express thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, and thermostable glucose-6-phosphate phosphatase to produce cultured cells that express thermostable enzymes;

(b) lysing the cultured cells to obtain a cell lysate;

(c) heating the cell lysate to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(d) incubating the heat-inactivated lysate in the presence of starch and inorganic phosphate to produce glucose.

10. A cell-free method for producing glucose, the method comprising:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, and thermostable glucose-6-phosphate phosphatase, to obtain at least two cultured cell populations expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to obtain a mixture of cell lysates that contains thermostable α-glucan phosphorylase, thermostable phosphoglucomutase and thermostable glucose-6-phosphate phosphatase;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(e) incubating the reaction mixture in the presence of starch and inorganic phosphate to obtain glucose.

11. A cell-free method for producing glucose, the method comprising:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, and thermostable glucose-6-phosphate phosphatase, to obtain at least two cultured cell populations expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to obtain a mixture of cell lysates;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate;

(e) adding to the heat-inactivated lysate at least one purified enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, and thermostable glucose-6-phosphate phosphatases to obtain a reaction mixture containing α-glucan phosphorylase, phosphoglucomutase and glucose- 6-phosphate phosphatase; and

(f) incubating the reaction mixture in the presence of starch and inorganic phosphate to obtain glucose.

12. Cell-free method for obtaining fructose, and the method includes:

(a) culturing cells engineered to express thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, thermostable phosphoglucoisomerase, and thermostable fructose-6-phosphate phosphatase to obtain cultured cells that express thermostable enzymes;

(b) lysing the cultured cells to obtain a cell lysate;

(c) heating the cell lysate to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(d) incubating the heat-inactivated lysate in the presence of starch and inorganic phosphate to produce fructose.

13. A cell-free method for obtaining fructose, and the method includes:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable phosphoglucoisomers, and thermostable fructose-6-phosphate phosphatases, to obtain at least two cultured cell populations expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to obtain a mixture of cell lysates that contains a thermostable α-glucan phosphorylase, a thermostable phosphoglucomutase, a thermostable phosphoglucoisomerase, and a thermostable fructose-6-phosphate phosphatase;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(e) incubating the reaction mixture in the presence of starch and inorganic phosphate to obtain fructose.

14. A cell-free method for obtaining fructose, and the method includes:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable phosphoglucoisomers, and thermostable fructose-6-phosphate phosphatases, to obtain at least two cultured cell populations expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to obtain a mixture of cell lysates;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate;

(e) adding to the heat-inactivated lysate at least one purified enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable phosphoglucoisomerases, and thermostable fructose-6-phosphate phosphatases to form a reaction mixture containing α-glucan phosphorylase, phosphoglucomutase , phosphoglucoisomerase and fructose-6-phosphate phosphatase; and

(f) incubating the reaction mixture in the presence of starch and inorganic phosphate to obtain fructose.

15. Cell-free method for obtaining sorbitol, and the method includes:

(a) culturing cells engineered to express thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, thermostable aldose dehydrogenase, and thermostable sorbitol-6-phosphate phosphatase to obtain cultured cells that express thermostable enzymes;

(b) lysing the cultured cells to obtain a cell lysate;

(c) heating the cell lysate to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(d) incubating the heat-inactivated lysate in the presence of starch and inorganic phosphate to obtain sorbitol.

16. Cell-free method for obtaining sorbitol, and the method includes:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable aldose dehydrogenases, and thermostable sorbitol-6-phosphate phosphatases, to obtain at least two cultured cell populations expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to obtain a mixture of cell lysates that contains thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, thermostable aldose dehydrogenase and thermostable sorbitol-6-phosphate phosphatase;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(e) incubating the reaction mixture in the presence of starch and inorganic phosphate to obtain sorbitol.

17. Cell-free method for obtaining sorbitol, and the method includes:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable aldose dehydrogenases, and thermostable sorbitol-6-phosphate phosphatases, to obtain at least two cultured cell populations expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to obtain a mixture of cell lysates;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate;

(e) adding to the heat-inactivated lysate at least one purified enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable aldose dehydrogenases, and thermostable sorbitol-6-phosphate phosphatases to form a reaction mixture containing α-glucan phosphorylase, phosphoglucomutase , aldose dehydrogenase and sorbitol-6-phosphate phosphatase; and

(f) incubating the reaction mixture in the presence of starch and inorganic phosphate to obtain sorbitol.

18. A cell-free method for producing ribulose, the method comprising:

(a) culturing cells engineered to express thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, thermostable glucose-6-phosphate dehydrogenase, thermostable 6-phosphogluconolactonase, thermostable 6-phosphogluconate dehydrogenase, and thermostable ribulose-5-phosphate phosphatase to obtain cultured cells that express thermostable enzymes ;

(b) lysing the cultured cells to obtain a cell lysate;

(c) heating the cell lysate to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(d) incubating the heat-inactivated lysate in the presence of starch and inorganic phosphate to produce ribulose.

19. A cell-free method for producing ribulose, the method comprising:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable glucose-6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases, thermostable 6-phosphogluconate dehydrogenases and thermostable ribulose-5-phosphate phosphatases, producing at least two cultured cell populations expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to obtain a mixture of cell lysates that contains thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, thermostable glucose-6-phosphate dehydrogenase, thermostable 6-phosphogluconolactonase, thermostable 6-phosphogluconate dehydrogenase and thermostable ribulose-5-phosphate phosphatase;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(e) incubating the reaction mixture in the presence of starch and inorganic phosphate to obtain ribulose.

20. A cell-free method for producing ribulose, the method comprising:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable glucose-6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases, thermostable 6-phosphogluconate dehydrogenases and thermostable ribulose-5-phosphate phosphatases, producing at least two cultured cell populations expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to obtain a mixture of cell lysates;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate;

(e) adding to the heat-inactivated lysate at least one purified enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable glucose-6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases, thermostable 6-phosphogluconate dehydrogenases, and thermostable ribulose-5 -phosphate phosphatases, to obtain a reaction mixture containing α-glucan phosphorylase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase and ribulose-5-phosphate phosphatase; and

(f) incubating the reaction mixture in the presence of starch and inorganic phosphate to obtain ribulose.

21. A cell-free method for obtaining ribose, and the method includes:

(a) culturing cells engineered to express thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, thermostable glucose-6-phosphate dehydrogenase, thermostable 6-phosphogluconolactonase, thermostable 6-phosphogluconate dehydrogenase, thermostable ribose-5-phosphate isomerase and thermostable ribose-5-phosphate phosphatase, to obtain cultured cells that express thermostable enzymes;

(b) lysing the cultured cells to obtain a cell lysate;

(c) heating the cell lysate to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(d) incubating the heat-inactivated lysate in the presence of starch and inorganic phosphate to produce ribose.

22. A cell-free method for obtaining ribose, and the method includes:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable glucose-6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases, thermostable 6-phosphogluconate dehydrogenases, thermostable ribose-5-phosphate isomerases, and thermostable ribose-5-phosphate phosphatases to produce at least two cultured cell populations expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to form a mixture of cell lysates that contains a thermostable α-glucan phosphorylase, a thermostable phosphoglucomutase, a thermostable glucose-6-phosphate dehydrogenase, a thermostable 6-phosphogluconolactonase, a thermostable 6-phosphogluconate dehydrogenase, a thermostable ribose-5-phosphate isomerase, and thermostable ribose-5-phosphate phosphatase;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(e) incubating the reaction mixture in the presence of starch and inorganic phosphate to obtain ribose.

23. A cell-free method for obtaining ribose, and the method includes:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable glucose-6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases, thermostable 6-phosphogluconate dehydrogenases, thermostable ribose-5-phosphate isomerases, and thermostable ribose-5-phosphate phosphatases to produce at least two cultured cell populations expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to obtain a mixture of cell lysates;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate;

(e) adding to the heat-inactivated lysate at least one purified enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable glucose-6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases, thermostable 6-phosphogluconate dehydrogenases, thermostable ribose-5 -phosphate isomerases and thermostable ribose-5-phosphate phosphatases, to obtain a reaction mixture containing α-glucan phosphorylase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, ribose-5-phosphate isomerase and ribose-5-phosphate phosphatase; and

(f) incubating the reaction mixture in the presence of starch and inorganic phosphate to obtain ribose.

24. A cell-free method for obtaining arabinose, and the method includes:

(a) culturing cells engineered to express thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, thermostable glucose-6-phosphate dehydrogenase, thermostable 6-phosphogluconolactonase, thermostable 6-phosphogluconate dehydrogenase, thermostable arabinose-5-phosphate isomerase and thermostable arabinose-5-phosphate phosphatase, to obtain cultured cells that express thermostable enzymes;

(b) lysing the cultured cells to obtain a cell lysate;

(c) heating the cell lysate to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(d) incubating the heat-inactivated lysate in the presence of starch and inorganic phosphate to produce arabinose.

25. A cell-free method for obtaining arabinose, and the method includes:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable glucose-6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases, thermostable 6-phosphogluconate dehydrogenases, thermostable arabinose-5-phosphate isomerases, and thermostable arabinose-5-phosphate phosphatases to produce at least two cultured cell populations expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to form a cell lysate mixture that contains a thermostable α-glucan phosphorylase, a thermostable phosphoglucomutase, a thermostable glucose-6-phosphate dehydrogenase, a thermostable 6-phosphogluconolactonase, a thermostable 6-phosphogluconate dehydrogenase, a thermostable arabinose-5-phosphate isomerase, and thermostable arabinose-5-phosphate phosphatase;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(e) incubating the reaction mixture in the presence of starch and inorganic phosphate to obtain arabinose.

26. A cell-free method for producing arabinose, the method comprising:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable glucose-6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases, thermostable 6-phosphogluconate dehydrogenases, thermostable arabinose-5-phosphate isomerases, and thermostable arabinose-5-phosphate phosphatases to produce at least two cultured cell populations expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to obtain a mixture of cell lysates;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate;

(e) adding to the heat inactivated lysate at least one purified enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable glucose-6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases, thermostable 6-phosphogluconate dehydrogenases, thermostable arabinose-5 -phosphate isomerases and thermostable arabinose-5-phosphate phosphatases, to obtain a reaction mixture containing α-glucan phosphorylase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, arabinose-5-phosphate isomerase and arabinose-5-phosphate phosphatase; and

(f) incubating the reaction mixture in the presence of starch and inorganic phosphate to obtain arabinose.

27. A cell-free method for obtaining mannose, and the method includes:

(a) culturing cells engineered to express thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, thermostable phosphoglucoisomerase, thermostable mannose-6-phosphate epimerase, and thermostable mannose-6-phosphate phosphatase to obtain cultured cells that express thermostable enzymes;

(b) lysing the cultured cells to obtain a cell lysate;

(c) heating the cell lysate to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(d) incubating the heat-inactivated lysate in the presence of starch and inorganic phosphate to produce mannose.

28. A cell-free method for producing mannose, the method comprising:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable phosphoglucoisomerases, thermostable mannose-6-phosphate epimerases, and thermostable mannoses -6-phosphate phosphatase, with obtaining at least two cultured populations of cells expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to form a mixture of cell lysates that contains a thermostable α-glucan phosphorylase, a thermostable phosphoglucomutase, a thermostable phosphoglucoisomerase, a thermostable mannose-6-phosphate epimerase, and a thermostable mannose-6-phosphate phosphatase;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(e) incubating the reaction mixture in the presence of starch and inorganic phosphate to obtain mannose.

29. A cell-free method for producing mannose, the method comprising:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable phosphoglucoisomerases, thermostable mannose-6-phosphate epimerases, and thermostable mannoses -6-phosphate phosphatase, with obtaining at least two cultured populations of cells expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to obtain a mixture of cell lysates;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate;

(e) adding to the heat-inactivated lysate at least one purified enzyme selected from the group consisting of thermostable α-glucan phosphorylases, thermostable phosphoglucomutases, thermostable phosphoglucoisomerases, thermostable mannose-6-phosphate epimerases, and thermostable mannose-6-phosphate phosphatases to form a reaction mixture containing α-glucan phosphorylase, phosphoglucomutase, phosphoglucoisomerase, mannose-6-phosphate epimerase and mannose-6-phosphate phosphatase; and

(f) incubating the reaction mixture in the presence of starch and inorganic phosphate to obtain mannose.

30. The method of any one of embodiments 1-26, wherein the cells comprise bacterial cells.

31. The method of any one of embodiments 1-26 wherein the cells comprise yeast cells.

32. The method of any one of embodiments 1-31, wherein at least one of the enzymes is heterologous to cells.

33. The method of any one of embodiments 1-32, wherein step (b) of lysing comprises mechanical, chemical, or enzymatic lysing of the cultured cells.

34. The method of any one of embodiments 1-33, wherein step (c) of heating comprises heating the cell lysate to a temperature of at least 50°C.

35. The method of any one of embodiments 1-34, wherein the starch contains amylose, amylopectin, or both amylose and amylopectin.

36. The method of any one of embodiments 1-35, wherein the thermostable α-glucan phosphorylase and the thermostable phosphoglucomutase are expressed as a single fusion protein or bifunctional protein.

37. Cell lysate obtained by the method of any one of embodiments 1-36.

38. An engineered cell containing α-glucan phosphorylase, phosphoglucomutase and glucose-6-phosphate phosphatase, optionally wherein at least one of the above enzymes is a thermostable enzyme.

39. An engineered cell containing α-glucan phosphorylase, phosphoglucomutase, phosphoglucoisomerase and fructose-6-phosphate phosphatase, optionally wherein at least one of the above enzymes is a thermostable enzyme.

40. An engineered cell containing α-glucan phosphorylase, phosphoglucomutase, phosphoglucoisomerase, allulose-6-phosphate epimerase and allulose-6-phosphate phosphatase, optionally wherein at least one of the above enzymes is a thermostable enzyme.

41. Constructed cell containing α-glucan phosphorylase, phosphoglucomutase, aldose dehydrogenase and sorbitol-6-phosphate phosphatase, optionally, at least one of the above enzymes is a thermostable enzyme.

42. An engineered cell containing α-glucan phosphorylase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, and ribulose-5-phosphate phosphatase, optionally wherein at least one of the above enzymes is a thermostable enzyme.

43. Constructed cell containing α-glucan phosphorylase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, ribose-5-phosphate isomerase, and ribose-5-phosphate phosphatase, optionally wherein at least one of the above enzymes is a thermostable enzyme .

44. Constructed cell containing α-glucan phosphorylase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, arabinose-5-phosphate isomerase and arabinose-5-phosphate phosphatase, optionally wherein at least one of the above enzymes is a thermostable enzyme .

45. An engineered cell containing α-glucan phosphorylase, phosphoglucomutase, phosphoglucoisomerase, mannose-6-phosphate epimerase, and mannose-6-phosphate phosphatase, optionally wherein at least one of the above enzymes is a thermostable enzyme.

46. An engineered cell according to any one of embodiments 38-45, wherein the cell is a bacterial cell or a yeast cell.

47. A single cell lysate, a mixture of cell lysates derived from at least two cell populations, or a reaction mixture that contains α-glucan phosphorylase, phosphoglucomutase and glucose-6-phosphate phosphatase, optionally wherein at least one of the above enzymes is a thermostable enzyme.

48. A single cell lysate, a mixture of cell lysates obtained from at least two cell populations, or a reaction mixture that contains α-glucan phosphorylase, phosphoglucomutase, phosphoglucoisomerase and fructose-6-phosphate phosphatase, optionally wherein at least one of the above enzymes is thermostable enzyme.

49. A single cell lysate, a mixture of cell lysates obtained from at least two cell populations, or a reaction mixture that contains α-glucan phosphorylase, phosphoglucomutase, phosphoglucoisomerase, allulose-6-phosphate epimerase and allulose-6-phosphate phosphatase, optionally at least one of the above enzymes is a thermostable enzyme.

50. A single cell lysate, a mixture of cell lysates derived from at least two cell populations, or a reaction mixture that contains α-glucan phosphorylase, phosphoglucomutase, aldose dehydrogenase, and sorbitol-6-phosphate phosphatase, optionally wherein at least one of the above enzymes is thermostable enzyme.

51. A single cell lysate, a mixture of cell lysates obtained from at least two cell populations, or a reaction mixture that contains α-glucan phosphorylase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase and ribulose-5-phosphate phosphatase , optionally wherein at least one of the above enzymes is a thermostable enzyme.

52. A single cell lysate, a mixture of cell lysates obtained from at least two cell populations, or a reaction mixture that contains α-glucan phosphorylase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, ribose-5-phosphate isomerase and ribose-5-phosphate phosphatase, optionally wherein at least one of the above enzymes is a thermostable enzyme.

53. A single cell lysate, a mixture of cell lysates obtained from at least two cell populations, or a reaction mixture that contains α-glucan phosphorylase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, arabinose-5-phosphate isomerase and arabinose-5-phosphate phosphatase, optionally wherein at least one of the above enzymes is a thermostable enzyme.

54. A single cell lysate, a mixture of cell lysates obtained from at least two cell populations, or a reaction mixture that contains α-glucan phosphorylase, phosphoglucomutase, phosphoglucoisomerase, mannose-6-phosphate epimerase and mannose-6-phosphate phosphatase, optionally at least one of the above enzymes is a thermostable enzyme.

55. A cell-free method for producing allulose, the method comprising:

(a) culturing cells engineered to express thermostable cellodextrin phosphorylase, thermostable phosphoglucomutase, thermostable phosphoglucoisomerase, thermostable allulose-6-phosphate epimerase, and thermostable allulose-6-phosphate phosphatase to obtain cultured cells that express thermostable enzymes;

(b) lysing the cultured cells to obtain a cell lysate;

(c) heating the cell lysate to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(d) incubating the heat-inactivated lysate in the presence of cellodextrin and inorganic phosphate to obtain allulose.

56. A cell-free method for producing allulose, the method comprising:

(a) culturing at least two cell populations, the cells of each population being engineered to express at least one enzyme selected from the group consisting of thermostable cellodextrin phosphorylases, thermostable phosphoglucomutases, thermostable phosphoglucoisomerases, thermostable allulose-6-phosphate epimerases, and thermostable allulose-6 -phosphate phosphatase, with obtaining at least two cultured populations of cells expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to obtain a mixture of cell lysates that contains thermostable cellodextrinphosphorylase, thermostable phosphoglucomutase, thermostable phosphoglucoisomerase, thermostable allulose-6-phosphate epimerase and thermostable allulose-6-phosphate phosphatase;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(e) incubating the reaction mixture in the presence of cellodextrin and inorganic phosphate to obtain allulose.

57. A cell-free method for producing allulose, the method comprising:

(a) culturing at least two cell populations, the cells of each population being engineered to express at least one enzyme selected from the group consisting of thermostable cellodextrin phosphorylases, thermostable phosphoglucomutases, thermostable phosphoglucoisomerases, thermostable allulose-6-phosphate epimerases, and thermostable allulose-6 -phosphate phosphatase, with obtaining at least two cultured populations of cells expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to obtain a mixture of cell lysates;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate;

(e) adding to the heat-inactivated lysate at least one purified enzyme selected from the group consisting of thermostable cellodextrin phosphorylases, thermostable phosphoglucomutases, thermostable phosphoglucoisomerases, thermostable allulose-6-phosphate epimerases, and thermostable allulose-6-phosphate phosphatases, to obtain a reaction mixture containing cellodextrinphosphorylase, phosphoglucomutase, phosphoglucoisomerase, allulose-6-phosphate epimerase and allulose-6-phosphate phosphatase; and

(f) incubating the reaction mixture in the presence of cellodextrin and inorganic phosphate to obtain allulose.

58. The cell-free method of any one of embodiments 55-57, wherein the one or more thermostable cellodextrin phosphorylases are selected from the group consisting of Aquifex aeolicus, Thermocrinis minervae, Thermosulfidibacter takaii, Thermosulfurimonas dismutans, Thermococcus litoralis, Palaeococcus pacificus, Thermotoga necellapolitana, Ruminiclostridium cellodextrin phosphorylases. . _

59. The cell-free method of any one of embodiments 55-58, wherein the one or more thermostable phosphoglucomutases are selected from the group consisting of Thermococcus kodakaraensis, Pyrococcus kukulkanii, Ammonifex degensii, Methanothermobacter wolfeii, Methanothermus fervidus, Sulfolobus acidocaldarius, Archaeoglobus fulgidus, Ferroglobus placidus phosphoglucomutases. , Geoglobus ahangari, Archaeoglobus veneficus, Archaeoglobus sulfaticallidus, Aciduliprofundum boonie, Clostridium thermocellum, Defluviitalea phaphyphila, Caminicella sporogenes, Caloranaerobacter ferrireducens, Thermosipho malanesiensis, Fervidobacterium pennivorans, Symbiobacterium thermophilum, Spirochaeta thermophila and Thermoanagelobacter w .

60. The cell-free method of any one of embodiments 55-59, wherein the one or more thermostable phosphoglucomutases are selected from the group consisting of Thermus thermophilus, Meiothermus timidus, Thermus filiformis, Marinithermus hydrothermalis, Thermosipho africanus, Sulfurihydrogenibium azorense, Persephonella marina, Marinitoga piezophila phosphoglucomutases. , Kosmotoga olearia, Thermotoga maritima, Geobacillus stearothermophilus, Anoxybacillus flavithermus, Thermosulfidibacter takaii, Fervidobacterium nodosum, Clostridium thermocellum, Thermoanaerobacterium thermosaccharolyticum, Methanococcus jannaschii, Methanotorris igneus, Methanocaldococcus villosus, Methanothermococcus okinawensis, Pseudothermotoga thermarum, Deferribacter desulfuricans и Thermovibrio ammonificans .

61. A cell-free method according to any one of embodiments 55-60, wherein one or more thermostable allulose-6-phosphate epimerases are selected from the group consisting of allulose-6-phosphate epimerases of Thermobacterium thermosaccharolyticum, Thermoanaerobacter brockii, Caldanaerobacter subterraneus, Deferribacter desulfuricans, Thermocrinis ruber, Hydrogenivirga sp. 128-5-R1-1, Brevibacillus thermoruber, Thermosipho atlanticus and Thermosulfidibacter takaii .

62. The cell-free method of any one of embodiments 55-61, wherein the one or more thermostable allulose-6-phosphate phosphatases are selected from the group consisting of allulose-6-phosphate phosphatases Thermoanaerobacter wiegelii , Thermoanaerobacter ethanolicus, Thermus islandicus, Deinococcus geothermalis DSM 11300, Thermosphaera aggregans, Crenarchaeota archaeon, Pyrococcus horikoshii Ot3, Aquifex aeolicus, Ruminiclostridium thermocellum, Desulfotomaculum kuznetsovii, Caldanaerobacter subterraneus, Acidothermus cellulolyticus, Methanothermobacter thermautotrophicus, Thermobifida fusca, Thermotoga neapolitana, Petrotoga mobilus, and Ther- modesulfat

63. A cell-free method for producing glucose, the method comprising:

(a) culturing cells engineered to express thermostable cellodextrinphosphorylase, thermostable phosphoglucomutase, and thermostable glucose-6-phosphate phosphatase to produce cultured cells that express thermostable enzymes;

(b) lysing the cultured cells to obtain a cell lysate;

(c) heating the cell lysate to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(d) incubating the heat-inactivated lysate in the presence of cellodextrin and inorganic phosphate to produce glucose.

64. A cell-free method for producing glucose, the method comprising:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable cellodextrin phosphorylases, thermostable phosphoglucomutases, and thermostable glucose-6-phosphate phosphatase, to obtain at least two cultured populations of cells expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to obtain a mixture of cell lysates that contains thermostable cellodextrinphosphorylase, thermostable phosphoglucomutase and thermostable glucose-6-phosphate phosphatase;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(e) incubating the reaction mixture in the presence of cellodextrin and inorganic phosphate to obtain glucose.

65. A cell-free method for producing glucose, the method comprising:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable cellodextrin phosphorylases, thermostable phosphoglucomutases, and thermostable glucose-6-phosphate phosphatase, to obtain at least two cultured populations of cells expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to obtain a mixture of cell lysates;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate;

(e) adding to the heat-inactivated lysate at least one purified enzyme selected from the group consisting of thermostable cellodextrin phosphorylases, thermostable phosphoglucomutases, and thermostable glucose-6-phosphate phosphatases to form a reaction mixture containing cellodextrin phosphorylase, phosphoglucomutase, and glucose-6-phosphate phosphatase; and

(f) incubating the reaction mixture in the presence of cellodextrin and inorganic phosphate to obtain glucose.

66. A cell-free method for obtaining fructose, and the method includes:

(a) culturing cells designed to express thermostable cellodextrinphosphorylase, thermostable phosphoglucomutase, thermostable phosphoglucoisomerase, and thermostable fructose-6-phosphate phosphatase to obtain cultured cells that express thermostable enzymes;

(b) lysing the cultured cells to obtain a cell lysate;

(c) heating the cell lysate to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(d) incubating the heat-inactivated lysate in the presence of cellodextrin and inorganic phosphate to produce fructose.

67. A cell-free method for obtaining fructose, and the method includes:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable cellodextrin phosphorylases, thermostable phosphoglucomutases, thermostable phosphoglucoisomers, and thermostable fructose-6-phosphate phosphatases, to obtain at least at least two cultured populations of cells expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to obtain a mixture of cell lysates that contains a thermostable cellodextrin phosphorylase, a thermostable phosphoglucomutase, a thermostable phosphoglucoisomerase, and a thermostable fructose-6-phosphate phosphatase;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(e) incubating the reaction mixture in the presence of cellodextrin and inorganic phosphate to obtain fructose.

68. A cell-free method for obtaining fructose, and the method includes:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable cellodextrin phosphorylases, thermostable phosphoglucomutases, thermostable phosphoglucoisomers, and thermostable fructose-6-phosphate phosphatases, to obtain at least at least two cultured populations of cells expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to obtain a mixture of cell lysates;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate;

(e) adding to the heat-inactivated lysate at least one purified enzyme selected from the group consisting of thermostable cellodextrin phosphorylases, thermostable phosphoglucomutases, thermostable phosphoglucoisomerases, and thermostable fructose-6-phosphate phosphatases to form a reaction mixture containing cellodextrin phosphorylase, phosphoglucomutase, phosphoglucoisomerase and fructose -6-phosphate phosphatase; and

(f) incubating the reaction mixture in the presence of cellodextrin and inorganic phosphate to obtain fructose.

69. Cell-free method for obtaining sorbitol, and the method includes:

(a) culturing cells engineered to express thermostable cellodextrin phosphorylase, thermostable phosphoglucomutase, thermostable aldose dehydrogenase, and thermostable sorbitol-6-phosphate phosphatase to obtain cultured cells that express thermostable enzymes;

(b) lysing the cultured cells to obtain a cell lysate;

(c) heating the cell lysate to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(d) incubating the heat-inactivated lysate in the presence of cellodextrin and inorganic phosphate to obtain sorbitol.

70. A cell-free method for obtaining sorbitol, and the method includes:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable cellodextrin phosphorylases, thermostable phosphoglucomutases, thermostable aldose dehydrogenases, and thermostable sorbitol-6-phosphate phosphatases, to obtain at least at least two cultured populations of cells expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to form a mixture of cell lysates that contains a thermostable cellodextrin phosphorylase, a thermostable phosphoglucomutase, a thermostable aldose dehydrogenase, and a thermostable sorbitol-6-phosphate phosphatase;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(e) incubating the reaction mixture in the presence of cellodextrin and inorganic phosphate to obtain sorbitol.

71. A cell-free method for obtaining sorbitol, and the method includes:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable cellodextrin phosphorylases, thermostable phosphoglucomutases, thermostable aldose dehydrogenases, and thermostable sorbitol-6-phosphate phosphatases, to obtain at least at least two cultured populations of cells expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to obtain a mixture of cell lysates;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate;

(e) adding to the heat-inactivated lysate at least one purified enzyme selected from the group consisting of thermostable cellodextrin phosphorylases, thermostable phosphoglucomutases, thermostable aldose dehydrogenases, and thermostable sorbitol-6-phosphate phosphatases to form a reaction mixture containing cellodextrin phosphorylase, phosphoglucomutase, aldose dehydrogenase and sorbitol -6-phosphate phosphatase; and

(f) incubating the reaction mixture in the presence of cellodextrin and inorganic phosphate to obtain sorbitol.

72. A cell-free method for obtaining ribulose, and the method includes:

(a) culturing cells engineered to express thermostable cellodextrin phosphorylase, thermostable phosphoglucomutase, thermostable glucose-6-phosphate dehydrogenase, thermostable 6-phosphogluconolactonase, thermostable 6-phosphogluconate dehydrogenase, and thermostable ribulose-5-phosphate phosphatase to obtain cultured cells that express thermostable enzymes;

(b) lysing the cultured cells to obtain a cell lysate;

(c) heating the cell lysate to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(d) incubating the heat inactivated lysate in the presence of cellodextrin and inorganic phosphate to produce ribulose.

73. A cell-free method for obtaining ribulose, and the method includes:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable cellodextrin phosphorylases, thermostable phosphoglucomutases, thermostable glucose-6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases, thermostable 6 α-phosphogluconate dehydrogenases and thermostable ribulose-5-phosphate phosphatases to produce at least two cultured cell populations expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to form a mixture of cell lysates that contains a thermostable cellodextrin phosphorylase, a thermostable phosphoglucomutase, a thermostable glucose-6-phosphate dehydrogenase, a thermostable 6-phosphogluconolactonase, a thermostable 6-phosphogluconate dehydrogenase, and a thermostable ribulose-5-phosphate phosphatase;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(e) incubating the reaction mixture in the presence of cellodextrin and inorganic phosphate to obtain ribulose.

74. A cell-free method for obtaining ribulose, and the method includes:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable cellodextrin phosphorylases, thermostable phosphoglucomutases, thermostable glucose-6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases, thermostable 6 α-phosphogluconate dehydrogenases and thermostable ribulose-5-phosphate phosphatases to produce at least two cultured cell populations expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to obtain a mixture of cell lysates;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate;

(e) adding to the heat-inactivated lysate at least one purified enzyme selected from the group consisting of thermostable cellodextrin phosphorylases, thermostable phosphoglucomutases, thermostable glucose-6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases, thermostable 6-phosphogluconate dehydrogenases, and thermostable ribulose-5-phosphate phosphatases to obtain a reaction mixture containing cellodextrinphosphorylase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase and ribulose-5-phosphate phosphatase; and

(f) incubating the reaction mixture in the presence of cellodextrin and inorganic phosphate to obtain ribulose.

75. A cell-free method for obtaining ribose, and the method includes:

(a) culturing cells engineered to express thermostable cellodextrin phosphorylase, thermostable phosphoglucomutase, thermostable glucose-6-phosphate dehydrogenase, thermostable 6-phosphogluconolactonase, thermostable 6-phosphogluconate dehydrogenase, thermostable ribose-5-phosphate isomerase, and thermostable ribose-5-phosphate phosphatase to obtain cultured cells , which express thermostable enzymes;

(b) lysing the cultured cells to obtain a cell lysate;

(c) heating the cell lysate to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(d) incubating the heat-inactivated lysate in the presence of cellodextrin and inorganic phosphate to produce ribose.

76. A cell-free method for obtaining ribose, and the method includes:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable cellodextrin phosphorylases, thermostable phosphoglucomutases, thermostable glucose-6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases, thermostable 6 -phosphogluconate dehydrogenases, thermostable ribose-5-phosphate isomerase and thermostable ribose-5-phosphate phosphatase, with obtaining at least two cultured populations of cells expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to form a mixture of cell lysates that contains a thermostable cellodextrin phosphorylase, a thermostable phosphoglucomutase, a thermostable glucose-6-phosphate dehydrogenase, a thermostable 6-phosphogluconolactonase, a thermostable 6-phosphogluconate dehydrogenase, a thermostable ribose-5-phosphate isomerase, and a thermostable riboso -5-phosphate phosphatase;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(e) incubating the reaction mixture in the presence of cellodextrin and inorganic phosphate to obtain ribose.

77. A cell-free method for obtaining ribose, and the method includes:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable cellodextrin phosphorylases, thermostable phosphoglucomutases, thermostable glucose-6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases, thermostable 6 -phosphogluconate dehydrogenases, thermostable ribose-5-phosphate isomerase and thermostable ribose-5-phosphate phosphatase, with obtaining at least two cultured populations of cells expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to obtain a mixture of cell lysates;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate;

(e) adding to the heat-inactivated lysate at least one purified enzyme selected from the group consisting of thermostable cellodextrin phosphorylases, thermostable phosphoglucomutases, thermostable glucose-6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases, thermostable 6-phosphogluconate dehydrogenases, thermostable ribose-5-phosphate isomerases and thermostable ribose-5-phosphate phosphatase, to obtain a reaction mixture containing cellodextrinphosphorylase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, ribose-5-phosphate isomerase and ribose-5-phosphate phosphatase; and

(f) incubating the reaction mixture in the presence of cellodextrin and inorganic phosphate to obtain ribose.

78. A cell-free method for obtaining arabinose, and the method includes:

(a) culturing cells engineered to express thermostable cellodextrin phosphorylase, thermostable phosphoglucomutase, thermostable glucose-6-phosphate dehydrogenase, thermostable 6-phosphogluconolactonase, thermostable 6-phosphogluconate dehydrogenase, thermostable arabinose-5-phosphate isomerase and thermostable arabinose-5-phosphate phosphatase to obtain cultured cells , which express thermostable enzymes;

(b) lysing the cultured cells to obtain a cell lysate;

(c) heating the cell lysate to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(d) incubating the heat-inactivated lysate in the presence of cellodextrin and inorganic phosphate to produce arabinose.

79. Cell-free method for obtaining arabinose, and the method includes:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable cellodextrin phosphorylases, thermostable phosphoglucomutases, thermostable glucose-6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases, thermostable 6 -phosphogluconate dehydrogenases, thermostable arabinose-5-phosphate isomerase and thermostable arabinose-5-phosphate phosphatases, to obtain at least two cultured cell populations expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to form a mixture of cell lysates that contains a thermostable cellodextrin phosphorylase, a thermostable phosphoglucomutase, a thermostable glucose-6-phosphate dehydrogenase, a thermostable 6-phosphogluconolactonase, a thermostable 6-phosphogluconate dehydrogenase, a thermostable arabinose-5-phosphate isomerase, and a thermostable arabinose -5-phosphate phosphatase;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(e) incubating the reaction mixture in the presence of cellodextrin and inorganic phosphate to obtain arabinose.

80. Cell-free method for obtaining arabinose, and the method includes:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable cellodextrin phosphorylases, thermostable phosphoglucomutases, thermostable glucose-6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases, thermostable 6 -phosphogluconate dehydrogenases, thermostable arabinose-5-phosphate isomerase and thermostable arabinose-5-phosphate phosphatases, to obtain at least two cultured cell populations expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to obtain a mixture of cell lysates;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate;

(e) adding to the heat-inactivated lysate at least one purified enzyme selected from the group consisting of thermostable cellodextrin phosphorylases, thermostable phosphoglucomutases, thermostable glucose-6-phosphate dehydrogenases, thermostable 6-phosphogluconolactonases, thermostable 6-phosphogluconate dehydrogenases, thermostable arabinose-5-phosphate isomerase and thermostable arabinose-5-phosphate phosphatases, to obtain a reaction mixture containing cellodextrinphosphorylase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, arabinose-5-phosphate isomerase and arabinose-5-phosphate phosphatase; and

(f) incubating the reaction mixture in the presence of cellodextrin and inorganic phosphate to obtain arabinose.

81. A cell-free method for producing mannose, and the method includes:

(a) culturing cells engineered to express thermostable cellodextrin phosphorylase, thermostable phosphoglucomutase, thermostable phosphoglucoisomerase, thermostable mannose-6-phosphate epimerase, and thermostable mannose-6-phosphate phosphatase to obtain cultured cells that express thermostable enzymes;

(b) lysing the cultured cells to obtain a cell lysate;

(c) heating the cell lysate to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(d) incubating the heat-inactivated lysate in the presence of cellodextrin and inorganic phosphate to produce mannose.

82. A cell-free method for obtaining mannose, and the method includes:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable cellodextrin phosphorylases, thermostable phosphoglucomutases, thermostable phosphoglucoisomerases, thermostable mannose-6-phosphate epimerases, and thermostable mannose-6 -phosphate phosphatase, with obtaining at least two cultured populations of cells expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to obtain a mixture of cell lysates that contains a thermostable cellodextrin phosphorylase, a thermostable phosphoglucomutase, a thermostable phosphoglucoisomerase, a thermostable mannose-6-phosphate epimerase, and a thermostable mannose-6-phosphate phosphatase;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(e) incubating the reaction mixture in the presence of cellodextrin and inorganic phosphate to obtain mannose.

83. A cell-free method for obtaining mannose, and the method includes:

(a) culturing at least two cell populations, wherein the cells of each population are engineered to express at least one enzyme selected from the group consisting of thermostable cellodextrin phosphorylases, thermostable phosphoglucomutases, thermostable phosphoglucoisomerases, thermostable mannose-6-phosphate epimerases, and thermostable mannose-6 -phosphate phosphatase, with obtaining at least two cultured populations of cells expressing different enzymes;

(b) lysing the cells of at least two cultured populations to obtain at least two cell lysates;

(c) combining at least two cell lysates to obtain a mixture of cell lysates;

(d) heating the mixture of cell lysates to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate;

(e) adding to the heat-inactivated lysate at least one purified enzyme selected from the group consisting of thermostable cellodextrin phosphorylases, thermostable phosphoglucomutases, thermostable phosphoglucoisomerases, thermostable mannose-6-phosphate epimerases, and thermostable mannose-6-phosphate phosphatases, to obtain a reaction mixture containing cellodextrinphosphorylase, phosphoglucomutase, phosphoglucoisomerase, mannose-6-phosphate epimerase and mannose-6-phosphate phosphatase; and

(f) incubating the reaction mixture in the presence of cellodextrin and inorganic phosphate to obtain mannose.

84. The method of any one of embodiments 1-80, wherein the cells comprise bacterial cells.

85. The method of any one of embodiments 1-80, wherein the cells comprise yeast cells.

86. The method of any one of embodiments 1-85, wherein at least one of the enzymes is heterologous to cells.

87. The method of any one of embodiments 1-86, wherein step (b) of lysing comprises mechanical, chemical, or enzymatic lysing of the cultured cells.

88. The method of any one of embodiments 1-87 wherein step (c) of heating comprises heating the cell lysate to a temperature of at least 50°C.

89. The method of any one of embodiments 1-88, wherein the cellodextrin contains amylose, amylopectin, or both amylose and amylopectin.

90. The method of any one of embodiments 1-89, wherein the thermostable cellodextrin phosphorylase and the thermostable phosphoglucomutase are expressed as a single fusion protein or a bifunctional protein.

91. Cell lysate obtained by the method of any one of embodiments 1-90.

92. An engineered cell containing cellodextrin phosphorylase, phosphoglucomutase and glucose-6-phosphate phosphatase, optionally wherein at least one of the above enzymes is a thermostable enzyme.

93. An engineered cell containing cellodextrin phosphorylase, phosphoglucomutase, phosphoglucoisomerase, and fructose-6-phosphate phosphatase, optionally wherein at least one of the above enzymes is a thermostable enzyme.

94. An engineered cell containing cellodextrin phosphorylase, phosphoglucomutase, phosphoglucoisomerase, allulose-6-phosphate epimerase, and allulose-6-phosphate phosphatase, optionally wherein at least one of the above enzymes is a thermostable enzyme.

95. An engineered cell containing cellodextrinphosphorylase, phosphoglucomutase, aldose dehydrogenase, and sorbitol-6-phosphate phosphatase, optionally wherein at least one of the above enzymes is a thermostable enzyme.

96. An engineered cell containing cellodextrin phosphorylase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, and ribulose-5-phosphate phosphatase, optionally wherein at least one of the above enzymes is a thermostable enzyme.

97. An engineered cell containing cellodextrin phosphorylase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, ribose-5-phosphate isomerase, and ribose-5-phosphate phosphatase, optionally wherein at least one of the above enzymes is a thermostable enzyme.

98. An engineered cell containing cellodextrin phosphorylase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, arabinose-5-phosphate isomerase, and arabinose-5-phosphate phosphatase, optionally wherein at least one of the above enzymes is a thermostable enzyme.

99. An engineered cell containing cellodextrin phosphorylase, phosphoglucomutase, phosphoglucoisomerase, mannose-6-phosphate epimerase, and mannose-6-phosphate phosphatase, optionally wherein at least one of the above enzymes is a thermostable enzyme.

100. An engineered cell according to any one of embodiments 35-41, wherein the cell is a bacterial cell or a yeast cell.

101. A single cell lysate, a mixture of cell lysates derived from at least two cell populations, or a reaction mixture that contains cellodextrin phosphorylase, phosphoglucomutase, and glucose-6-phosphate phosphatase, optionally wherein at least one of the above enzymes is a thermostable enzyme.

102. A single cell lysate, a mixture of cell lysates derived from at least two cell populations, or a reaction mixture that contains cellodextrin phosphorylase, phosphoglucomutase, phosphoglucoisomerase, and fructose-6-phosphate phosphatase, optionally wherein at least one of the above enzymes is a thermostable enzyme.

103. A single cell lysate, a mixture of cell lysates obtained from at least two cell populations, or a reaction mixture that contains cellodextrinphosphorylase, phosphoglucomutase, phosphoglucoisomerase, allulose-6-phosphate epimerase and allulose-6-phosphate phosphatase, optionally at least one of of the above enzymes is a thermostable enzyme.

104. A single cell lysate, a mixture of cell lysates derived from at least two cell populations, or a reaction mixture that contains cellodextrin phosphorylase, phosphoglucomutase, aldose dehydrogenase, and sorbitol-6-phosphate phosphatase, optionally wherein at least one of the above enzymes is a thermostable enzyme.

105. A single cell lysate, a mixture of cell lysates obtained from at least two cell populations, or a reaction mixture that contains cellodextrinphosphorylase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase and ribulose-5-phosphate phosphatase, optionally and at least one of the above enzymes is a thermostable enzyme.

106. A single cell lysate, a mixture of cell lysates obtained from at least two cell populations, or a reaction mixture that contains cellodextrinphosphorylase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, ribose-5-phosphate isomerase and ribose -5-phosphate phosphatase, optionally wherein at least one of the above enzymes is a thermostable enzyme.

107. A single cell lysate, a mixture of cell lysates obtained from at least two cell populations, or a reaction mixture that contains cellodextrinphosphorylase, phosphoglucomutase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, arabinose-5-phosphate isomerase and arabinose -5-phosphate phosphatase, optionally wherein at least one of the above enzymes is a thermostable enzyme.

108. A single cell lysate, a mixture of cell lysates obtained from at least two cell populations, or a reaction mixture that contains cellodextrinphosphorylase, phosphoglucomutase, phosphoglucoisomerase, mannose-6-phosphate epimerase and mannose-6-phosphate phosphatase, optionally at least one of of the above enzymes is a thermostable enzyme.

109. A cell-free method for obtaining sugar, and the method includes:

(a) culturing cells engineered to express a thermostable α-glucan phosphorylase, a thermostable phosphoglucomutase, and at least one thermostable enzyme selected from the group consisting of isomerases, epimerases, dehydrogenases, and sugar phosphatases to obtain cultured cells that express such enzymes;

(b) lysing the cultured cells from step (a) to obtain a cell lysate;

(c) heating the cell lysate to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate the thermostable enzymes from step (a), to obtain a heat-inactivated lysate; and

(d) incubating the heat-inactivated lysate in the presence of starch and inorganic phosphate to produce sugar.

110. The method of Embodiment 109, wherein the at least one thermostable enzyme is an isomerase selected from the group consisting of phosphoglucoisomerase, ribose-5-phosphate isomerase, and arabinose-5-phosphate isomerase.

111. The method of embodiment 109 or 110 wherein the at least one thermostable enzyme is allulose 6-phosphate epimerase.

112. The method of any one of embodiments 109-111, wherein the at least one thermostable enzyme is a dehydrogenase selected from the group consisting of aldose dehydrogenase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase.

113. The method of any one of embodiments 109-112, wherein at least one thermostable enzyme is a sugar phosphatase selected from the group consisting of glucose-6-phosphate phosphatase, fructose-6-phosphate phosphatase, allulose-6-phosphate phosphatase, sorbitol- 6-phosphate phosphatase, ribulose-5-phosphate phosphatase, ribose-5-phosphate phosphatase and arabinose-5-phosphate phosphatase.

114. The method of embodiment 109 wherein the sugar is selected from the group consisting of glucose, fructose, allulose, sorbitol, ribulose, ribose, and arabinose.

115. The method of embodiment 114 wherein the sugar is glucose and at least one thermostable enzyme comprises glucose-6-phosphate phosphatase.

116. The method of embodiment 115 wherein cells are engineered to express thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, and thermostable glucose-6-phosphate phosphatase.

117. The method of embodiment 114 wherein the sugar is fructose and at least one thermostable enzyme is selected from phosphoglucoisomerases and fructose-6-phosphate phosphatases.

118. The method of embodiment 117 wherein cells are engineered to express thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, thermostable phosphoglucoisomerase, and thermostable fructose-6-phosphate phosphatase.

119. The method of embodiment 114 wherein the sugar is allulose and at least one thermostable enzyme is selected from phosphoglucoisomerases, allulose-6-phosphate epimerases, and allulose-6-phosphate phosphatases.

120. The method of embodiment 119 wherein cells are engineered to express thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, thermostable phosphoglucoisomerase, thermostable allulose-6-phosphate epimerase, and thermostable allulose-6-phosphate phosphatase.

121. The method of embodiment 114 wherein the sugar is sorbitol and at least one thermostable enzyme is selected from aldose dehydrogenases and sorbitol-6-phosphate phosphatases.

122. The method of embodiment 121 wherein cells are engineered to express thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, thermostable aldose dehydrogenase, and thermostable sorbitol-6-phosphate phosphatase.

123. The method of embodiment 114 wherein the sugar is ribulose and at least one thermostable enzyme is selected from glucose-6-phosphate dehydrogenases, 6-phosphogluconolactonases, 6-phosphogluconate dehydrogenases, and ribulose-5-phosphate phosphatases.

124. The method of embodiment 123 wherein cells are engineered to express thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, thermostable glucose-6-phosphate dehydrogenase, thermostable 6-phosphogluconolactonase, thermostable 6-phosphogluconate dehydrogenase, and thermostable ribulose-5-phosphate phosphatase.

125. The method of embodiment 114, wherein the sugar is ribose and at least one thermostable enzyme is selected from glucose-6-phosphate dehydrogenases, 6-phosphogluconolactonases, 6-phosphogluconate dehydrogenases, ribose-5-phosphate isomerase, and ribose-5-phosphate phosphatases.

126. The method of embodiment 125 wherein cells are engineered to express thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, thermostable glucose-6-phosphate dehydrogenase, thermostable 6-phosphogluconolactonase, thermostable 6-phosphogluconate dehydrogenase, thermostable ribose-5-phosphate isomerase, and thermostable ribose-5 -phosphate phosphatase.

127. The method of Embodiment 114, wherein the sugar is arabinose and at least one thermostable enzyme is selected from glucose-6-phosphate dehydrogenases, 6-phosphogluconolactonases, 6-phosphogluconate dehydrogenases, arabinose-5-phosphate isomerases, and arabinose-5-phosphate phosphatases.

128. The method of embodiment 127 wherein cells are engineered to express thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, thermostable glucose-6-phosphate dehydrogenase, thermostable 6-phosphogluconolactonase, thermostable 6-phosphogluconate dehydrogenase, thermostable arabinose-5-phosphate isomerase, and thermostable arabinose-5 -phosphate phosphatase.

129. The method of any one of embodiments 109-128, wherein the cells are bacterial cells.

130. The method of any one of embodiments 109-128, wherein the cells are yeast cells.

131. The method of any one of embodiments 109-130, wherein the thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, and/or at least one thermostable enzyme is/are heterologous to cells.

132. The method of any one of embodiments 109-131 wherein step (b) of lysing comprises mechanical, chemical, or enzymatic lysing of the cultured cells.

133. The method of any one of embodiments 109-132, wherein step (c) of heating comprises heating the cell lysate to a temperature of at least 50°C.

134. The method of any one of embodiments 109-133, wherein the starch contains amylose, amylopectin, or both amylose and amylopectin.

135. The method of any one of embodiments 109-134, wherein the thermostable α-glucan phosphorylase and the thermostable phosphoglucomutase are expressed as a single fusion protein.

136. A cell-free method for obtaining sugar, and the method includes:

(a) culturing cells engineered to express α-glucan phosphorylase, phosphoglucomutase, and at least one enzyme selected from the group consisting of isomerases, epimerases, dehydrogenases, and sugar phosphatases to obtain cultured cells that express such enzymes;

(b) lysing the cultured cells from step (a) to obtain a cell lysate; and

(c) incubating the lysate in the presence of starch and inorganic phosphate to produce sugar.

137. A cell-free method for obtaining sugar, and the method includes:

(a) culturing cells engineered to express (i) a fusion protein that contains α-glucan phosphorylase hybridized to phosphoglucomutase and (ii) at least one enzyme selected from the group consisting of isomerases, epimerases, dehydrogenases, and sugar phosphatases, with obtaining cultured cells that express such enzymes;

(b) lysing the cultured cells from step (a) to obtain a cell lysate; and

(c) incubating the lysate in the presence of starch and inorganic phosphate to produce sugar.

138. A cell-free method for obtaining sugar, and the method includes:

(a) culturing cells engineered to express a fusion protein that contains α-glucan phosphorylase hybridized to phosphoglucomutase;

(b) culturing cells designed to express at least one enzyme selected from the group consisting of isomerases, epimerases, dehydrogenases, and sugar phosphatases to obtain cultured cells that express such enzymes;

(c) lysing the cultured cells from step (a) from step (b) to obtain cell lysates; and

(d) incubating the lysates in the presence of starch and inorganic phosphate to produce sugar.

139. The method of embodiment 137 or 138, wherein the enzymes from steps (a) and/or (b) are thermostable enzymes.

140. The method of embodiment 139, wherein the method further comprises heating one or more cell lysates to a temperature that inactivates naturally occurring molecules with enzymatic activity but does not inactivate thermostable enzymes to produce one or more heat-inactivated lysates.

141. Cell lysate obtained by the method of any one of embodiments 109-138.

142. An engineered cell containing a thermostable α-glucan phosphorylase, a thermostable phosphoglucomutase, and at least one thermostable enzyme selected from the group consisting of isomerases, epimerases, dehydrogenases, and sugar phosphatases.

143. The engineered cell of embodiment 142, comprising:

(a) thermostable α-glucan phosphorylase, thermostable phosphoglucomutase and thermostable glucose-6-phosphate phosphatase;

(b) thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, phosphoglucoisomerase and fructose-6-phosphate phosphatase;

(c) thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, phosphoglucoisomerase, allulose-6-phosphate epimerase and allulose-6-phosphate phosphatase;

(d) thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, aldose dehydrogenase and sorbitol-6-phosphate phosphatase;

(e) thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, and ribulose-5-phosphate phosphatase;

(f) thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, ribose-5-phosphate isomerase, and ribose-5-phosphate phosphatase; or

(g) thermostable α-glucan phosphorylase, thermostable phosphoglucomutase, glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, 6-phosphogluconate dehydrogenase, arabinose-5-phosphate isomerase, and arabinose-5-phosphate phosphatase.

144. The engineered cell of embodiment 142 or 143, wherein the cell is a bacterial cell or a yeast cell.

EXAMPLES

Example 1

Cell-free conversion of starch to allulose

This example describes the conversion of starch to allulose. Cells (eg, bacterial or yeast cells) engineered to express at least one heterologous gene encoding at least one enzyme for converting starch to allulose were grown in liquid cultures to high cell density. Examples of heterologous enzymes that could be used in this example include thermostable variants of α-glucan phosphorylase (EC 2.4.1.1), phosphoglucomutase (EC 5.4.2.2, 5.4.2.5 or 5.4.2.6), phosphoglucoisomerase (EC 5.3.1.9), alluloso -6-phosphate epimerase (EC 5.3.1.-) and allulose-6-phosphate phosphatase (EC 5.3.1.-). At the end of the growth step, the expression of one or more heterologous enzymes was induced and then the cell biomass was collected. The harvested biomass was then lysed mechanically, chemically or enzymatically. The cell lysate is then heated to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate one or more heterologous enzymes. To the heat-inactivated lysate, starch-based feedstock, inorganic phosphate, and optionally other additional nutrients were added to convert starch to allulose (FIG. 1).

Example 2

Cellular conversion of starch to glucose

This example describes the conversion of starch to glucose. Cells (eg, bacterial or yeast cells) engineered to express at least one heterologous gene encoding at least one enzyme for converting starch to glucose were grown in liquid cultures to high cell density. Examples of heterologous enzymes that could be used in this example include thermostable variants of α-glucan phosphorylase (EC 2.4.1.1), phosphoglucomutase (EC 5.4.2.2, 5.4.2.5 or 5.4.2.6), and glucose-6-phosphate phosphatase (EC 5.3. one.-). At the end of the growth step, the expression of one or more heterologous enzymes was induced and then the cell biomass was collected. The harvested biomass was then lysed mechanically, chemically or enzymatically. The cell lysate is then heated to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate one or more heterologous enzymes. To the heat-inactivated lysate, starch-based raw materials, inorganic phosphate, and optionally other additional nutrients were added to ensure the conversion of starch to glucose (FIG. 2A).

This example also describes another way of converting starch to glucose. Cells (eg, bacterial or yeast cells) engineered to express at least one heterologous gene encoding at least one enzyme for converting starch to glucose were grown in liquid cultures to high cell density. Examples of heterologous enzymes that could be used in this example include thermostable variants of α-glucan phosphorylase (EC 2.4.1.1) and glucose-1-phosphate phosphatase (EC 3.1.3.10). At the end of the growth step, the expression of one or more heterologous enzymes was induced and then the cell biomass was collected. The harvested biomass was then lysed mechanically, chemically or enzymatically. The cell lysate is then heated to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate one or more heterologous enzymes. To the heat-inactivated lysate, starch-based raw materials, inorganic phosphate, and optionally other additional nutrients were added to ensure the conversion of starch to glucose (FIG. 2B).

Example 3

Cellular conversion of starch to fructose

This example describes the conversion of starch to fructose. Cells (eg, bacterial or yeast cells) engineered to express at least one heterologous gene encoding at least one enzyme for converting starch to fructose were grown in liquid cultures to high cell density. Examples of heterologous enzymes that could be used in this example include thermostable variants of α-glucan phosphorylase (EC 2.4.1.1), phosphoglucomutase (EC 5.4.2.2, 5.4.2.5 or 5.4.2.6), phosphoglucoisomerase (EC 5.3.1.9) and fructose -6-phosphate phosphatase (EC 5.3.1.-). At the end of the growth step, the expression of one or more heterologous enzymes was induced and then the cell biomass was collected. The harvested biomass was then lysed mechanically, chemically or enzymatically. The cell lysate is then heated to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate one or more heterologous enzymes. To the heat-inactivated lysate, starch-based raw materials, inorganic phosphate, and optionally other additional nutrients were added to ensure the conversion of starch to fructose (FIG. 3).

Example 4

Cell-free conversion of starch to sorbitol

This example describes the conversion of starch to sorbitol. Cells (eg, bacterial or yeast cells) engineered to express at least one heterologous gene encoding at least one enzyme for converting starch to sorbitol were grown in liquid cultures to high cell density. Examples of heterologous enzymes that could be used in this example include thermostable variants of α-glucan phosphorylase (EC 2.4.1.1), phosphoglucomutase (EC 5.4.2.2, 5.4.2.5 or 5.4.2.6), aldose dehydrogenase (EC 1.1.1.200) and sorbitol -6-phosphate phosphatase (EC 5.3.1.-). At the end of the growth step, the expression of one or more heterologous enzymes was induced and then the cell biomass was collected. The harvested biomass was then lysed mechanically, chemically or enzymatically. The cell lysate is then heated to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate one or more heterologous enzymes. To the heat-inactivated lysate, starch-based feedstock, inorganic phosphate, and optionally other additional nutrients were added to convert starch to sorbitol (FIG. 4).

Example 5

Cellular conversion of starch to ribulose

This example describes the conversion of starch to ribulose. Cells (eg, bacterial or yeast cells) engineered to express at least one heterologous gene encoding at least one enzyme for converting starch to ribulose were grown in liquid cultures to high cell density. Examples of heterologous enzymes that could be used in this example include thermostable variants of α-glucan phosphorylase (EC 2.4.1.1), phosphoglucomutase (EC 5.4.2.2, 5.4.2.5 or 5.4.2.6), glucose-6-phosphate dehydrogenase (EC 1.1. 1.49), 6-phosphogluconolactonase (EC 3.1.1.31), 6-phosphogluconate dehydrogenase (EC 1.1.1.44) and ribulose-5-phosphate phosphatase (EC 5.3.1.-) and ribulose-6-phosphate phosphatase (EC 5.3.1. -). At the end of the growth step, the expression of one or more heterologous enzymes was induced and then the cell biomass was collected. The harvested biomass was then lysed mechanically, chemically or enzymatically. The cell lysate is then heated to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate one or more heterologous enzymes. To the heat-inactivated lysate, starch-based feedstock, inorganic phosphate, and optionally other additional nutrients were added to convert starch to ribulose (FIG. 5).

Example 6

Cell-free conversion of starch to ribose

This example describes the conversion of starch to ribose. Cells (eg, bacterial or yeast cells) engineered to express at least one heterologous gene encoding at least one enzyme for converting starch to ribose were grown in liquid cultures to high cell density. Examples of heterologous enzymes that could be used in this example include thermostable variants of α-glucan phosphorylase (EC 2.4.1.1), phosphoglucomutase (EC 5.4.2.2, 5.4.2.5 or 5.4.2.6), glucose-6-phosphate dehydrogenase (EC 1.1. 1.49), 6-phosphogluconolactonase (EC 3.1.1.31), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), ribose-5-phosphate isomerase (EC 5.3.1.6) and ribose-5-phosphate phosphatase (EC 5.3.1.-). At the end of the growth step, the expression of one or more heterologous enzymes was induced and then the cell biomass was collected. The harvested biomass was then lysed mechanically, chemically or enzymatically. The cell lysate is then heated to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate one or more heterologous enzymes. To the heat-inactivated lysate, starch-based feedstock, inorganic phosphate, and optionally other additional nutrients were added to convert starch to ribose (FIG. 6).

Example 7

Cell-free conversion of starch to arabinose

This example describes the conversion of starch to arabinose. Cells (eg, bacterial or yeast cells) engineered to express at least one heterologous gene encoding at least one enzyme for converting starch to arabinose were grown in liquid cultures to high cell density. Examples of heterologous enzymes that could be used in this example include thermostable variants of α-glucan phosphorylase (EC 2.4.1.1), phosphoglucomutase (EC 5.4.2.2, 5.4.2.5 or 5.4.2.6), glucose-6-phosphate dehydrogenase (EC 1.1. 1.49), 6-phosphogluconolactonase (EC 3.1.1.31), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), arabinose-5-phosphate isomerase (EC 5.3.1.6) and arabinose-5-phosphate phosphatase (EC 5.3.1.-). At the end of the growth step, the expression of one or more heterologous enzymes was induced and then the cell biomass was collected. The harvested biomass was then lysed mechanically, chemically or enzymatically. The cell lysate is then heated to a temperature that inactivates natural molecules with enzymatic activity but does not inactivate one or more heterologous enzymes. To the heat-inactivated lysate, starch-based feedstock, inorganic phosphate, and optionally other additional nutrients were added to convert starch to arabinose (FIG. 7).

Example 8

Cell-free conversion of cellulose/cellodextrin to allulose

This example describes the conversion of cellulose/cellodextrin to allulose. Cells (eg, bacterial or yeast cells) engineered to express at least one heterologous gene encoding at least one enzyme for converting cellulose/cellodextrin to allulose were grown in liquid cultures to high cell density. Examples of heterologous enzymes that could be used in this example include thermostable variants of cellodextrin phosphorylase (EC 2.4.1.49), phosphoglucomutase (EC 5.4.2.2, 5.4.2.5 or 5.4.2.6), phosphoglucoisomerase (EC 5.3.1.9), allulose-6 -phosphate epimerase (EC 5.3.1.-) and allulose-6-phosphate phosphatase (EC 5.3.1.-). At the end of the growth step, the expression of one or more heterologous enzymes was induced and then the cell biomass was collected. The harvested biomass was then lysed mechanically, chemically or enzymatically. The cell lysate is then heated to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate one or more heterologous enzymes. Cellulose/cellodextrin feedstock, inorganic phosphate and optionally other additional nutrients were added to the heat inactivated lysate to convert cellulose/cellodextrin to allulose.

Example 9

Cell-free conversion of cellulose/cellodextrin to glucose

This example describes the conversion of cellulose/cellodextrin to glucose. Cells (eg, bacterial or yeast cells) engineered to express at least one heterologous gene encoding at least one enzyme for converting cellulose/cellodextrin to glucose were grown in liquid cultures to high cell density. Examples of heterologous enzymes that could be used in this example include thermostable variants of cellodextrin phosphorylase (EC 2.4.1.49), phosphoglucomutase (EC 5.4.2.2, 5.4.2.5 or 5.4.2.6), and glucose-6-phosphate phosphatase (EC 5.3.1. -). At the end of the growth step, the expression of one or more heterologous enzymes was induced and then the cell biomass was collected. The harvested biomass was then lysed mechanically, chemically or enzymatically. The cell lysate is then heated to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate one or more heterologous enzymes. Cellulose/cellodextrin feedstock, inorganic phosphate, and optionally other additional nutrients were added to the heat-inactivated lysate to allow the cellulose/cellodextrin to be converted to glucose.

This example also describes another route for converting cellulose/cellodextrin to glucose. Cells (eg, bacterial or yeast cells) engineered to express at least one heterologous gene encoding at least one enzyme for converting cellulose/cellodextrin to glucose were grown in liquid cultures to high cell density. Examples of heterologous enzymes that could be used in this example include thermostable variants of cellodextrinphosphorylase (EC 2.4.1.49) and glucose-1-phosphate phosphatase (EC 3.1.3.10). At the end of the growth step, the expression of one or more heterologous enzymes was induced and then the cell biomass was collected. The harvested biomass was then lysed mechanically, chemically or enzymatically. The cell lysate is then heated to a temperature that inactivates natural molecules with enzymatic activity, but does not inactivate one or more heterologous enzymes. Cellulose/cellodextrin feedstock, inorganic phosphate and optionally other additional nutrients were added to the heat inactivated lysate to allow the cellulose/cellodextrin to be converted to glucose.

Example 10

Cell-free conversion of cellulose/cellodextrin to fructose

This example describes the conversion of cellulose/cellodextrin to fructose. Cells (eg bacterial or yeast cells) engineered to express at least one heterologous gene encoding at least one enzyme for converting cellulose/cellodextrin to fructose were grown in liquid cultures to high cell density. Examples of heterologous enzymes that could be used in this example include thermostable variants of cellodextrin phosphorylase (EC 2.4.1.49), phosphoglucomutase (EC 5.4.2.2, 5.4.2.5 or 5.4.2.6), phosphoglucoisomerase (EC 5.3.1.9), and fructose-6 -phosphate phosphatase (EC 5.3.1.-). At the end of the growth step, the expression of one or more heterologous enzymes was induced and then the cell biomass was collected. The harvested biomass was then lysed mechanically, chemically or enzymatically. The cell lysate is then heated to a temperature that inactivates natural molecules with enzymatic activity but does not inactivate one or more heterologous enzymes. Cellulose/cellodextrin feedstock, inorganic phosphate and optionally other additional nutrients were added to the heat-inactivated lysate to convert the cellulose/cellodextrin to fructose.

Example 11

Cell-free conversion of cellulose/cellodextrin to sorbitol

This example describes the conversion of cellulose/cellodextrin to sorbitol. Cells (eg, bacterial or yeast cells) engineered to express at least one heterologous gene encoding at least one enzyme for converting cellulose/cellodextrin to sorbitol were grown in liquid cultures to high cell density. Examples of heterologous enzymes that could be used in this example include thermostable variants of cellodextrin phosphorylase (EC 2.4.1.49), phosphoglucomutase (EC 5.4.2.2, 5.4.2.5 or 5.4.2.6), aldose dehydrogenase (EC 1.1.1.200), and sorbitol-6 -phosphate phosphatase (EC 5.3.1.-). At the end of the growth step, the expression of one or more heterologous enzymes was induced and then the cell biomass was collected. The harvested biomass was then lysed mechanically, chemically or enzymatically. The cell lysate is then heated to a temperature that inactivates natural molecules with enzymatic activity but does not inactivate one or more heterologous enzymes. Cellulose/cellodextrin feedstock, inorganic phosphate and optionally other additional nutrients were added to the heat inactivated lysate to convert the cellulose/cellodextrin to sorbitol.

Example 12

Cell-free conversion of cellulose/cellodextrin to ribulose

This example describes the conversion of cellulose/cellodextrin to ribulose. Cells (eg, bacterial or yeast cells) engineered to express at least one heterologous gene encoding at least one enzyme for converting cellulose/cellodextrin to ribulose were grown in liquid cultures to high cell density. Examples of heterologous enzymes that could be used in this example include thermostable variants of cellodextrin phosphorylase (EC 2.4.1.49), phosphoglucomutase (EC 5.4.2.2, 5.4.2.5 or 5.4.2.6), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) . . At the end of the growth step, the expression of one or more heterologous enzymes was induced and then the cell biomass was collected. The harvested biomass was then lysed mechanically, chemically or enzymatically. The cell lysate is then heated to a temperature that inactivates natural molecules with enzymatic activity but does not inactivate one or more heterologous enzymes. Cellulose/cellodextrin feedstock, inorganic phosphate and optionally other additional nutrients were added to the heat inactivated lysate to convert cellulose/cellodextrin to ribulose.

Example 13

Cell-free conversion of cellulose/cellodextrin to ribose

This example describes the conversion of cellulose/cellodextrin to ribose. Cells (eg, bacterial or yeast cells) engineered to express at least one heterologous gene encoding at least one enzyme for converting cellulose/cellodextrin to ribose were grown in liquid cultures to high cell density. Examples of heterologous enzymes that could be used in this example include thermostable variants of cellodextrin phosphorylase (EC 2.4.1.49), phosphoglucomutase (EC 5.4.2.2, 5.4.2.5 or 5.4.2.6), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) , 6-phosphogluconolactonase (EC 3.1.1.31), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), ribose-5-phosphate isomerase (EC 5.3.1.6) and ribose-5-phosphate phosphatase (EC 5.3.1.-). At the end of the growth step, the expression of one or more heterologous enzymes was induced and then the cell biomass was collected. The harvested biomass was then lysed mechanically, chemically or enzymatically. The cell lysate is then heated to a temperature that inactivates natural molecules with enzymatic activity but does not inactivate one or more heterologous enzymes. Cellulose/cellodextrin feedstock, inorganic phosphate and optionally other additional nutrients were added to the heat-inactivated lysate to convert the cellulose/cellodextrin to ribose.

Example 14

Cell-free conversion of cellulose/cellodextrin to arabinose

This example describes the conversion of cellulose/cellodextrin to arabinose. Cells (eg, bacterial or yeast cells) engineered to express at least one heterologous gene encoding at least one enzyme for converting cellulose/cellodextrin to arabinose were grown in liquid cultures to high cell density. Examples of heterologous enzymes that could be used in this example include thermostable variants of cellodextrin phosphorylase (EC 2.4.1.49), phosphoglucomutase (EC 5.4.2.2, 5.4.2.5 or 5.4.2.6), glucose-6-phosphate dehydrogenase (EC 1.1.1.49) , 6-phosphogluconolactonase (EC 3.1.1.31), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), arabinose-5-phosphate isomerase (EC 5.3.1.6) and arabinose-5-phosphate phosphatase (EC 5.3.1.-). At the end of the growth step, the expression of one or more heterologous enzymes was induced and then the cell biomass was collected. The harvested biomass was then lysed mechanically, chemically or enzymatically. The cell lysate is then heated to a temperature that inactivates natural molecules with enzymatic activity but does not inactivate one or more heterologous enzymes. Cellulose/cellodextrin feedstock, inorganic phosphate, and optionally other additional nutrients were added to the heat-inactivated lysate to convert the cellulose/cellodextrin to arabinose.

All references, patents and patent applications disclosed in this document are incorporated by reference with respect to the subject matter for which each is mentioned, which in some cases may cover the entire document.

Unless expressly stated otherwise, the singular forms used in the context of the description and claims are to be understood as meaning "at least one".

It should also be understood that, unless expressly stated to the contrary, in any methods claimed herein that include more than one step or more than one action, the order of the steps or steps of such a method is not necessarily limited to the order in which the steps or steps of such a method are indicated.

In the claims, as well as in the above description, all transitional phrases such as "containing", "comprising", "carrying", "having", "comprising", "encompassing", "providing", "having in its composition" and the like should be understood as open, that is, meaning including without limitation. And only the transition phrases "consisting of" and "actually consisting of" should be considered as closed or semi-closed transition phrases, respectively, as specified in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. 2111.03).

--->

SEQUENCE LIST

<110> GREENLIGHT BIOSIENCES, INC.

<120> CELL-FREE SUGAR PRODUCTION

<130> G0830.70022WO00

<140> PCT/US2018/012516

<141> 2018-01-05

<150> US 62/443,447

<151> 2017-01-06

<150> US 62/538,181

<151> 2017-07-28

<160> 10

<170>PatentIn version 3.5

<210> 1

<211> 26

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 1

Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr

1 5 10 15

Thr Met Met Phe Ser Ala Ser Ala Leu Ala

20 25

<210> 2

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 2

Met Lys Gln Ser Thr Ile Ala Leu Ala Leu Leu Pro Leu Leu Phe Thr

1 5 10 15

Pro Val Thr Lys Ala

twenty

<210> 3

<211> 25

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 3

Met Met Ile Thr Leu Arg Lys Leu Pro Leu Ala Val Ala Val Ala Ala

1 5 10 15

Gly Val Met Ser Ala Gln Ala Met Ala

20 25

<210> 4

<211> 23

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 4

Met Asn Lys Lys Val Leu Thr Leu Ser Ala Val Met Ala Ser Met Leu

1 5 10 15

Phe Gly Ala Ala Ala His Ala

twenty

<210> 5

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 5

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala

1 5 10 15

Ala Gln Pro Ala Met Ala

twenty

<210> 6

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 6

Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser

1 5 10 15

Ala Ser Ala

<210> 7

<211> 23

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 7

Met Met Thr Lys Ile Lys Leu Leu Met Leu Ile Ile Phe Tyr Leu Ile

1 5 10 15

Ile Ser Ala Ser Ala His Ala

twenty

<210> 8

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 8

Met Lys Gln Ala Leu Arg Val Ala Phe Gly Phe Leu Ile Leu Trp Ala

1 5 10 15

Ser Val Leu His Ala

twenty

<210> 9

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 9

Met Arg Val Leu Leu Phe Leu Leu Leu Ser Leu Phe Met Leu Pro Ala

1 5 10 15

Phe Ser

<210> 10

<211> 39

<212> PRT

<213> Artificial sequence

<220>

<223> Synthetic polypeptide

<400> 10

Met Ala Asn Asn Asp Leu Phe Gln Ala Ser Arg Arg Arg Phe Leu Ala

1 5 10 15

Gln Leu Gly Gly Leu Thr Val Ala Gly Met Leu Gly Pro Ser Leu Leu

20 25 30

Thr Pro Arg Arg Ala Thr Ala

35

<---

Claims (18)

1. A method for producing allulose, comprising the following steps: converting polymeric glucose carbohydrate to glucose-1-phosphate (G1P) using α-glucan or cellodextrinphosphorylase; conversion of G1P to glucose-6-phosphate (G6P) using phosphoglucomutase; conversion of G6P to fructose-6-phosphate (F6P) using phosphoglucoisomerase; conversion of F6P to allulose-6-phosphate (A6P), using allulose-6-phosphate epimerase (A6PE); and converting A6P to allulose using allulose-6-phosphate phosphatase (A6PP); where A6PP is A6PP Ruminiclostridium thermocellum. 2. A method for producing allulose, comprising the following steps: converting polymeric glucose carbohydrate to glucose-1-phosphate (G1P) using α-glucan or cellodextrinphosphorylase; converting G1P to produce glucose-6-phosphate (G6P) using phosphoglucomutase; conversion of G6P to fructose-6-phosphate (F6P) using phosphoglucoisomerase; conversion of F6P to give allulose-6-phosphate (A6P) using allulose-6-phosphate epimerase (A6PE); and converting A6P to allulose using allulose-6-phosphate phosphatase (A6PP); where A6PE is and A6PE Brevibacillus thermoruber. 3. The method according to claim 1 or 2, wherein at least one of the enzymes is thermostable, or at least two of the enzymes are thermostable. 4. The method according to any one of paragraphs. 1-3 wherein A6PE is, A6PP is, or both A6PE and A6PP are thermally stable or are designed to be thermally stable. 5. The method according to any one of paragraphs. 1-4, wherein the polymeric glucose carbohydrate is selected from starch, cellodextrin, maltodextrins and glycogen. 6. The method according to any one of paragraphs. 1-4 further comprising treating the polymeric glucose carbohydrate with a debranching enzyme; and wherein the polymeric glucose carbohydrate is selected from glycogen, starch and hydrolysed maltodextrins. 7. The method of claim 6 wherein the debranching enzyme is selected from (i) isoamylases optionally selected from Sulfolobus tokodaii, Metallosphaera hakonensis, Sphaerobacter thermophiles and Bacillus lentus isoamylases, and (ii) pullulanases optionally selected from Fervidobacterium pennavorans pullulanases. Thermotoga sp. RQ5, Bacillus flavocaldarius, Thermosipho africanus and Kosmotoga olearia. 8. The method according to any one of paragraphs. 1 and 3-7, in which A6PE is selected from the group consisting of allulose-6-phosphate epimerases of Thermobacterium thermosaccharolyticum, Thermoanaerobacter brockii, Caldanaerobacter subterraneus, Deferribacter desulfuricans, Thermocrinis ruber, Hydrogenivirga sp. 128-5-R1-1, Brevibacillus thermoruber, Thermosipho atlanticus and Thermosulfidibacter takaii. 9. The method according to claim 8, wherein A6PE is selected from the group consisting of allulose-6-phosphate epimerases from Thermobacterium thermosaccharolyticum, Hydrogenivirga sp. 128-5-R1-1, and Brevibacillus thermoruber. 10. The method according to any one of paragraphs. 1-9, wherein the α-glucan phosphorylase is selected from the group consisting of α-glucan phosphorylases from Aquifex aeolicus, Thermocrinis minervae, Thermosulfidibacter takaii, Thermosulfurimonas dismutans, Thermococcus litoralis, Palaeococcus pacificus, Thermotoga neapolitana, Ruminiclostridium thermocellum, Pyrococcus abyssi, Thermococcus thioreducens, Deinococcus radiodurans, Sulfolobus acidocaldarius, Thermus caldophilus, Meiothermus silvanus, Oceanithermus profundus, Ardenticatena maritima, Thermococcus barophilus, Pseudothermotoga thermarum, Hydrogenobacter thermophilus , Thermus oshimai, Meiothermus ruber and Marinitoga piezophila. 11. The method according to any one of paragraphs. 1-10, wherein the cellodextrinphosphorylase is selected from the group consisting of Clostridium thermocellum, Clostridium straminisolvens, Thermotoga RQ2 cellodextrinphosphorylases; Ignisphaera aggregans, Thermotoga maritima, Spirochaeta thermophila, Caldicellulosiruptor bescii, Dictyoglomus thermophilum, Thermoanaerobacterium thermosaccharolyticum, Thermosipho africanus, Caldisalinibacter kiritimatiensis, Defluviitalea phaphyphila, Caldicellulosiruptor kronotskyensis, Thermococcus sibiricus and Thermosphaeragregans. 12. The method according to any one of paragraphs. 1-11, при котором фосфоглюкомутаза выбрана из группы, состоящей из фосфоглюкомутаз Thermococcus kodakaraensis, Pyrococcus kukulkanii, Ammonifex degensii, Methanothermobacter wolfeii, Methanothermus fervidus, Sulfolobus acidocaldarius, Archaeoglobus fulgidus, Ferroglobus placidus, Geoglobus ahangari, Archaeoglobus veneficus, Archaeoglobus sulfaticallidus, Aciduliprofundum boonie, Clostridium thermocellum, Defluviitalea phaphyphila, Caminicella sporogenes, Caloranaerobacter ferrireducens, Thermosipho malanesiensis, Fervidobacterium pennivorans, Symbiobacterium thermophilum, Spirochaeta thermophila and Thermoanaerobacter wiegelii. 13. The method according to any one of paragraphs. 1-12, in which the phosphoglucoisomerase is selected from the group consisting of phosphoglucoisomerases Thermus thermophilus, Meiothermus timidus, Thermus filiformis, Marinithermus hydrothermalis, Thermosipho africanus, Sulfurihydrogenibium azorense, Persephonella marina, Marinitoga piezophila, Kosmotoga olearia, Thermotoga maritima, Geobacillus stearoxybacillus, Anviothermoma Thermosulfidibacter takaii, Fervidobacterium nodosum, Clostridium thermocellum, Thermoanaerobacterium thermosaccharolyticum, Methanococcus jannaschii, Methanotorris igneus, Methanocaldococcus villosus, Methanothermococcus okinawensis, Pseudothermotoga thermarum, Deferribacter desulfuricans, and Thermovibrio ammonificans. 14. The method according to any one of paragraphs. 2-7 and 9-13, wherein A6PP is selected from the group consisting of allulose-6-phosphate phosphatases Thermoanaerobacter wiegelii, Thermoanaerobacter ethanolicus, Thermus islandicus, Deinococcus geothermalis DSM 11300, Thermosphaera aggregans, Crenarchaeota archaeon, Pyrococcus horikoshii Ot3, Aquifex aeolicus, Ruminiclostridium thermocellum, Desulfotomaculum kuznetsovii, Caldanaerobacter subterraneus, Acidothermus cellulolyticus, Methanothermobacter thermautotrophicus, Thermobifida fusca, Thermotoga neapolitana, Petrotoga mobilis and Thermodesulfatator indicus, Thermus thermophilus, Bacteroides vulgatus and Bacteroides fragilus. 15. The method according to any one of paragraphs. 1-14, in which allulose-6-phosphate phosphatase is specific for allulose-6-phosphate.

Images