Microorganisms for producing low calorie sugars

Abstract

The present disclosure relates to microorganisms for psicose biosynthesis. Methods of producing the disclosed microorganisms and methods of producing psicose are also provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 405,208, filed September 9, 2022, and U.S. Provisional Patent Application Serial No. 63 / 450,582, filed March 7, 2023, the contents of each of which are incorporated herein by reference in their entireties and claim the benefit of their respective priority.

[0003] Sequence Listing

[0004] This application contains a sequence listing submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy was created on September 5, 2023, is named 081906-1401075-232710PC_SL, and is 284,563 bytes in size. Technical Field

[0005] The subject matter disclosed herein relates to compositions and methods for producing low-calorie sugars in microorganisms. Background Art

[0006] Currently, industrial production of D-psicose (allulose) is carried out through a two-step in vitro enzymatic synthesis, firstly by xylose isomerase (EC 5.3.1.5) to convert glucose into fructose. The ΔG of this reaction is o The isomerization reaction is then epimerized to psicose by D-tagatose-3-epimerase (EC 5.1.3.31) or D-psicose-3-epimerase (EC 5.1.3.30). The main disadvantage of in vitro synthesis is the predicted ΔG of the reaction. o The reaction is thermodynamically unfavorable, with a maximum reaction pressure of +5 kJ / mol. Since both reactions are reversible, this in vitro system ultimately produces a mixture of glucose, fructose, and psicose, which increases the cost of downstream separation and purification processes. Summary of the Invention

[0007] In one aspect, the present disclosure relates to a recombinant microorganism comprising an exogenous epimerase and an exogenous phosphatase, wherein the recombinant microorganism produces an increased amount of psicose compared to a naturally-occurring microorganism.

[0008] In some embodiments, the epimerase is psicose-6-phosphate 3-epimerase (AlsE). In some embodiments, the epimerase is Escherichia coli AlsE. In some embodiments, the epimerase comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the epimerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the epimerase consists of the amino acid sequence of SEQ ID NO: 1.

[0009] In some embodiments, the phosphatase is hexitol phosphatase B (HxpB). In some embodiments, the phosphatase is Escherichia coli HxpB. In some embodiments, the phosphatase comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the phosphatase comprises the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the phosphatase consists of the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

[0010] In some embodiments, the recombinant microorganism further comprises exogenous galactose:H + Symporter (Galp) and glucokinase (Glk). In some embodiments, the GalP is E. coli GalP and the Glk is E. coli Glk. In some embodiments, the GalP comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 38, and the Glk comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, the GalP comprises the amino acid sequence set forth in SEQ ID NO: 38, and the Glk comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0011] In some embodiments, compared with the naturally occurring microorganism, the recombinant microorganism also comprises a sudden change in the gene of an enzyme of the pentose phosphate pathway of encoding. In some embodiments, the enzyme of the pentose phosphate pathway is glucose-6-phosphate 1-dehydrogenase (Zwf). In some embodiments, compared with the naturally occurring microorganism, the recombinant microorganism also comprises a sudden change in the gene of an enzyme of the glycolytic enzyme of encoding.

[0012] In some embodiments, the glycolytic enzyme is phosphofructokinase-1 (PfkA), phosphofructokinase-2 (PfkB) or pyruvate kinase (PykF). In some embodiments, the glycolytic enzyme is phosphofructokinase-1 (PfkA).

[0013] In some embodiments, the recombinant microorganism further comprises a mutation in a gene encoding an enzyme of the allose degradation pathway. In some embodiments, the enzyme of the allose degradation pathway is allose-6-phosphate isomerase (RpiB). In some embodiments, the recombinant microorganism further comprises a mutation in a gene encoding an enzyme of the mannose biosynthetic pathway. In some embodiments, the enzyme of the mannose biosynthetic pathway is mannose-6-phosphate isomerase (ManA).

[0014] In some embodiments, the microorganism further comprises an exogenous nuclease and an sgRNA. In some embodiments, the nuclease is dCas9. In some embodiments, the sgRNA targets a gene encoding a glycolytic enzyme. In some embodiments, the glycolytic enzyme is phosphofructokinase-2 (PfkB). In some embodiments, the exogenous epimerase and exogenous phosphatase are expressed from a stationary promoter. In some embodiments, the exogenous nuclease is expressed from an inducible promoter.

[0015] In some embodiments, the microorganism further comprises a mutation in a gene encoding an enzyme of glycogen biosynthesis selected from the group consisting of phosphoglucomutase (Pgm), UDP-glucose pyrophosphorylase, glycogen synthase, glycogen branching enzyme, and glycogenin. In some embodiments, the enzyme of glycogen biosynthesis is phosphoglucomutase (Pgm).

[0016] In another aspect, the present disclosure relates to a microorganism comprising recombinant polynucleotides encoding an epimerase and a phosphatase, wherein expression of the epimerase and the phosphatase results in increased production of psicose compared to a microorganism lacking the recombinant polynucleotides.

[0017] In some embodiments, the epimerase is psicose-6-phosphate 3-epimerase (AlsE). In some embodiments, the epimerase is Escherichia coli AlsE. In some embodiments, the epimerase comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the epimerase comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the epimerase consists of the amino acid sequence of SEQ ID NO: 1.

[0018] In some embodiments, the phosphatase is hexitol phosphatase B (HxpB). In some embodiments, the phosphatase is Escherichia coli HxpB. In some embodiments, the phosphatase comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the phosphatase comprises the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the phosphatase consists of the amino acid sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

[0019] In some embodiments, the microorganism also comprises a mutation in a gene encoding an enzyme of the pentose phosphate pathway. In some embodiments, the enzyme of the pentose phosphate pathway is glucose-6-phosphate 1-dehydrogenase (Zwf). In some embodiments, the microorganism also comprises a mutation in a gene encoding an enzyme of glycolysis. In some embodiments, the enzyme of glycolysis is phosphofructokinase-1 (PfkA), phosphofructokinase-2 (PfkB) or pyruvate kinase (PykF). In some embodiments, the enzyme of glycolysis is phosphofructokinase-1 (PfkA).

[0020] In some embodiments, the microorganism further comprises a mutation in a gene encoding an enzyme of an allose degradation pathway. In some embodiments, the enzyme of the allose degradation pathway is allose-6-phosphate isomerase (RpiB). In some embodiments, the microorganism further comprises a mutation in a gene encoding an enzyme of a mannose biosynthetic pathway. In some embodiments, the enzyme of the mannose biosynthetic pathway is mannose-6-phosphate isomerase (ManA).

[0021] In some embodiments, the microorganism further comprises an exogenous nuclease and an sgRNA. In some embodiments, the nuclease is dCas9. In some embodiments, the sgRNA targets a gene encoding a glycolytic enzyme. In some embodiments, the glycolytic enzyme is phosphofructokinase-2 (PfkB). In some embodiments, the exogenous epimerase and exogenous phosphatase are expressed from a stationary promoter. In some embodiments, the exogenous nuclease is expressed from an inducible promoter.

[0022] In some embodiments, the microorganism further comprises a mutation in a gene encoding an enzyme of glycogen biosynthesis selected from the group consisting of phosphoglucomutase (Pgm), UDP-glucose pyrophosphorylase, glycogen synthase, glycogen branching enzyme, and glycogenin. In some embodiments, the enzyme of glycogen biosynthesis is phosphoglucomutase (Pgm).

[0023] In one aspect, the present disclosure relates to a microorganism comprising recombinant polynucleotides encoding an epimerase and a phosphatase; mutations in genes encoding enzymes of the pentose phosphate pathway; mutations in genes encoding enzymes of glycolysis; mutations in genes encoding enzymes of the allose degradation pathway; and mutations in genes encoding enzymes of the mannose biosynthetic pathway; and, optionally, recombinant polynucleotides encoding GalP, Glk, or both.

[0024] In other aspects, the present disclosure relates to a microorganism comprising recombinant polynucleotides encoding psicose-6-phosphate 3-epimerase (AlsE) and hexitol phosphatase B (HxpB); a mutation of glucose-6-phosphate-1-dehydrogenase (Zwf); a mutation of phosphofructokinase-1 (PfkA); a mutation of allose-6-phosphate isomerase (RpiB); a mutation of mannose-6-phosphate isomerase (ManA); and, optionally, a recombinant polynucleotide encoding GalP, Glk, or both.

[0025] In addition, in one aspect, the present disclosure relates to a microorganism comprising a recombinant polynucleotide encoding psicose-6-phosphate-3-epimerase (AlsE); a recombinant polynucleotide encoding hexitol phosphatase B (HxpB); a mutation of glucose-6-phosphate-1-dehydrogenase (Zwf); a mutation of phosphofructokinase-1 (PfkA); a mutation of allose-6-phosphate isomerase (RpiB); a mutation of mannose-6-phosphate isomerase (ManA); and, optionally, a recombinant polynucleotide encoding GalP, Glk, or both.

[0026] In some embodiments, the recombinant polynucleotide is stably integrated into the genome. In some embodiments, the microorganism comprises an increased intracellular fructose-6-phosphate content compared to a naturally occurring microorganism. In some embodiments, the microorganism is Escherichia coli, Bacillus subtilis, or Lactococcus lactis.

[0027] In some embodiments, the mutation is a deletion. In some embodiments, the mutation reduces or eliminates the expression or activity of an enzyme.

[0028] In one aspect, the present disclosure relates to a method for producing psicose, comprising culturing the microorganism disclosed herein under conditions suitable for converting a substrate into psicose. In some embodiments, the substrate comprises glucose. In some embodiments, the psicose has a purity of at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%. In some embodiments, the purity is 100%. In some embodiments, the purity is determined by the following formula:

[0029]

[0030] Additionally, the present disclosure relates to psicose produced by a method comprising culturing a microorganism disclosed herein. In some embodiments, the psicose has a purity of at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%. In some embodiments, the purity is 100%. In some embodiments, the purity is determined by a formula disclosed herein.

[0031] Furthermore, the present disclosure relates to a method for producing a food product containing psicose, the method comprising culturing the microorganism disclosed herein under conditions suitable for converting a substrate into psicose; purifying the psicose; and combining the psicose with a food product to form the food product containing psicose. In some embodiments, the food product is chewing gum, candy, chocolate, or a savory food product. In some embodiments, the food product is a beverage, yogurt, ice cream, baked goods, or a nutritional bar. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This demonstrates the ability of E. coli to produce psicose. AL3601 is a product of MG1655 plus the Z1 fragment (lacI q tetR spec R ) and T7 RNA polymerase gene (P lacUV5 : T7 RNAP) (see Table 1). When grown at 30°C in M9P medium (M9 minimal medium containing 5 g / L yeast extract) containing 10 g / L glucose, AL3601 was not observed to produce psicose. However, after pfkA deletion, 0.15 g / L psicose was produced.

[0033] Figure 2 The biosynthetic pathway of psicose is described. Glucose is input and phosphorylated to glucose-6-phosphate (G6P) by a phosphotransferase system (PTS) or GalP / Glk. G6P is then isomerized to fructose-6-phosphate (F6P) via glucose-6-phosphate isomerase. F6P is epimerized to psicose-6-phosphate via D-psicose-6-phosphate 3-epimerase (AlsE), and then dephosphorylated to free psicose via hexitol phosphatase B (HxpB). Ultimately, free psicose can diffuse across the cell membrane into the supernatant. Competing pathways include the pentose phosphate pathway catalyzed by glucose-6-phosphate dehydrogenase (Zwf), glycolysis catalyzed by phosphofructokinase A and B (PfkA and B), the allose degradation pathway catalyzed by allose-6-phosphate isomerase (RpiB), and the mannose biosynthesis pathway catalyzed by mannose-6-phosphate isomerase (ManA).

[0034] Figure 3 A comparison of the production of psicose using the phosphatase HxpB and YbiV is shown. E. coli encodes the phosphatase HxpB and YbiV. Two plasmids, pAL1946 and pAL1947 (Table 2), were constructed and expressed under the inducible promoter P T7 AL3601 carrying either pAL1946 or pAL1947 was overexpressed in M9P medium containing 10 g / L glucose at 30°C and induced with 1 mM IPTG. Cultures containing pAL1946 produced 1.0 g / L psicose after 24 hours, while cultures containing pAL1947 produced 0.4 g / L. Cultures induced with IPTG reached lower densities than uninduced cultures.

[0035] Figure 4 The psicose production in AL3601 and triple knockout (TKO) strains is shown. AL3729 was generated by deleting the genes pfkA, zwf, and rpiB in AL3601 (Table 1). T7 Plasmid pAL1946, carrying the alsE-hxpB gene, was introduced into AL3601 and AL3729. Cultures were grown in M9P medium supplemented with 10 g / L glucose at 30°C and induced with 25 mM IPTG. The uninduced TKO strain produced 1.5 g / L psicose after 24 hours, while the induced TKO strain produced 0.6 g / L.

[0036] Figure 5 The promoter P LlacO1 and P T7 Comparison of allulose production. Genes alsE and hxpB in P T7 or P LlacO1 AL3601 (Table 1) was introduced with P T7 : alsE-hxpB pAL1946 (Table 2), while strain AL1050 was introduced with P LlacO1 : pAL2001 of alsE-hxpB (Table 2). Cultures were grown at 30°C in M9P medium supplemented with 10 g / L glucose and induced with 1 mM IPTG. LlacO1 The culture of P produced 0.5 g / L psicose after 24 h, while the culture of T7 The culture produced 0.5 g / L.

[0037] Figure 6 The promoter P in the TKO strain is shown. LlacO1 and P T7 AL3729 (AL3601+TKO, Table 1) was introduced with P T7: alsE-hxpB pAL1946 (Table 2), AL3756 (AL1050+TKO, Table 1) containing P LlacO1 : pAL2001 of alsE-hxpB (Table 2). Cultures were grown in M9P medium supplemented with 10 g / L glucose at 30°C and induced with 1 mM IPTG for 24 hours. T7 The uninduced TKO strain produced 1.8 g / L psicose, while the induced strain produced 0.6 g / L. LlacO1 The uninduced TKO strain produced 0.6 g / L psicose, while the induced strain produced 1.4 g / L.

[0038] Figure 7 GC / MS analysis for identifying by-products is shown. Gas chromatography-mass spectrometry (GC-MS) analysis was used to identify by-products in the production of psicose. The analysis determined that the by-product was mannose. The left side of the upper figure shows the GC elution peak (green) of the by-product compared to a mannose standard (brown). Both the by-product and the mannose standard eluted approximately 667.5-668.0 seconds after injection. The image on the right shows the mass spectrometric analysis of the by-product peak (upper figure) at 667.567 seconds compared to the mass spectrum of mannose (lower figure).

[0039] Figure 8 The identification and elimination of mannose byproducts are shown. To reduce mannose production, manA was deleted from AL3756 to generate the quadruple knockout (QKO) strain AL3990 (Table 1). The manA gene encodes the enzyme mannose-6-phosphate isomerase (ManA), which catalyzes the reversible isomerization of mannose-6-phosphate and F6P. LlacO1 The alsE-hxpB production plasmid was introduced into the QKO and TKO strains. Cultures were grown in M9P medium supplemented with 10 g / L glucose at 30°C and induced with 1 mM IPTG. After 24 hours, the QKO strain produced 3.5 g / L psicose and 0.7 g / L mannose, while the TKO strain produced 2.3 g / L psicose and 2.5 g / L mannose. D indicates gene deletion.

[0040] Figure 9 The psicose production in TKO and QKO strains is shown. The psicose production in AL3756 (ΔpfkAΔzwfΔrpiB) and AL3990 (ΔpfkAΔzwfΔrpiBΔmanA) strains (Table 1) was compared. LlacO1alsE-hxpB was transformed with pAL2001. The culture was grown in M9P medium supplemented with 10 g / L glucose at 30°C and induced with 1 mM IPTG. After 24 hours, the induced QKO strain produced 3.5 g / L psicose, a 34% yield. The TKO strain produced 2.3 g / L psicose, a 25% yield.

[0041] Figure 10 The production of psicose from 15 g / L glucose in AL3990 using pAL200 (Tables 1 and 2) is shown. AL3990 (ΔpfkAΔzwfΔrpiBΔmanA) (Table 1) was used to generate psicose from 15 g / L glucose. LlacO1 : pAL2001 transformation of alsE-hxpB. The culture was grown in M9P medium supplemented with 15 g / L glucose at 30°C and induced with 1 mM IPTG. After 24 hours, the induced strain produced 2.3 g / L, a 40% yield.

[0042] Figure 11 Dynamic regulation of carbon flux using high-density CRISPRi is shown. CRISPRi was used to knock down pfkB, the gene responsible for converting F6P to fructose-1,6-bisphosphate in glycolysis in the QKO strain. The gRNA was designed to target the promoter region of pfkB. Cells were grown in M9P medium supplemented with 10 g / L glucose at 37°C until the OD 600 The cells were then centrifuged and resuspended in 3.0 mL of M9P medium supplemented with 10 g / L glucose, 1 mM IPTG, and 100 ng / mL aTC. After 24 hours of growth at 30°C, the inducible strain containing the gRNA sequence for the pfkB promoter produced 1.7 g / L of psicose, a 46% yield. Figure 11 (Left panel) shows the produced psicose yield. Figure 11 (Middle panel) shows consumed glucose. Figure 11 (Right panel) shows the change in optical density.

[0043] Figure 12 The use of the stationary active promoter P gadB Inducer-free psicose production was performed. LlacO1 : alsE-hxpB plasmid pAL2001 (Table 2) and containing P gaaB pAL2247 (Table 2) containing alsE-hxpB was introduced into QKO strain AL3990 (Table 1). The cells were grown at 30°C in M9P medium supplemented with 30 g / L glucose and treated with 1 mM IPTG (for P LlacO1 The induced culture containing pAL2001 produced 6.8 g / L psicose, with a yield of 57%, while the culture containing PgadB The culture of alsE-hxpB produced 8.8 g / L psicose, with a yield of 63%. Figure 12 (Right panel) shows the produced psicose. Figure 12 (Middle panel) shows consumed glucose. Figure 12 (Left panel) shows the change in optical density.

[0044] Figure 13 The effect of the sugar symporter GalP on psicose production is shown. The galactose proton symporter GalP can be used to supplement glucose input. Once glucose is transported across the cell membrane by GalP, it can be phosphorylated by the glucokinase Glk and assimilated into central carbon metabolism. LtetO1 : galP-glk plasmid pAL2274 and pAL2001 (P LlacO1 : alsE-hxpB) or pAL2247 (P gadB : alsE-hxpB) were co-introduced into QKO strain AL3990 (Tables 1 and 2). Cultures were grown at 30°C in M9P medium supplemented with 30 g / L glucose and 1 mM IPTG (for P LlacO1 strain) were induced for 24 hours. LtetO1 It was not induced because full induction of galP-glk would impair cell growth. The culture containing pAL2247 and pAL2274 achieved the highest titer and yield, producing 10.7 g / L of psicose, a yield of 61%.

[0045] Figures 14A-14C The biosynthetic strategy of D-psicose is shown. Figure 14A The current industrial method for producing D-psicose is shown. o This results in limited yield (approximately 50%). Figure 14B The proposed D-psicose biosynthesis pathway is shown. Due to the large negative ΔG′ at 1 mM cell reactant concentration m , the dephosphorylation step thermodynamically drives production. Figure 14C Shown is the proposed pathway for D-psicose biosynthesis in Escherichia coli. Deleted steps are indicated in blue. Overexpressed steps are indicated in red. PTS, phosphotransferase system; AlsE, D-psicose 6-phosphate 3-epimerase; HxpB, hexitol phosphatase B.

[0046] Figures 15A-15D The D-psicose production capacity of E. coli was shown. -1 Grow in M9P medium containing glucose at 37°C until OD 600 The OD was about 0.4, and then grown at 30 °C for 24 hours.600 When the temperature is about 0.4, add 1mM IPTG ( Figures 15B-15D ). Figure 15A D-psicose production in MG1655 and AL3601 with and without pfkA and / or alsE deletion is shown (Table 5). Figure 15B Shown is the D-psicose assay of various sugar phosphatases with AlsE in AL3601. Figure 15C The P-terminal positions of the alsE and hxpB operons in AL3601 and AL1050, respectively, are shown. T7 and P LlacO1 The expression of ΔOD was 0.04477 / 0.0847 (Table 5). 600 Indicates 0 hour and 24 hour OD 600 difference. Figure 15D Comparison of the effects of gene deletion on D-psicose production is shown. Error bars represent standard deviation (n=3 biological replicates).

[0047] Figures 16A-16D The enhanced production capacity of D-psicose in Escherichia coli was demonstrated. Figure 16A Cells were grown in M9P medium containing different glucose concentrations to OD 600 About 0.4 (37 ° C), and then grown at 30 ° C for 24 hours. 600 When it is about 0.4. LlacO1 The constructs were supplemented with 1 mM IPTG. The alsE and hxpB operons were expressed in AL3756 and AL3990 (strains 1 and 2, Table 4), respectively. LlacO1 (pAL2001) and P gadB (pAL2247). Figure 16B It was shown that strains 1 and 2 were -1 Grow in M9P medium containing glucose at 37°C until OD 600 is about 0 (no culture at 37°C), about 0.4 or about 1, and then grown at 30°C for 24 hours. When the temperature is changed to 30°C, P LlacO1 constructs were supplemented with 1 mM IPTG. Figure 16C The operons of galP and glk are shown to be in P LlacO1 Strain 4 (AL3990 containing pAL2264 and pAL2247, Table 4) was expressed in the presence of 40 g L -1 Grow in M9P medium containing glucose to an OD 600 The OD was about 1, and then grown at 30 ° C for 24 hours. 600 When the pH was about 1, 1 mM IPTG was added to induce P LlacO1: galP-glk. Specific titer (g - 1 L -1 OD 600 -1 ) indicates relative to the final OD 600 titer. Figure 16D Comparison of the effects of gene deletion on D-psicose production is shown. ptsG, ptsH, and / or pgm were deleted in strain 4. D-psicose production was as shown in FIG. Figure 16C Error bars represent standard deviation (n=3 biological replicates).

[0048] Figure 17A and 17B Dynamic control of glycolysis using CRISPRi is shown. Figure 17A CRISPRi was used to knock down pfkB. sgRNAs targeting the pfkB promoter region or without targeting sequences were expressed from a constitutive promoter. dCas9 was induced by aTc. tet Strains 5, 6, and 7 (Table 4) were expressed in the presence of 40 g L -1 Grow in M9P medium containing glucose at 37°C until OD 600 The pH was about 1, after which 1 mM IPTG and 100 ng / mL aTc were added, and the cells were cultured at 30°C for 24 hours. Figure 17B The strain 7 showed the production of D-psicose at high cell density. -1 Glucose-free M9P medium was grown at 37°C until the OD 600 1, and then 1 mM IPTG and 100 ng mL -1 aTc induction was followed by growth for another 30 minutes. The culture was then centrifuged and resuspended in 40 g L -1 , 1 mM IPTG and 100 ng mL -1 The cells were cultured in M9P medium with aTC until the OD600 reached approximately 8 and grown at 30° C. for 24 hours. Error bars indicate standard deviation (n=3 biological replicates).

[0049] Figures 18A-18C GC-MS identification of the D-mannose by-product is shown. Figure 18A GC elution peaks are shown: by-product (green) and mannose standard (brown). Figure 18B and 18C The mass spectrum is shown: the by-product peak ( Figure 18B ) and mannose standards ( Figure 18C ).

[0050] Figure 19The effect of manA knockout on D-mannose production is shown. To reduce mannose production, manA was knocked out in AL3756 (Table 5) to generate AL3990 (Table 5). LlacO1 : Production of alsE-hxpB Plasmid pAL2001 (Table 6) was introduced into AL3756 and AL3990 to generate strains 1 and 2 (Table 4). Cultures were grown at 30°C in M9P medium supplemented with 10 g / L glucose and induced with 1 mM IPTG. After 24 hours, strain 1 produced 2.5 g L -1 D-mannose, while strain 2 produced 0.7 g L -1 Error bars represent standard deviation (n=3 biological replicates).

[0051] Figures 20A-20C The characteristics of the stationary phase promoter are shown. gadB 、P cbpA2 、P dps and P ihfA4 Fluorescence and OD of strains expressing sfGFP 600 time and activity, and compared with P induced with 1 mM IPTG LlacO1 Error bars represent standard deviation (n=3 biological replicates).

[0052] Figure 21A and 21B Inhibition of fluorescence by CRISPRi is shown. Figure 21A shows that the LlacO1 : Three sgRNAs for sfgfp. The figure discloses SEQ ID NO: 58. Figure 21B The aTc-inducible promoter P tet The dcas9 gene was cloned under the following conditions to generate plasmid pAL1952 (Table 6). Constitutively expressed sgRNAs A (pAL2066), B (pAL2173) and C (pAL2174) or sgRNA without targeting sequence (pAL2063) were each cloned into a plasmid containing P LlacO1 : sfgfp plasmid (Table 6). AL1050 and CRISPRi system were cultured at 30°C with or without the addition of 100 ng mL -1 In the case of aTc, fluorescence was measured at 0 and 4 hours after induction with 1 mM IPTG. Error bars represent standard deviation (n=3 biological replicates).

[0053] Figure 22A and 22BShown is the growth inhibition of CRISPRi. Two different CRISPRi systems were tested in AL4186 for their effects on growth (Table 5). In the first CRISPRi system, dcas9 and sgRNA were located on medium copy (p15A ori) and high copy (ColE ori) plasmids, respectively. In the second CRISPRi system, dcas9 and sgRNA were both located on the same plasmid (p15A ori). Figure 22A The pfkB promoter region was designed to target the middle region of the pfkB promoter. pfkB1 and P pfkB2 The figure discloses SEQ ID NO: 59. Figure 22B Cells were grown at 30°C and OD600 was measured at 0 and 24 hours. ΔOD 600 Indicates 0 hour and 24 hour OD 600 Error bars represent standard deviation (n=3 biological replicates).

[0054] Figures 23A-23C Glucose consumption and allulose production over time are shown. -1 ( Figure 23A ), 5g L -1 ( Figure 23B ) and 10 g L -1 ( Figure 23C The strain 7 (Table 4) was cultured in M9P medium containing 5% (v / v) glucose at 30°C for 10 h, and the D-glucose consumption and D-psicose production of the strain 7 were monitored. Error bars represent standard deviations (n=3 biological replicates).

[0055] Figure 24 High cell density D-psicose production in strain AL4186 was shown. P g adB : alsE-hxpB), pAL2264 (P LlacO1 :galP-glk) and pAL2188 (P tet :dcas9 pTargetF-pfkB) was transformed into strain AL4186 (MG1655ΔpfkAΔrpiBΔmanAΔpgm). The culture was carried out in the presence of 40 g L -1 Glucose-free M9P medium was grown at 37°C until the OD 600 1, and then 1 mM IPTG and 100 ng mL -1 aTc induction was followed by growth for another 30 minutes. The culture was then centrifuged and resuspended in 40 g L -1 , 1 mM IPTG and 100 ng mL -1 aTC in M9P medium to OD600 The culture was approximately 10 and grown at 30°C for 8 hours with samples taken at 0, 4, and 8 hours. During the 8 hours, the culture produced an average of 15.3 g L -1 D-psicose, specific titer 1.4 g L -1 OD 600 -1 , with a yield of 43% and a productivity of 1.9 g L -1 hr -1 Although the titers of D-psicose produced during 0-4 h and 4-8 h were similar, 7.3 and 8.0 g L -1 , but the yield at 4-8 h (53%) was higher than that at 0-4 h (35%). Error bars represent standard deviation (n=3 biological replicates).

[0056] Figure 25 The HPLC chromatogram of a high cell density experiment of 24 hours of culture of strain 7 (see Table 4) is shown. The elution of D-glucose occurs at about 3.36 minutes, and the elution of D-psicose occurs at about 5.45 minutes. Figure 26A and 26B The biochemical characteristics of phosphate are shown. Figure 26A The AlphaFold predicted structure of HxpB is shown, where P6P Located in the active site pocket In , the volumes are calculated using MoloVol and CAVER respectively. Figure 26B ASP173 is shown to interact with the magnesium ion, thereby positioning the phosphate in P6P for nucleophilic attack by ASP15. Residues GLU22, TRP25, LEU52, and SER117 are predicted to form hydrogen bonds with the hydroxyl group (shown as yellow dashed lines) and position P6P for hydrolysis. DETAILED DESCRIPTION

[0057] The market for rare sugars is expanding as foods, dietary supplements, and health products. Among rare sugars, D-psicose has attracted particular attention. However, current methods for producing D-psicose are costly, inefficient, and thermodynamically unfavorable, limiting its potential for widespread use. Notably, the present disclosure addresses various obstacles in D-psicose production, including thermodynamic barriers, limited yields, the need for enzyme purification, and the addition of cofactors. Another important discovery disclosed herein is that Escherichia coli naturally possesses a thermodynamically favorable pathway for D-psicose production, which allows for improved D-psicose yield by increasing expression of native genes and eliminating competing pathways without the need for introducing heterologous genes. The subject matter of the present disclosure facilitates the industrial-scale production of D-psicose without the need for expensive enzyme purification or difficult separation of raw materials and products.

[0058] This disclosure is based in part on the discovery that microorganisms comprising specific genetic modifications (e.g., gene deletions) can be used to produce low-calorie sugars. In certain embodiments, the low-calorie sugar is psicose. For clarity and not limitation, the detailed description of the subject matter disclosed herein is divided into the following subsections:

[0059] 1. Definition;

[0060] 2. Microorganisms that produce allulose;

[0061] 3. Production and generation of microorganisms;

[0062] 4. A method for producing psicose; and

[0063] 5. Food.

[0064] 1. Definition

[0065] The terms used in this specification generally have their ordinary meaning in the art within the context of the present invention and the specific context in which each term is used. Certain terms are discussed below or elsewhere in this specification to provide additional guidance to practitioners in describing the methods and compositions of the present invention and how to make and use them.

[0066] As used herein, when used with the term "comprising" in the claims and / or the specification, the expression "a" or "an" may refer to "one", but is also consistent with the meaning of "one or more", "at least one", and "one or more than one".

[0067] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend on how the value is measured or determined, i.e., the limits of the measurement system. For example, according to practice in the art, "about" can mean within 3 or more standard deviations. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and still more preferably up to 1% above or below a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, more preferably within 2-fold of a value.

[0068] As used herein, the terms "include," "comprising," "having," "having," "may," "containing," and variations thereof are intended to be open transitional phrases, terms, or words that do not exclude the possibility of other functions or configurations. The present disclosure also encompasses other embodiments "comprising," "consisting of," and "consisting essentially of" the embodiments or elements set forth herein, whether or not explicitly stated.

[0069] As used herein, the term "microorganism" refers to any organism that exists in the form of a microscopic cell, which is included in the scope of archaea, bacteria or eukaryotes, the latter including yeast and filamentous fungi, protozoa, algae or more advanced protozoa. In some embodiments, the term includes prokaryotic or eukaryotic cells or organisms with microscopic size, including but not limited to bacteria, archaea and eubacteria of all species and eukaryotic microorganisms (such as yeast and fungi). In some embodiments, the term microorganism includes cells that can be cultured to produce chemical substances (such as sugars). In some embodiments, the microorganism is a prokaryotic microorganism. In some embodiments, the prokaryotic microorganism is a bacterium.

[0070] As used herein, the terms "bacteria," "bacteria," or "eubacteria" refer to the domain of prokaryotes. In certain embodiments, bacteria include gram-negative bacteria, gram-positive bacteria, proteobacteria, cyanobacteria, spirochetes and related species, planctomyces, bacteroidetes, chlamydiae, green sulfur bacteria, green non-sulfur bacteria, radioresistant micrococci, thermotoga, and Thermosipho thermophiles.

[0071] As used herein, the term "gram-negative bacteria" includes cocci, nonentericrods, and enteric rods. Gram-negative bacteria include, but are not limited to, for example, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, and the like. ibrio), Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium.

[0072] As used herein, the term "Gram-positive bacteria" includes cocci, nonsporulating rods, and sporulating rods. Gram-positive bacterial genera include, but are not limited to, for example, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

[0073] As used herein, the term "recombinant microorganism" refers to a microorganism that contains one or more recombinant polynucleotides.

[0074] As used herein, the term "exogenous" refers to a molecule that is naturally present and / or produced in a given yeast, bacteria, organism, microorganism, or cell in nature. As used herein, the term "endogenous" refers to a molecule that is naturally present and / or produced in a given yeast, bacteria, organism, microorganism, or cell in nature.

[0075] As used herein, the terms "nucleic acid molecule," "nucleotide sequence," or "polynucleotide" refer to a single-stranded or double-stranded sequence of covalently linked nucleotides in which the 3' and 5' ends of the nucleotides are linked by phosphodiester bonds. Nucleic acid molecules can include deoxyribonucleotide bases or ribonucleotide bases and can be synthesized in vitro or isolated from natural sources.

[0076] As used herein, "recombinant polynucleotide" refers to a polynucleotide whose exact nucleotide sequence is foreign (i.e., not naturally occurring) to a given host. In some embodiments, the recombinant polynucleotide sequence is naturally present in a given host, but in an amount that is non-natural (e.g., greater than or less than expected), or additionally, the sequence of the polynucleotide is comprised of two or more subsequences that do not have the same relationship to each other in nature. For example, but not limited to, a recombinant polynucleotide can have two or more sequences from unrelated polynucleotides or from endogenous nucleotides to arrange into a new polynucleotide. In some embodiments, the present disclosure provides for introducing a recombinant polynucleotide into a microorganism, wherein the polynucleotide encodes a polypeptide that is not normally present in the microorganism. The polynucleotide sequence encoding the polypeptide is then recombinant or heterologous with reference to the genome of the microorganism.

[0077] As used herein, "gene" refers to a DNA region (including exons and introns) encoding a gene product, as well as a DNA region that regulates the production of a gene product, regardless of whether such regulatory sequences are adjacent to coding and / or transcribed sequences. In certain non-limiting embodiments, a gene includes a promoter sequence, a terminator, translation regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions.

[0078] The terms "polypeptide," "peptide," "amino acid sequence," and "protein" are used interchangeably herein to refer to a molecule formed by the linkage of at least two amino acids. The linkage between one amino acid residue and the next is an amide bond, sometimes referred to as a peptide bond. Polypeptides can be obtained by any suitable method known in the art, including isolation from natural sources, expression in a recombinant expression system, chemical synthesis, or enzymatic synthesis. The term applies to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0079] As used herein, the term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that act in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are amino acids encoded by the genetic code, as well as later modified amino acids such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs and derivatives can refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., a carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid but that act in a manner similar to naturally occurring amino acids. Non-limiting examples of amino acids include tryptophan, phenylalanine, histidine, glycine, cysteine, alanine, tyrosine, serine, methionine, asparagine, leucine, asparagine, threonine, isoleucine, proline, glutamic acid, aspartic acid, hydroxyproline, arginine, cystine, glutamine, lysine, valine, ornithine, taurine, and combinations thereof.

[0080] As used herein, the term "isolated" refers to material that is removed from at least one component with which it is naturally associated (eg, removed from its original environment).

[0081] As used herein, the terms "reduce" and "lower" refer to a measurable decrease in an endpoint (e.g., enzymatic activity, compound production, protein expression) by at least about 10%, at least about 50%, at least about 75%, or at least about 90%. In certain embodiments, the decrease can be from about 10% to about 100%.

[0082] As used herein, the terms "increase," "enhance," and "elevate" refer to a measurable increase in an endpoint (e.g., enzymatic activity, compound production, protein expression) by at least about 10%, at least about 50%, at least about 75%, or at least about 90%. In certain embodiments, the increase is from about 10% to about 100%. In certain embodiments, the increase can be at least about 10-fold, about 100-fold, or about 1000-fold or more. In certain embodiments, the increase can be about 100-fold or more, about 1000-fold or more, or about 10,000-fold or more.

[0083] The technology of determining nucleic acid and amino acid sequence identity is known in the art. Generally, such technology comprises determining the nucleotide sequence of the mRNA of a gene and / or determining the amino acid sequence encoded thereby, and comparing these sequences with a second nucleotide or amino acid sequence. It is also possible to determine and compare genomic sequences in this way. Generally speaking, identity / homogeneity refers to the precise correspondence of nucleotides and nucleotides or amino acids and amino acids between two polynucleotides or polypeptide sequences. They can be compared by determining the percentage identity of two or more sequences (polynucleotides or amino acids). The percentage identity of two sequences (no matter whether it is a nucleic acid sequence or an amino acid sequence) is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence and multiplied by 100. Unless otherwise stated, when using BLAST or BLAST 2.0 sequence comparison algorithms with default parameters to measure the maximum correspondence on a comparison window or a specified region, the percentage identity of two sequences is determined. See, for example, the NCBI website at cbi.nlm.nih.gov / BLAST. For example, BLASTN and BLASTP can use the following default parameters: genetic code = standard; filter = none; chain = two; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sort by = HIGH SCORE; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Detailed information on these programs can be found on the GenBank website.

[0084] "Mutations" in genes can include nucleotide changes, deletions of one or more nucleotides (which can include the entire coding sequence and / or promoter or other regulatory sequences), and insertions of one or more nucleotides, which can occur in the coding sequence of a gene or its regulatory components (e.g., the promoter of a gene). Mutations can include, for example, mutations that reduce or eliminate function (e.g., nonsense mutations) and genomic changes that reduce or knock out expression of a gene product.

[0085] As used herein, the term "yield" refers to the amount of a product recovered from a process or chemical reaction. For example, but not limited to, yield refers to the amount of psicose recovered from culturing one of the microorganisms disclosed herein. In certain embodiments, yield is expressed as a fraction or percentage based on the raw materials used, or as a ratio of the final product to the starting material, without taking into account any side reactions. As used herein, the term "yield coefficient" is a measure of the ratio of the amount of product to the amount of raw materials consumed. In certain embodiments, the yield coefficient refers to the yield of psicose relative to the amount of substrate (e.g., glucose).

[0086] 2. Microorganisms that produce allulose

[0087] The present disclosure provides genetically engineered microorganisms. In certain embodiments, the microorganisms of the present disclosure can produce increased amounts of psicose, for example, an increased amount compared to a naturally-producing control microorganism.

[0088] D-Psicose, also known as D-allulose, is a naturally occurring but rare monosaccharide. It is a ketohexose with the same empirical formula as common monosaccharides such as glucose and fructose. It is a diastereomer with fructose at the 3-position. Its enantiomer, L-psicose, is not known in nature but has been synthesized. D-Psicose has the following formula:

[0089]

[0090] D-psicose is 70% as sweet as sucrose (e.g., table sugar), but contains only about 10% of the nutritional energy (e.g., calories) of sucrose. Therefore, D-psicose can be used as a substitute for sucrose and artificial sweeteners.

[0091] The present disclosure provides genetically engineered microorganisms having increased production of psicose compared to naturally occurring microorganisms. Figure 2 The biochemical pathways regulated in exemplary microorganisms of the present disclosure are shown. In this exemplary microorganism, glucose is input and phosphorylated to glucose-6-phosphate (G6P) by a phosphotransferase system (PTS) or GalP / Glk. G6P is then isomerized to fructose-6-phosphate (F6P) via glucose-6-phosphate isomerase. Next, F6P is epimerized to psicose-6-phosphate via D-psicose-6-phosphate 3-epimerase (AlsE), and then dephosphorylated to free psicose via hexitol phosphatase B (HxpB). Finally, free psicose can diffuse across the cell membrane into the supernatant. In some embodiments, the microorganisms of the present disclosure further comprise gene editing (e.g., knockout) of certain competing pathways, including the pentose phosphate pathway catalyzed by glucose-6-phosphate dehydrogenase (Zwf), glycolysis catalyzed by phosphofructokinase A and B (PfkA and PfkB), the allose degradation pathway catalyzed by allose-6-phosphate isomerase (RpiB), and the mannose biosynthesis pathway catalyzed by mannose-6-phosphate isomerase (ManA).

[0092] 2.1. Psicose-producing enzyme

[0093] In certain embodiments, the microorganisms of the present disclosure include overexpression of at least one gene encoding an enzyme that catalyzes a reaction that produces psicose. In certain embodiments, the microorganisms of the present disclosure include a recombinant polynucleotide encoding at least one enzyme that catalyzes a reaction that produces psicose. In certain embodiments, the enzyme is an epimerase, such as an epimerase that converts fructose-6-phosphate (F6P) to psicose-6-phosphate. As used herein, the term "epimerase" refers to a class of enzymes that catalyze the inversion of asymmetric groups in substrates having multiple asymmetric centers. Non-limiting examples of epimerases include D-psicose-6-phosphate 3-epimerase, methylmalonyl-CoA epimerase, UDPgalactose 4-epimerase, UDPglucose 4-epimerase, UDPglucuronic acid 4-epimerase, UDPglucuronic acid 5'-epimerase, ribose-5-phosphate epimerase, GDP-mannose 3,5-epimerase, L-ribulose phosphate 4-epimerase, UDP-N- Acetyl glucosamine 2-epimerase, UDP-N-acetylglucosamine 4-epimerase, UDP galactose 4-epimerase, UDP glucose 4-epimerase, UDP glucuronic acid 4-epimerase, UDP glucuronic acid 5'-epimerase, GDP mannose 3,5-epimerase, methylmalonyl-CoA epimerase, ribose-5-phosphate epimerase, and UDP-N-acetylglucosamine 2-epimerase.

[0094] In some embodiments, the epimerase is D-psicose-6-phosphate 3-epimerase (AlsE) (UniProt No. P32719). AlsE catalyzes the reversible epimerization of D-psicose 6-phosphate to D-fructose 6-phosphate. In certain embodiments, AlsE is Escherichia coli AlsE. In certain embodiments, AlsE comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, AlsE comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, AlsE consists of the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO: 1 is as follows:

[0095]

[0096] In certain embodiments, the gene alsE is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the nucleotide sequence set forth in SEQ ID NO: 2. In certain embodiments, alsE comprises the nucleotide sequence set forth in SEQ ID NO: 2. In certain embodiments, alsE consists of the nucleotide sequence set forth in SEQ ID NO: 2. SEQ ID NO: 2 is as follows:

[0097]

[0098] In certain embodiments, the enzyme is a phosphatase, for example, a phosphatase that dephosphorylates psicose-6-phosphate to form free psicose. As used herein, the term "phosphatase" refers to a class of enzymes that catalyze the removal of phosphate groups from organic compounds. In certain embodiments, the phosphatase catalyzes the removal of phosphate groups from sugars. In certain embodiments, the sugar is a hexose.

[0099] In certain embodiments, the phosphatase is hexitol phosphatase B (HxpB) (UniProt No. P77247 or UniProt No. Q7ADF8). HxpB catalyzes the dephosphorylation of D-psicose 6-phosphate. In certain embodiments, HxpB is Escherichia coli HxpB. In certain embodiments, HxpB comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, HxpB comprises the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, HxpB consists of the amino acid sequence set forth in SEQ ID NO: 3. SEQ ID NO: 3 is as follows:

[0100]

[0101]

[0102] In certain embodiments, HxpB comprises the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, HxpB consists of the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO: 4 is as follows:

[0103]

[0104] In certain embodiments, the gene hxpB is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the nucleotide sequence set forth in SEQ ID NO: 5. In certain embodiments, hxpB comprises the nucleotide sequence set forth in SEQ ID NO: 5. In certain embodiments, hxpB consists of the nucleotide sequence set forth in SEQ ID NO: 5. SEQ ID NO: 5 is as follows:

[0105]

[0106] In certain embodiments, the phosphatase is a sugar phosphatase, such as YbiV, G6425 (EcoCyc) P75792 (UniProt), and catalyzes the dephosphorylation of D-psicose 6-phosphate. In certain embodiments, YbiV is Escherichia coli YbiV. In certain embodiments, YbiV comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence of SEQ ID NO: 36. In certain embodiments, YbiV comprises the amino acid sequence of SEQ ID NO: 36. In certain embodiments, YbiV consists of the amino acid sequence of SEQ ID NO: 36. SEQ ID NO: 36 is as follows:

[0107]

[0108] In certain embodiments, the gene ybiV is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the nucleotide sequence set forth in SEQ ID NO: 37. In certain embodiments, ybiV comprises the nucleotide sequence set forth in SEQ ID NO: 37. In certain embodiments, ybiV consists of the nucleotide sequence set forth in SEQ ID NO: 37. SEQ ID NO: 37 is as follows:

[0109]

[0110]

[0111] In certain embodiments, one or more transporters that transport glucose into the cell can be expressed (ie, overexpressed) in the cell, thereby increasing glucose in the cell. In some embodiments, galactose:H +One or both of the symporter (Galp) and glucokinase (Glk) are expressed in the microorganism. For example, in some embodiments, GalP transports glucose into the cell where it is phosphorylated to glucose-6-phosphate by Glk and assimilated into the central carbon metabolism.

[0112] In certain embodiments, galactose:H + Symporter (Galp) is described in EG12148 (EcoCyc) or POAEP 1 (UniProt). In certain embodiments, GalP is E. coli GalP. In certain embodiments, GalP comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO:38. In certain embodiments, GalP comprises the amino acid sequence set forth in SEQ ID NO:38. In certain embodiments, GalP consists of the amino acid sequence set forth in SEQ ID NO:38. SEQ ID NO:38 is as follows:

[0113]

[0114] In certain embodiments, the gene galP is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the nucleotide sequence set forth in SEQ ID NO: 39. In certain embodiments, galP comprises the nucleotide sequence set forth in SEQ ID NO: 39. In certain embodiments, galP consists of the nucleotide sequence set forth in SEQ ID NO: 39. SEQ ID NO: 39 is as follows:

[0115]

[0116]

[0117] In certain embodiments, the glucokinase (Glk) is as described in EG 12957 (EcoCyc) or POA6V8 (UniProt). In certain embodiments, the Glk is E. coli Glk. In certain embodiments, the Glk comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO:40. In certain embodiments, the Glk comprises the amino acid sequence set forth in SEQ ID NO:40. In certain embodiments, the Glk consists of the amino acid sequence set forth in SEQ ID NO:40. SEQ ID NO:40 is as follows:

[0118]

[0119] In certain embodiments, the gene glk is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the nucleotide sequence set forth in SEQ ID NO:41. In certain embodiments, glk comprises the nucleotide sequence set forth in SEQ ID NO:41. In certain embodiments, glk consists of the nucleotide sequence set forth in SEQ ID NO:41. SEQ ID NO:41 is as follows:

[0120]

[0121]

[0122] Without wishing to be bound by theory, the inventors of the present disclosure believe that any enzyme that performs a similar function to the above-mentioned enzymes can be used in the microorganisms of the present disclosure. For example, but not limited to, the microorganisms of the present disclosure can include any enzyme that can catalyze the reversible epimerization of D-fructose 6-phosphate to D-psicose 6-phosphate. In another non-limiting example, the microorganisms of the present disclosure can include any enzyme that can dephosphorylate psicose-6-phosphate to free psicose.

[0123] 2.2. Competitive Path

[0124] In certain embodiments, the microorganisms of the present disclosure include mutations in genes encoding one or more enzymes that regulate a biochemical pathway that can reduce the production of psicose. In certain embodiments, the microorganisms of the present disclosure include reduced expression of genes encoding enzymes that regulate a biochemical pathway that can reduce the production of psicose. Physiologically, cells catalyze sugars to produce energy (e.g., ATP) through the pentose phosphate pathway and glycolysis. The inventors of the present disclosure have discovered that the absence or reduced expression of genes encoding enzymes in certain metabolic pathways can lead to increased production of psicose.

[0125] In certain embodiments, the microorganisms of the present disclosure include mutations in genes encoding enzymes of the pentose phosphate pathway. In certain embodiments, the microorganisms of the present disclosure include reduced expression of genes encoding enzymes of the pentose phosphate pathway. In certain embodiments, the enzymes of the pentose phosphate pathway are selected from the following groups: glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, phosphogluconate dehydrogenase, pentose phosphate isomerase, pentose phosphate epimerase, transketolase, and transaldolase. In certain embodiments, the enzyme of the pentose phosphate pathway is glucose-6-phosphate dehydrogenase (Zwf) (Entrez Gene ID: 946370). Zwf catalyzes the oxidation of glucose 6-phosphate to 6-phosphogluconolactone. In certain embodiments, Zwf is Escherichia coli Zwf. The representative nucleotide sequence of gene zwf is shown in SEQ ID NO: 6, or is at least 90%, 95%, 95% or 99% identical to SEQ ID NO: 6. SEQ ID NO: 6 is as follows:

[0126]

[0127]

[0128] In certain embodiments, the E. coli Zwf comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 20. In certain embodiments, the E. coli Zwf comprises the amino acid sequence set forth in SEQ ID NO: 20. SEQ ID NO: 20 is as follows:

[0129]

[0130]

[0131] In certain embodiments, Zwf is Bacillus subtilis Zwf. A representative amino acid sequence of Bacillus subtilis Zwf is P54547 (Uniprot) / BSU23850 (KEGG) or as set forth in SEQ ID NO: 10, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 10. SEQ ID NO: 10 is as follows:

[0132]

[0133] A representative nucleotide sequence of the Bacillus subtilis zwf gene is shown in SEQ ID NO: 26, or is at least 90%, 95%, 95% or 99% identical to SEQ ID NO: 26. SEQ ID NO: 26 is as follows:

[0134]

[0135]

[0136] In certain embodiments, Zwf is Lactococcus lactis Zwf. A representative amino acid sequence of Lactococcus lactis Zwf is LLA12_RS12225: glucose-6-phosphate dehydrogenase, EC 1.1.1.49, or as set forth in SEQ ID NO: 11, or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 11. SEQ ID NO: 11 is as follows:

[0137]

[0138] A representative nucleotide sequence of the zwf gene of Lactococcus lactis is shown in SEQ ID NO: 33, or is at least 90%, 95%, 95% or 99% identical to SEQ ID NO: 33. SEQ ID NO: 33 is as follows:

[0139]

[0140]

[0141] In certain embodiments, the microorganisms of the present disclosure include mutations in genes encoding enzymes of glycogen biosynthesis. In certain embodiments, the microorganisms of the present disclosure include reduced expression of genes encoding enzymes of glycogen biosynthesis. In certain embodiments, the enzymes of glycogen biosynthesis are selected from the following groups: phosphoglucomutase (Pgm), UDP-glucose pyrophosphorylase, glycogen synthase, glycogen branching enzyme, and glycogenin. In some embodiments, the enzyme of glycogen biosynthesis is phosphoglucomutase (Pgm) (Entrez Gene ID: 946370). Pgm (EC5.4.2.2) is an enzyme that transfers a phosphate group on an α-D-glucose monomer from position 1 to position 6 or from position 6 to position 1. In certain embodiments, Pgm is Escherichia coli Pgm. A representative nucleotide sequence of gene pgm is shown in SEQ ID NO: 42, or is at least 90%, 95%, 95% or 99% identical to SEQ ID NO: 47. SEQ ID NO: 47 is as follows:

[0142]

[0143]

[0144] In certain embodiments, the E. coli Pgm comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 48. In certain embodiments, the E. coli Pgm comprises the amino acid sequence set forth in SEQ ID NO: 48. SEQ ID NO: 48 is as follows:

[0145]

[0146] In certain embodiments, Pgm is Bacillus subtilis Pgm. A representative nucleotide sequence of the gene pgm is set forth in SEQ ID NO:49, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO:49. SEQ ID NO:49 is as follows:

[0147]

[0148]

[0149] In certain embodiments, the Bacillus subtilis Pgm comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 50. In certain embodiments, the Bacillus subtilis Pgm comprises the amino acid sequence set forth in SEQ ID NO: 50. SEQ ID NO: 50 is as follows:

[0150]

[0151]

[0152] In certain embodiments, Pgm is Lactococcus lactis Pgm. A representative nucleotide sequence of the gene pgm is set forth in SEQ ID NO: 51, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 51. SEQ ID NO: 51 is as follows:

[0153]

[0154] In certain embodiments, the Lactococcus lactis Pgm comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 52. In certain embodiments, the Lactococcus lactis Pgm comprises the amino acid sequence set forth in SEQ ID NO: 52. SEQ ID NO: 52 is as follows:

[0155]

[0156] In certain embodiments, the microorganisms of the present disclosure include mutations in genes encoding enzymes of glycolysis. In certain embodiments, the microorganisms of the present disclosure include reduced expression of genes encoding enzymes of glycolysis. In certain embodiments, the enzymes of glycolysis are selected from the group consisting of phosphofructokinase A, phosphofructokinase B, fructose-bisphosphate aldolase, triose phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, and pyruvate kinase. In certain embodiments, the enzyme of glycolysis is phosphofructokinase B (PfkB). In certain embodiments, the enzyme of glycolysis is pyruvate kinase. In certain embodiments, the enzyme of glycolysis is phosphofructokinase A (PfkA) (Entrez Gene ID: 948412). PfkA catalyzes the phosphorylation of D-fructose 6-phosphate with ATP to fructose 1,6-bisphosphate, which is the first step in glycolysis. In certain embodiments, PfkA is Escherichia coli PfkA. The representative nucleotide sequence of the gene pfkA is shown in SEQ ID NO: 7. SEQ ID NO: 7 is as follows:

[0157]

[0158] In certain embodiments, the E. coli PfkA comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the E. coli PfkA comprises the amino acid sequence set forth in SEQ ID NO: 21. SEQ ID NO: 21 is as follows:

[0159]

[0160] In certain embodiments, the PfkA is Bacillus subtilis PfkA. A representative amino acid sequence of Bacillus subtilis PfkA is O34529 (Uniprot) / BSU29190 (KEGG) or as set forth in SEQ ID NO: 12, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 12. SEQ ID NO: 12 is as follows:

[0161]

[0162] A representative nucleotide sequence of the Bacillus subtilis gene pfkA is shown in SEQ ID NO: 27, or is at least 90%, 95%, 95% or 99% identical to SEQ ID NO: 27. SEQ ID NO: 27 is as follows:

[0163]

[0164]

[0165] In certain embodiments, the PfkA is a Lactococcus lactis PfkA. A representative amino acid sequence of a Lactococcus lactis PfkA is LLA12_RS07020: ATP-dependent 6-phosphofructokinase, EC 2.7.1.11, or as set forth in SEQ ID NO: 13, or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 13, which is as follows:

[0166]

[0167] A representative nucleotide sequence of the pfkA gene of Lactococcus lactis is shown in SEQ ID NO: 32, or is at least 90%, 95%, 95% or 99% identical to SEQ ID NO: 32. SEQ ID NO: 32 is as follows:

[0168]

[0169]

[0170] In certain embodiments, PfkB is E. coli PfkB. A representative nucleotide sequence of the gene pfkB is shown in SEQ ID NO: 22. SEQ ID NO: 22 is as follows:

[0171]

[0172] In certain embodiments, the E. coli PfkB comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 23. In certain embodiments, the E. coli PfkB comprises the amino acid sequence set forth in SEQ ID NO: 23. SEQ ID NO: 23 is as follows:

[0173]

[0174] In certain embodiments, the microorganisms of the present disclosure do not include the absence or reduced expression of hexokinase. In certain embodiments, the microorganisms of the present disclosure do not include the absence, destruction or reduced expression of glucokinase. In certain embodiments, the microorganisms of the present disclosure do not include the absence or reduced expression of glucose-6-phosphate isomerase.

[0175] In certain embodiments, the microorganisms of the present disclosure include mutations in genes encoding enzymes of the allose degradation pathway. In certain embodiments, the microorganisms of the present disclosure include reduced expression of genes encoding enzymes of the allose degradation pathway. In certain embodiments, the enzyme of the allose degradation pathway is allose-6-phosphate isomerase (RpiB) (Entrez Gene ID: 948602). RpiB catalyzes the interconversion of ribulose-5-P and ribose-5-P, as well as the interconversion of D-allose-6-phosphate (All6P) and D-allose-6-phosphate. In certain embodiments, RpiB is Escherichia coli RpiB. A representative nucleotide sequence of gene rpiB is shown in SEQ ID NO: 8, or is at least 90%, 95%, 95% or 99% identical to SEQ ID NO: 8. SEQ ID NO: 8 is as follows:

[0176]

[0177] In certain embodiments, the E. coli RpiB comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence of SEQ ID NO: 24. In certain embodiments, the E. coli RpiB comprises the amino acid sequence of SEQ ID NO: 24. SEQ ID NO: 24 is as follows:

[0178]

[0179] In certain embodiments, RpiB is the Bacillus subtilis gene rpiB. A representative amino acid sequence of RpiB is A0A6M4JQ63 (Uniprot) / BSU36920 (KEGG) or as set forth in SEQ ID NO: 14, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 14. SEQ ID NO: 14 is as follows:

[0180]

[0181] A representative nucleotide sequence of the Bacillus subtilis gene rpiB is shown in SEQ ID NO: 28, or is at least 90%, 95%, 95% or 99% identical to SEQ ID NO: 28. SEQ ID NO: 28 is as follows:

[0182]

[0183] In certain embodiments, RpiB is Lactococcus lactis RpiB. A representative amino acid sequence of the gene rpiB is LLA12_RS12460: ribose-5-phosphate isomerase, EC 5.3.1.6 or as set forth in SEQ ID NO: 15, or at least 90%, 95%, 95% or 99% identical to SEQ ID NO: 15, as set forth below.

[0184]

[0185] A representative nucleotide sequence of the rpiB gene of Lactococcus lactis is shown in SEQ ID NO: 34, or is at least 90%, 95%, 95% or 99% identical to SEQ ID NO: 34. SEQ ID NO: 34 is as follows:

[0186]

[0187] In certain embodiments, the microorganisms of the present disclosure include mutations in genes encoding enzymes of the mannose biosynthetic pathway. In certain embodiments, the microorganisms of the present disclosure include reduced expression of genes encoding enzymes of the mannose biosynthetic pathway. In certain embodiments, the enzyme of the mannose biosynthetic pathway is mannose-6-phosphate isomerase (ManA) (Entrez Gene ID: 944840). ManA is involved in the synthesis of GDP-mannose and dolichol-phosphate-mannose, which are required for many key mannosyl transfer reactions. ManA also catalyzes the interconversion of fructose-6-phosphate and mannose-6-phosphate. In certain embodiments, ManA is Escherichia coli ManA. A representative nucleotide sequence of the gene manA is set forth in SEQ ID NO: 9, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 9, which is set forth below.

[0188]

[0189]

[0190] In certain embodiments, the E. coli ManA comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 25. In certain embodiments, the E. coli ManA comprises the amino acid sequence set forth in SEQ ID NO: 25. SEQ ID NO: 25 is as follows:

[0191]

[0192] In certain embodiments, ManA is Bacillus subtilis ManA. A representative amino acid sequence of ManA is O31646 (Uniprot) / BSU12020 (KEGG) or as set forth in SEQ ID NO: 16 or at least 90%, 95%, 95% or 99% identical to SEQ ID NO: 16.

[0193]

[0194] A representative nucleotide sequence of the Bacillus subtilis gene manA is shown in SEQ ID NO: 29, or is at least 90%, 95%, 95% or 99% identical to SEQ ID NO: 29.

[0195]

[0196] In certain embodiments, optionally in combination with the deletion of B. subtilis ManA in B. subtilis, one or both of the following are also deleted.

[0197] (i) YvyI of Bacillus subtilis. A representative amino acid sequence of YvyI is P39841 (Uniprot) / BSU35790 (KEGG) or as set forth in SEQ ID NO: 17, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 17. SEQ ID NO: 17 is as follows:

[0198]

[0199]

[0200] A representative nucleotide sequence of the Bacillus subtilis gene yvyI is shown in SEQ ID NO: 30, or is at least 90%, 95%, 95% or 99% identical to SEQ ID NO: 30. SEQ ID NO: 30 is as follows:

[0201]

[0202] (ii) GmuF of Bacillus subtilis. A representative amino acid sequence of GmuF is O05511 (Uniprot) / BSU05870 (KEGG) or as set forth in SEQ ID NO: 18, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 18. SEQ ID NO: 18 is as follows:

[0203]

[0204] A representative nucleotide sequence of the Bacillus subtilis gene gmuF is shown in SEQ ID NO: 31, or is at least 90%, 95%, 95% or 99% identical to SEQ ID NO: 31. SEQ ID NO: 31 is as follows:

[0205]

[0206]

[0207] In certain embodiments, ManA is Lactococcus lactis ManA. A representative amino acid sequence of Lactococcus lactis ManA is LLA12_RS03920: mannose-6-phosphate isomerase, EC 5.3.1.8, or as set forth in SEQ ID NO: 19, or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 19. SEQ ID NO: 19 is as follows:

[0208]

[0209] A representative nucleotide sequence of the Lactococcus lactis gene manA is shown in SEQ ID NO: 35, or is at least 90%, 95%, 95% or 99% identical to SEQ ID NO: 35. SEQ ID NO: 35 is as follows:

[0210]

[0211]

[0212] In some embodiments, the disappearance of gene comprises non-frameshift deletion, frameshift deletion or its combination.In some embodiments, the disappearance of gene can be realized by inserting (for example, non-frameshift insertion, frameshift insertion or its combination).In some embodiments, the disappearance of gene comprises nonsense mutation.

[0213] Cells

[0214] The present disclosure provides recombinant microorganisms. Any culturable microorganism is suitable for the compositions and methods described herein. In certain embodiments, the microorganism is a bacterium. In certain embodiments, the microorganism is selected from the group consisting of Acetobacter aceti, Achromobacter, Acidiphilium, Acinetobacter, Actinomadura, Actinoplanes, Aeropyrumpernix, Agrobacterium, Alealigenes, Ananascomosus (M), Arthrobacter, Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus lentus, Bacillus licheniformis, and Bacillus spp. lichiformix), Bacillus macerans, Bacillus stearothermophilus, Bacillus subtilis, Bifidobacterium, Brevibacillus brevis, Burkholderia cepacia, Candida cylindracea, Carica papaya (L), Cellulosimicrobium, Cephalosporium, Chaetomium erraticum, Chaetomium gracile, Clostridium, Clostridium butyricum, Clostridium acetobutylicum, Clostridium thermocellum thermocellum), Corynebacterium (glutamicum), Corynebacterium (glutamicum),efficiens), Escherichia coli, Enterococcus, Erwina chrysanthemi, Gliconobacter, Gluconacetobacter, Haloarcula, Humicola insolens, Kitasatospora setae, Klebsiella, Klebsiella oxytoca, Kocuria, Lactlactis, Lactobacillus, Lactobacillus fermentum, Lactobacillus sake, Lactococcus, Lactococcus lactis lactis), Leuconostoc, Methylocysis, Methanolobus siciliae, Methanogenium organophilum, Methanobacterium bryantii, Microbacterium imperiale, Micrococcus lysodeikticus, Microlunatus, Mucorjavanicus, Mycobacterium, Myrothecium, Nitrobacter, Nitrosomonas, Nocardia, Papayacarica, Pediococcus, Pediococcus halophilus, Heterotrophic nitrifying and aerobic denitrifying bacteria (Paracoccus pantotrophus), Propionibacterium, Pseudomonas, Pseudomonas fluorescensfluorescens), Pseudomonas denitrificans, Pyrococcus, Pyrococcus furiosus, Pyrococcus horikoshii, Rhizobium, Rhizomucor miehei, Rhizomucor pusillus Lindt, Rhizopus, Rhizopus delemar, Rhizopus japonicus, Rhizopus niveus, Rhizopus oryzae, Rhizopus oligosporus, Rhodococcus, Sclerotinalibertina, Sphingobacterium multivorum), Sphingobium, Sphingomonas, Streptococcus, Streptococcus thermophilus Y-1, Streptomyces, Streptomyces griseus, Streptomyces lividans, Streptomyces murinus, Streptomyces rubiginosus, Streptomyces violaceoruber, Streptoverticillium mobaraense, Tetragenococcus, Thermus, Thiosphaera In some embodiments, the microorganism is a bacterium selected from the group consisting of: pantotropha, Trametes, Vibrio alginolyticus, Xanthomonas, Zymomonas, and Zymomonas mobilis. In some embodiments, the microorganism is Escherichia coli (E. coli). In some embodiments, the microorganism is Bacillus subtilis. In some embodiments, the microorganism is Lactococcus lactis.

[0215] In certain embodiments, the E. coli is selected from the group consisting of enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enterohemorrhagic E. coli (EHEC), uropathogenic E. coli (UPEC), verotoxin-producing E. coli, E. coli O157:H7, E. coli O104:H4, E. coli O121, E. coli O104:H21, E. coli K1, and E. coli NC101. In certain embodiments, the E. coli is E. coli K12. In certain embodiments, the E. coli is E. coli B. In certain embodiments, the E. coli is E. coli C.

[0216] In certain embodiments, the E. coli is derived from a strain selected from the group consisting of NCTC 12757, NCTC 12779, NCTC 12790, NCTC 12796, NCTC 12811, ATCC 11229, ATCC 25922, ATCC 8739, DSM 30083, BC5849, BC 8265, BC 8267, BC 8268, BC 8270, BC8271, BC 8272, BC 8273, BC 8276, BC 8277, BC 8278, BC 8279, BC 8312, BC 8317, BC 8319, BC 8320, BC 8321, BC 8322, BC 8326, BC8327, BC 8331, BC 8335, BC 8338, BC 8341, BC 8344, BC 8345, BC 8346, BC 8347, BC 8348, BC 8863, and BC 8864.

[0217] In certain embodiments, the E. coli is derived from a strain selected from the group consisting of BC 4734 (O26: H11), BC4735 (O157: H-), BC 4736, BC 4737 (nd), BC 4738 (O157: H7), BC 4945 (O26: H-), BC 4946 (O157: H7), BC 4947 (O111: H-), BC 4948 (O157: H), BC 4949 (O5), BC 5579 (O157: H7), BC5580 (O157: H7), BC 5582 (O3: H), BC 5643 (O2: H5), BC 5644 (O128), BC 5645 (O55: H-), BC5646 (O69: H-), BC BC 5647 (O101: H9), BC 5648 (O103: H2), BC 5850 (O22: H8), BC 5851 (O55: H-), BC 5852 (O48: H21), BC 5853 (O26: H11), BC 5854 (O157: H7), BC 5855 (O157: H-), BC 5856 (O26: H-), BC 5857 (O103: H2), BC 5858 (O26: H11), BC 7832, BC 7833 (O antigen original form: H-), BC 7834 (ONT: H-), BC 7835 (O103: H2), BC 7836 (O57: H-), BC 7837 (ONT: H-), BC7838, BC7839 (O128: H2), BC 7840 (O157: H-), BC 7841 (O23: H-), BC 7842 (O157: H-), BC 7843, BC7844 (O157: H-), BC 7845 (O103: H2), BC 7846 (O26: H11), BC 7847 (O145: H-), BC 7848 (O157: H-), BC 7849 (O156: H47), BC 7850, BC 7851 (O157: H-), BC 7852 (O157: H-), BC 7853 (O5: H-), BC 7854 (O157: H7), BC BC 7855 (O157: H7), BC 7856 (O26: H-), BC 7857, BC 7858, BC7859 (ONT: H-), BC 7860 (O129: H-), BC 7861, BC 7862 (O103: H2), BC 7863, BC 7864 (O antigen original form: H-), BC 7865, BC 7866 (O26: H-), BC 7867 (O antigen original form: H-), BC 7868, BCBC 7869 (ONT: H-), BC 7870 (O113: H-), BC 7871 (ONT: H-), BC 7872 (ONT: H-), BC 7873, BC 7874 (O antigen original form: H-), BC 7875 (O157: H-), BC 7876 (O111: H-), BC 7877 (O146: H21), BC 7878 (O145: H-), BC 7879 (O22: H8), BC 7880 (O antigen original form: H-), BC 7881 (O145: H-), BC 8275 (O157: H7), BC 8318 (O55: K-: H-), BC 8325 (O157: H7), BC 8332 (ONT) and BC 8333.

[0218] In certain embodiments, the E. coli is derived from a strain selected from the group consisting of BC 8246 (O152: K-: H-), BC 8247 (O 124: K(72): H3), BC 8248 (O 124), BC 8249 (O 112), BC 8250 (O136: K(78): H-), BC 8251 (O124: H-), BC 8252 (O144: K-: H-), BC 8253 (O143: K: H-), BC 8254 (O143), BC 8255 (O112), BC 8256 (O28a.e), BC 8257 (O124: H-), BC 8258 (O143), BC 8259 (O167:K-:H5), BC8260 (O128a.c.:H35), BC 8261 (O164), BC 8262 (O164:K-:H-), BC 8263 (O164) and BC 8264 (O124).

[0219] In certain embodiments, the E. coli is derived from a strain selected from the group consisting of BC 5581(O78:H11), BC5583(O2:K1), BC 8221(O118), BC 8222(O148:H-), BC 8223(O111), BC 8224(O110:H-), BC8225(O148), BC 8226(O118), BC 8227(O25:H42), BC 8229(O6), BC 8231(O153:H45), BC8232(O9), BC 8233(O148), BC 8234(O128), BC 8235(O118), BC 8237(O111), BC 8238(O110:H17), BC 8240(O148), BC 8241(O6H16), BC 8243(O153), BC 8244(O15:H-), BC 8245(O20), BC 8269(O125a.c:H-), BC 8313(O6:H6), BC 8315(O153:H-), BC 8329, BC 8334(O118:H12) and BC 8339.

[0220] In certain embodiments, the E. coli is derived from a strain selected from the group consisting of BC 7567(O86), BC 7568(O128), BC 7571(O114), BC 7572(O119), BC 7573(O125), BC 7574(O124), BC7576(O127a), BC 7577(O126), BC 7578(O142), BC 7579(O26), BC 7580(OK26), BC 7581(O142), BC 7582(O55), BC 7583(O158), BC 7584(O-), BC 7585(O-), BC 7586(O-), BC 8330, BC 8550(O26), BC 8551(O55), BC 8552(O158), BC 8553(O26), BC 8554(O158), BC 8555(O86), BC 8556(O128), BC 8557(OK26), BC 8558(O55), BC 8560(O158), BC 8561(O158), BC 8562(O114), BC 8563(O86), BC 8564(O128), BC 8565(O158), BC 8566(O158), BC 8567(O158), BC 8568(O111), BC8569(O128), BC 8570(O114), BC 8571(O128), BC 8572(O128), BC 8573(O158), BC 8574(O158), BC 8575(O158), BC 8576(O158), BC 8577(O158), BC 8578(O158), BC 8581(O158), BC 8583(O128), BC 8584(O158), BC 8585(O128), BC8586(O158), BC 8588(O26), BC8589(O86), BC 8590(O127), BC 8591(O128), BC 8592(O114), BC 8593(O114), BC 8594(O114), BC 8595(O125), BC 8596(O158), BC 8597(O26), BC 8598(O26), BC 8599(O158), BC8605(O158), BC 8606(O158), BC 8607(O158), BC 8608(O128), BC 8609(O55), BC 8610(O114), BC8615(O158), BC 8616(O128), BC8617(O26), BC 8618(O86), BC 8619, BC 8620, BC 8621, BC 8622, BC 8623, BC 8624(O158) and BC 8625(O158).

[0221] In certain embodiments, the Bacillus subtilis is derived from strain 168.

[0222] In certain embodiments, the Lactococcus lactis is derived from strain A12.

[0223] In certain embodiments, the microorganism is a fungal cell. In certain embodiments, the fungal cell is selected from the group consisting of Aspergillus, Aspergillus nidulans, Aspergillus niger, Aspargillus oryze, Aspergillus melleus, Aspergillus pulverulentus, Aspergillus saitoi, Aspergillus sojea, Aspergillus terreus, Aspergillus pseudoterreus, Aspergillus usamii, Candida rugosa, Issatchenkia orientalis, Kluyveromyces, Kluyveromyces fragilis, Kluyveromyces lactis. lactis), Kluyveromyces marxianas, Penicillium, Penicillium camemberti, Penicillium citrinum, Enicillium emersonii, Penicillium roqueforti, Penicillium lilactinum, Penicillium multicolor, Rhodosporidium toruloides, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride, Trichosporon penicillaium, Yarrowia lipolytica lipolytica) and Zygosaccharomyces rouxii.

[0224] In certain embodiments, the microorganism is a yeast cell. In certain embodiments, the yeast cell is Saccharomyces cerevisiae.

[0225] 2.4. Exemplary Microorganisms

[0226] In certain embodiments, the present disclosure provides a recombinant microorganism comprising an increased psicose production compared to a naturally occurring microorganism. In certain embodiments, the recombinant microorganism comprises an exogenous epimerase and an exogenous phosphatase. In certain embodiments, the exogenous epimerase is psicose-6-phosphate 3-epimerase (AlsE). In certain embodiments, the exogenous phosphatase is hexitol phosphatase B (HxpB). In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is Escherichia coli.

[0227] In certain embodiments, the present disclosure provides a recombinant microorganism comprising an increased psicose production compared to a naturally occurring microorganism. In certain embodiments, the recombinant microorganism comprises an exogenous epimerase, an exogenous phosphatase, and deletions of one, two, three, or four (4) genes. In certain embodiments, the exogenous epimerase is psicose-6-phosphate 3-epimerase (AlsE). In certain embodiments, the exogenous phosphatase is hexitol phosphatase B (HxpB). In certain embodiments, the four deleted genes are glucose-6-phosphate 1-dehydrogenase, phosphofructokinase-1, psicose-6-phosphate isomerase, and mannose-6-phosphate isomerase. In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is Escherichia coli.

[0228] In certain embodiments, the present disclosure provides a microorganism comprising a recombinant polynucleotide, wherein the microorganism comprises an increased psicose production compared to a naturally occurring microorganism. In certain embodiments, the recombinant polynucleotide comprises a nucleotide sequence encoding an exogenous epimerase and an exogenous phosphatase. In certain embodiments, the exogenous epimerase is psicose-6-phosphate 3-epimerase (AlsE). In certain embodiments, the exogenous phosphatase is hexitol phosphatase B (HxpB). In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is Escherichia coli.

[0229] In certain embodiments, the present disclosure provides a microorganism comprising a recombinant polynucleotide, wherein the microorganism comprises an increased psicose production compared to a naturally occurring microorganism. In certain embodiments, the recombinant polynucleotide comprises a nucleotide sequence encoding an exogenous epimerase and an exogenous phosphatase. In certain embodiments, the exogenous epimerase is psicose-6-phosphate 3-epimerase (AlsE). In certain embodiments, the exogenous phosphatase is hexitol phosphatase B (HxpB). In certain embodiments, the microorganism further comprises a deletion of a first gene. In certain embodiments, the first gene is glucose-6-phosphate 1-dehydrogenase. In certain embodiments, the microorganism further comprises a deletion of a second gene. In certain embodiments, the second gene is phosphofructokinase-1. In certain embodiments, the microorganism further comprises a deletion of a third gene. In certain embodiments, the third gene is glucose-6-phosphate isomerase. In certain embodiments, the microorganism further comprises a deletion of a fourth gene. In certain embodiments, the fourth gene is mannose-6-phosphate isomerase. In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is Escherichia coli. In certain embodiments, the bacterium is Bacillus subtilis. In certain embodiments, the bacterium is Lactococcus lactis.

[0230] In certain embodiments, the present disclosure provides a recombinant microorganism comprising increased psicose production compared to a naturally occurring microorganism. In certain embodiments, the recombinant microorganism comprises an exogenous epimerase, an exogenous phosphatase, an exogenous nuclease, an sgRNA, and deletions of four (4) genes. In certain embodiments, the exogenous epimerase is psicose-6-phosphate 3-epimerase (AlsE). In certain embodiments, the exogenous phosphatase is hexitol phosphatase B (HxpB). In certain embodiments, the exogenous nuclease is dCas9. In certain embodiments, the four deleted genes are zwf, pfkA, RpiB, and ManA. In certain embodiments, the sgRNA targets pfkB. In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is Escherichia coli. In certain embodiments, the bacterium is Bacillus subtilis. In certain embodiments, the bacterium is Lactococcus lactis.

[0231] In certain embodiments, the present disclosure provides a microorganism comprising a recombinant polynucleotide, wherein the microorganism comprises an increased psicose production compared to a naturally occurring microorganism. In certain embodiments, the recombinant polynucleotide comprises a nucleotide sequence encoding an exogenous epimerase, a nucleotide sequence encoding an exogenous phosphatase, and a nucleotide sequence encoding a nuclease. In certain embodiments, the exogenous epimerase is psicose-6-phosphate 3-epimerase (AlsE). In certain embodiments, the exogenous phosphatase is hexitol phosphatase B (HxpB). In certain embodiments, the exogenous nuclease is dCas9. In certain embodiments, the microorganism further comprises a deletion of a first gene. In certain embodiments, the first gene is zwf. In certain embodiments, the microorganism further comprises a deletion of a second gene. In certain embodiments, the second gene is pfkA. In certain embodiments, the microorganism further comprises a deletion of a third gene. In certain embodiments, the third gene is rpiB. In certain embodiments, the microorganism further comprises a deletion of a fourth gene. In certain embodiments, the fourth gene is manA. In certain embodiments, the microorganism further comprises an sgRNA. In certain embodiments, the sgRNA targets pfkB. In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is Escherichia coli. In certain embodiments, the bacterium is Bacillus subtilis. In certain embodiments, the bacterium is Lactococcus lactis.

[0232] 3. Production and Method of Microbial Allulose

[0233] The present disclosure also provides methods for preparing and / or generating any microorganism disclosed herein. Many recombinant techniques well known in the art can be used to introduce one or more recombinant polynucleotides of the present disclosure into microorganisms, including but not limited to protoplast fusion, transfection, transformation, conjugation and transduction. These technologies include conventional molecular biology techniques (e.g., recombinant techniques), microbiology, cell biology and biochemistry, which are within the technical scope of this area. Additional information on these techniques can be found in: Molecular Cloning: A Laboratory Manual, 2nd edition (Sambrook et al., 1989); Oligonucleotide Synthesis (Gait, ed., 1984); Animal Cell Culture (Freshney, ed., 1987); Gene Transfer Vectors for Mammalian Cells (Miller and Calos, eds., 1987); Current Protocols in Molecular Biology (Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); and Current Protocols in Immunology (Coligan et al., eds., 1991).

[0234] 3.1. Recombinant polynucleotides

[0235] In certain embodiments, the recombinant polynucleotides disclosed herein can be stably integrated into the microbial chromosome. In certain embodiments, the recombinant polynucleotides disclosed herein are stably integrated into the microbial chromosome using homologous recombination, transposition-based chromosomal integration, recombinase-mediated cassette exchange (RMCE; For example, using the Cre-lox system) or an integration plasmid (for example, a yeast integration plasmid). A variety of integration techniques suitable for various microorganisms are known in the art (see, for example, Griffiths, AJF, Miller, JH, Suzuki, DT, etc. "An Introduction to Genetic Analysis". 7th edition. New York: WH Freeman; 2000). In certain embodiments, the recombinant polynucleotides disclosed herein are maintained on an extrachromosomal plasmid (for example, an expression plasmid or a vector) in the recombinant microorganism of the present disclosure. A variety of extrachromosomal plasmids suitable for use in a variety of microorganisms are known in the art, including but not limited to replicating plasmids (e.g., yeast replicating plasmids comprising an autonomously replicating sequence ARS), centromeric plasmids (e.g., yeast centromeric plasmids comprising an autonomously replicating sequence CEN), episomal plasmids (e.g., 2-pm plasmids) and / or artificial chromosomes (e.g., yeast artificial chromosomes YACs or bacterial artificial chromosomes BACs).

[0236] 3.1.1. Carrier

[0237] In certain embodiments, the present disclosure provides a vector comprising a nucleotide sequence disclosed herein. As used herein, the term "vector" refers to a polynucleotide construct designed for introducing nucleic acid into one or more microorganisms. Vectors may include, but are not limited to, cloning vectors, expression vectors, shuttle vectors, plasmids, and cassette vectors. As used herein, the term "plasmid" refers to a circular double-stranded DNA construct used as a clone and / or expression vector. In certain embodiments, when introducing a microorganism, a plasmid can be an extrachromosomal self-replicating genetic element (e.g., an episomal plasmid). In certain embodiments, a plasmid can be integrated into a microbial chromosome. In certain embodiments, a vector can guide the expression of a coding region operably connected thereto, such as an "expression vector." These expression vectors allow exogenous polynucleotides and / or polypeptides to be expressed in a microorganism. In certain embodiments, a vector allows one or more polynucleotides to be integrated into the genome of a microorganism.

[0238] In certain embodiments, the vectors disclosed herein include a promoter. In certain embodiments, the vector is a bacterial or prokaryotic expression vector. In certain embodiments, the vector is a yeast or fungal cell expression vector.

[0239] In certain embodiments, the vectors disclosed herein comprise nucleotide sequences in a single operon.

[0240] 3.1.2. Promoter

[0241] In certain non-limiting embodiments, the recombinant polynucleotides disclosed herein include a regulatory sequence, enhancer, or promoter. For example, but not limited to, the nucleotide sequence encoding the alsE gene and / or the hpxB gene can be operably linked to a regulatory sequence, enhancer, or promoter.

[0242] As used herein, the term "promoter" refers to any nucleotide sequence that regulates the initiation of transcription of a specific coding sequence under its control. From a biological point of view, a promoter does not transcribe, but coordinates the assembly of components that initiate transcription of other nucleotide sequences. In addition, a promoter can restrict this assembly and subsequent transcription to specific prerequisites. For example, but not limited to, a promoter can allow transcription in response to one or more environmental, temporal or developmental stimuli. Bacterial and fungal cells have a variety of proteins that can sense external or internal conditions and initiate signal cascades, ultimately leading to the binding of proteins to specific promoters, which then initiate transcription of nucleic acids under the control of the promoter. In some embodiments, the promoter is endogenous. In some embodiments, the promoter is exogenous. In some embodiments, the promoter is artificially designed for expression in a specific species.

[0243] In certain embodiments, the promoter is a constitutive promoter. A constitutive promoter is a promoter that drives the expression of a nucleotide sequence continuously and uninterruptedly in response to internal or external stimuli. Constitutive promoters are often used in recombineering to ensure continuous expression of a desired nucleotide sequence. Constitutive promoters result in the expression of large amounts of nucleic acids and, therefore, are used in many recombineering applications to achieve high levels of recombinant protein and enzyme activity. Non-limiting examples of constitutive promoters encompassed by the present disclosure include the E. coli promoter P spc 、P bla 、P RNAI 、P RNAII , P1 and P2 from rrnB and the lambda phage promoter P L (Liang, ST et al., J Moi. Biol., 292(1):19-37 (1999)). In some embodiments, the promoter is active during the stationary phase of the microorganism. Exemplary stationary phase promoters can be found, for example, in Shimada et al., JOURNAL OF BACTERIOLOGY, November 2004, pp. 7112-7122; Pletnev et al., ACTA NATURAE | Vol. 7, No. 4 (27) 2015.

[0244] In some embodiments, the promoter is an inducible promoter. An inducible promoter is a promoter that drives expression of a nucleotide sequence in response to a stimulus. An inducible promoter drives sustained expression when exposed to a specific stimulus (e.g., IPTG). In some embodiments, an inducible promoter drives a graded expression level related to the amount of stimulus. Non-limiting examples of stimuli for inducible promoters include heat shock, exogenous compounds or lack of exogenous compounds (e.g., sugars, metals, drugs, or phosphates), salt or osmotic shock, oxygen, and biological stimuli (e.g., growth factors or pheromones). Non-limiting examples of inducible promoters include the E. coli promoter P lac 、P taq ), P tac 、P T7 、P BAD , and P Lacuv .

[0245] In certain embodiments, a recombinant polynucleotide may include multiple promoters. In certain embodiments, the multiple promoters may be the same. For example, but not limited to, a recombinant polynucleotide may include a nucleotide sequence encoding an aslE gene operably linked to a first promoter and a nucleotide sequence encoding an hpxB gene operably linked to a second promoter, wherein the first and second promoters are the same. In certain embodiments, the multiple promoters may be different. For example, but not limited to, a recombinant polynucleotide may include a nucleotide sequence encoding an aslE gene operably linked to a first promoter and a nucleotide sequence encoding an hpxB gene operably linked to a second promoter, wherein the first and second promoters are different. In certain embodiments, the promoters are P LlacO1 In some embodiments, P LlacO1 The promoter comprises the nucleotide sequence shown in SEQ ID NO: 42. In certain embodiments, P LlacO1 The promoter consists of the nucleotide sequence shown in SEQ ID NO: 42. LlacO1 The promoter is a mixed regulatory region consisting of the bacteriophage lambda promoter P in which the CI binding site is replaced by lacO1. L The hybrid design allows for strong initiation, which can be inhibited by LacI, Lac inhibitors (i.e., repressors), or induced by IPTG.

[0246] In certain embodiments, the promoter is P LtetO1 In certain embodiments, P LTETO1 The promoter comprises the nucleotide sequence shown in SEQ ID NO: 43. In certain embodiments, P LTETO1 The promoter consists of the nucleotide sequence shown in SEQ ID NO:43.

[0247] In certain embodiments, the promoter is P T7 In certain embodiments, P T7 The promoter comprises the nucleotide sequence shown in SEQ ID NO: 44. In certain embodiments, P T7 The promoter consists of the nucleotide sequence shown in SEQ ID NO:44.

[0248] In certain embodiments, the promoter is P tet In certain embodiments, P tet The promoter comprises the nucleotide sequence shown in SEQ ID NO: 45. In certain embodiments, P tet The promoter consists of the nucleotide sequence shown in SEQ ID NO:45.

[0249] In certain embodiments, the promoter is P gadB In certain embodiments, P gadB The promoter comprises the nucleotide sequence shown in SEQ ID NO: 46. In certain embodiments, P gadB The promoter consists of the nucleotide sequence shown in SEQ ID NO:46.

[0250] P LlacO1 Promoter nucleotide sequence:

[0251]

[0252] P LtetO1 Promoter nucleotide sequence:

[0253]

[0254] P T7 Promoter nucleotide sequence:

[0255]

[0256] P tet Promoter nucleotide sequence:

[0257]

[0258] P gadB Promoter nucleotide sequence:

[0259]

[0260] In certain embodiments, the promoter is a stationary phase promoter. As used herein, the term "stationary phase promoter" refers to a promoter upstream of a gene that is transcribed during the stationary phase of microbial growth. The life cycle of an E. coli culture includes five different stages: a lag phase, a logarithmic phase, a stationary phase, a death phase, and a long-term stationary phase. When cells are inoculated into a culture medium and adjust their metabolic processes according to the new environment, a lag phase occurs. The cells will then grow and divide rapidly and enter the logarithmic phase. At this point, the enzymes associated with central carbon metabolism are most important, and the transcription of the corresponding genes will be upregulated. Once the cells experience environmental stress (such as scarcity of nutrients in the culture medium), their growth and division will slow down, and the culture will enter the stationary phase. Using a stationary phase promoter can prevent the production pathway from competing with central carbon metabolism for carbon flux during the logarithmic growth phase, when cells need carbon to strictly grow and divide.

[0261] In certain embodiments, the stationary phase promoter is P gadB In certain embodiments, P galB The promoter comprises the nucleotide sequence shown in SEQ ID NO: 46 or SEQ ID NO: 53. In certain embodiments, P galB The promoter consists of the nucleotide sequence shown in SEQ ID NO:53.

[0262] In certain embodiments, the stationary phase promoter is P cbpA2 In certain embodiments, P cbpA2 The promoter comprises the nucleotide sequence shown in SEQ ID NO: 54. In certain embodiments, P cbpA2 The promoter consists of the nucleotide sequence shown in SEQ ID NO:54.

[0263] In certain embodiments, the stationary phase promoter is P ihfA4 In certain embodiments, P ihfA4 The promoter comprises the nucleotide sequence shown in SEQ ID NO: 55. In certain embodiments, P ihfA4 The promoter consists of the nucleotide sequence shown in SEQ ID NO:55.

[0264] In certain embodiments, the stationary phase promoter is P dps In certain embodiments, P dps The promoter comprises the nucleotide sequence shown in SEQ ID NO: 56. In certain embodiments, P dps The promoter consists of the nucleotide sequence shown in SEQ ID NO:56.

[0265]

[0266]

[0267] 3.1.3. Genetic markers

[0268] In some embodiments, the recombinant polynucleotides of the present invention include genetic markers. These genetic markers allow the selection of microorganisms with one or more desired polynucleotides (e.g., recombinant polynucleotides). In some embodiments, the genetic marker is an antibiotic resistance marker selected from the following group: apramycin resistance, ampicillin resistance, kanamycin resistance, spectinomycin resistance, tetracycline resistance, neomycin resistance, chloramphenicol resistance, gentamicin resistance, erythromycin resistance, carbenicillin resistance, actinomycin D resistance, neomycin resistance, polymyxin resistance, bleomycin resistance, and streptomycin resistance. In some embodiments, the genetic marker includes a coding sequence for an antibiotic resistance protein (e.g., the beta-lactamase of some ampicillin resistance markers) and a promoter or enhancer element that drives the coding sequence to be expressed in the microorganism of the present invention. In some embodiments, the microorganism of the present invention grows under conditions where the antibiotic resistance marker is expressed and imparts resistance to the microorganism, thereby selecting the microorganism that successfully integrated the marker. In some embodiments, the genetic marker is an auxotrophic marker. In some embodiments, auxotrophic markers are genes that participate in vitamin, amino acid, fatty acid synthesis or carbohydrate metabolism. In some embodiments, auxotrophic markers are genes for synthesizing amino acids. In some embodiments, auxotrophic markers are genes for synthesizing glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, methionine, asparagine, glutamine, lysine, arginine, histidine, aspartic acid or glutamic acid. In some embodiments, auxotrophic markers are genes for synthesizing adenosine, biotin, thiamine, leucine, glucose, lactose or maltose. In some embodiments, microorganisms of the present disclosure express and give microbial growth (lack of the endogenous ability to produce nutrients) under the conditions of auxotrophic resistance markers lacking corresponding nutrients or culture medium, thereby selecting the microorganisms that successfully integrate markers.

[0269] 3.2. Gene deletion and reduced expression

[0270] In certain embodiments, the disclosure further provides methods for introducing deletions of any gene or enzyme disclosed herein. These deletions can be generated by any suitable gene editing method. In certain embodiments, the deletion is generated by a method including homologous recombination, zinc finger nucleases, meganucleases, transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR) systems or combinations thereof.

[0271] In certain embodiments, the deletion is produced by the CRISPR system. The clustered regularly interspaced short palindromic repeats (CRISPR) system is a genome editing tool found in prokaryotic cells. When used for genome editing, the system includes Cas9 (a protein that can modify DNA using crRNA as its guide), CRISPR RNA (crRNA, which contains the RNA used by Cas9 to guide it to the correct part of the host DNA and the region bound to tracrRNA (usually in the form of a hairpin loop) to form an active complex with Cas9), transactivating crRNA (tracrRNA, which binds to crRNA and forms an active complex with Cas9) and an optional DNA repair template portion (guiding the cell repair process to allow the insertion of DNA of a specific DNA sequence). Multiple crRNAs and tracrRNAs can be packaged together to form a single guide RNA (sgRNA). The sgRNA can be connected to the Cas9 gene and made into a plasmid for transfection into cells. In certain embodiments, the CRISPR system comprises a base editor. In certain embodiments, the CRISPR system comprises a transposase / recombinase. In certain embodiments, the CRISPR system comprises a guide editor. In certain embodiments, the CRISPR system comprises a genetic regulator. In certain embodiments, the CRISPR system comprises a CRISPRoff system. Additional details regarding the CRISPR system of the present disclosure can be found in Anzalone et al., Nature biotechnology 38.7 (2020): 824-844 and et al., Cell 184.9(2021):2503-2519 and Jiang et al., Appl Environ Microbiol. 2015 Apr;81(7):2506-14, the contents of each of which are incorporated herein by reference in their entirety.

[0272] In certain embodiments, the deletion is generated by a zinc finger nuclease. Zinc finger nuclease (ZFN) is an artificial restriction enzyme generated by combining a zinc finger DNA binding domain with a DNA cleavage domain. The zinc finger domain can be engineered to target a specific DNA sequence and allow the zinc finger nuclease to target the desired sequence within the genome. The DNA binding domain of an individual ZFN typically contains multiple individual zinc fingers repeated, and each can identify multiple base pairs. The most common method for generating new zinc finger domains is to combine smaller zinc finger "modules" of known specificity. The most common cleavage domain in ZFN is the non-specific cleavage domain from the type IIs restriction endonuclease FokI.

[0273] In certain embodiments, the deletion is generated by a TALEN system. Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cut specific DNA sequences. The TALEN system operates on almost the same principle as ZFNs. They are generated by combining a transcription activator-like effector DNA binding domain with a DNA cleavage domain. Transcription activator-like effectors (TALEs) contain a 33-34 amino acid repeat motif with two variable positions that strongly recognize specific nucleotides. By assembling an array of these TALEs, the TALE DNA binding domain can be engineered to bind to the desired DNA sequence, thereby guiding the nuclease to cut at a specific location in the genome.

[0274] In certain embodiments, the deletion is generated by a meganuclease. A meganuclease is an endodeoxyribonuclease that recognizes double-stranded DNA sites of about 12 to about 40 base pairs that occur only once in the genome. Meganucleases are some of the most specific naturally occurring restriction enzymes. Meganucleases are also defined as molecular DNA scissors because they can replace, eliminate, or modify sequences in a highly targeted manner. Protein engineering allows for modification of their recognition and target sequences.

[0275] In certain embodiments, the disclosure also provides methods for reducing the expression of any gene or enzyme disclosed herein. In certain embodiments, reducing the expression of genes and enzymes disclosed herein includes using oligonucleotides (e.g., zwf, manA, rpiB, pfkA, pfkB, etc.) with mRNA complementary sequences to genes disclosed herein. Non-limiting examples of these oligonucleotides include small interfering RNA (siRNA), short hairpin RNA (shRNA), and microRNA (miRNA). In certain embodiments, these oligonucleotides can be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to at least a portion of a zwfmRNA sequence. In certain embodiments, these oligonucleotides can be identical to at least a portion of a zwfmRNA sequence. In certain embodiments, these oligonucleotides may be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to at least a portion of a pfk mRNA sequence. In certain embodiments, these oligonucleotides may be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to at least a portion of a pfkA mRNA sequence. In certain embodiments, these oligonucleotides may be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to at least a portion of a pfkB mRNA sequence. In certain embodiments, these oligonucleotides may be at least about 99% identical to at least a portion of a pfkB mRNA sequence. In certain embodiments, these oligonucleotides may be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to at least a portion of the rpiB mRNA sequence. In certain embodiments, these oligonucleotides may be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to at least a portion of the manA mRNA sequence. In certain embodiments, these oligonucleotides may be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to at least a portion of the manA mRNA sequence. In certain embodiments, the antisense, shRNA, miRNA, or siRNA molecules may include DNA or atypical or non-naturally occurring residues, such as, but not limited to, phosphorothioate residues.

[0276] In some embodiments, reducing the expression of the genes and enzymes disclosed herein can include using CRISPR, which can mutate the coding sequence or promoter to reduce or eliminate the expression of the gene product, or CRISPRi can be targeted to the genes disclosed herein to reduce the expression of one or more genes. See, for example, Arroya-Olarte et al., Microorganisms, 2021 Apr; 9(4): 844. Zhang et al. Front. Microbiol. (2021 Mar 31).

[0277] In certain embodiments, the reduction in expression of the genes and enzymes disclosed herein comprises the use of a CRISPRi system. The CRISPRi system silences genes at the transcriptional level and has fewer sequence-specific off-target effects than RNAi. In certain embodiments, the CRISPRi system comprises a catalytically inactive Cas9 (dCas9). dCas9 is a programmable transcription factor that can target a promoter via sgRNA and act as a repressor in the promoter. In certain embodiments, dCas9 comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or at least about 100% identical to the amino acid sequence shown in SEQ ID NO: 57. In certain embodiments, dCas9 comprises the amino acid sequence shown in SEQ ID NO: 57. In certain embodiments, dCas9 consists of the amino acid sequence shown in SEQ ID NO: 57. SEQ ID NO: 57 is as follows:

[0278]

[0279]

[0280] In certain embodiments, dCas9 is regulated by a promoter (e.g., as described in Section 3.1.2). In certain embodiments, the promoter is an arabinose-inducible promoter. In certain embodiments, the promoter is a stationary phase promoter. In certain embodiments, the CRISPRi system includes a small guide RNA (sgRNA). In certain embodiments, the sgRNA of the CRISPRi system targets genes encoding enzymes of competitive pathways. For example, but not limited to, the sgRNA can target the zwf gene, the pgm gene, the pfkA gene, the pfkB gene, the ManA gene, or the RpiB gene. In certain embodiments, the sgRNA can target any part of a gene. For example, but not limited to, the sgRNA can target a promoter, an operon, or a sequence encoding a protein.

[0281] In certain embodiments, the CRISPRi system comprises dCas9 and sgRNA. In certain embodiments, dCas9 is regulated by an inducible promoter. In certain embodiments, the inducible promoter is P tet In certain embodiments, the sgRNA targets the pfkB gene. In certain embodiments, the sgRNA targets the promoter of the pfkB gene.

[0282] 3.3. Transformation and gene editing

[0283] In certain embodiments, the present disclosure provides transformation uses of the plasmids and vectors disclosed herein. The vectors and plasmids disclosed herein can be transformed into cells by any system known in the art. For example, but not limited to, the microorganisms of the present disclosure can be transformed by particle bombardment, chemical transformation, Agrobacterium transformation, nanospike transformation, electroporation, and viral transformation.

[0284] In certain embodiments, the vectors of the present disclosure can be introduced into microorganisms using a variety of techniques, including transformation, transfection, transduction, viral infection, gene gun or Ti-mediated gene transfer. Non-limiting examples of these methods include calcium phosphate transfection, DEAE-dextran-mediated transfection, lipofection and electroporation (see, for example, Davis, L, Dibner, M., Battey, I., 1986 "Basic Methods in Molecular Biology"; Gietz et al., Nucleic Acids Res. 27: 69-74 (1992); Ito et al., J. Bacterol. 153: 163-168 (1983); and Becker and Guarente, "Methods in Enzymology" 194: 182-187 (1991)). In certain embodiments, the transformed microorganism is referred to as a recombinant microorganism.

[0285] In certain embodiments, the present disclosure provides methods for introducing exogenous proteins (e.g., nucleases), RNA (e.g., gRNA), and DNA (e.g., recombinant polynucleotides disclosed herein) into microorganisms. Various methods for achieving this have been previously described, including direct transfection of proteins and nucleotide sequences or DNA transformation, followed by intracellular expression of RNA and protein (see, e.g., Dicarlo, JE et al. "Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems." Nucleic Acids Res (2013). doi: 10.1093 / nar / gkt135; Ren, ZJ, Baumann, RG, and Black, LW "Cloning of linear DNAs in vivo by overexpressed T4 DNA ligase: construction of a T4 phage hoc gene display vector." Gene 195, 303-311 (1997); Lin, S., Staahl, BT, Alla, RK, and Doudna, JA "Enhanced homology-directed human genome engineering by controlled timing of CRISPR / Cas9 delivery." Elife 3, e04766 (2014)).

[0286] 3.4. Reorganization system

[0287] In certain embodiments, the present disclosure also provides a homologous recombination system for editing (e.g., insertion, deletion) in a microorganism. In certain embodiments, the homologous recombination system can be native to the host cell or introduced into a cell host. For example, but not limited to, the gene of the homologous recombination system can be introduced into a plasmid, introduced into a linear DNA fragment, introduced as an RNA or RNA group and translated from an RNA or RNA group, or introduced as a protein or protein group. In certain embodiments, the method includes a recombinant polynucleotide disclosed herein. In certain embodiments, the polynucleotide includes a sequence (e.g., a left homology arm and a right homology arm) homologous to a region in a nucleic acid (e.g., a genome, a plasmid, etc.), so that the left homology arm and the right homology arm are separated via designed genetic editing (e.g., promoter, insertion, replacement, SNP, terminator, degradation sub, tag sequence, degradation signal sequence or deletion). In certain embodiments, the recombinant polynucleotide includes a genetic marker, a reverse selective genetic marker (e.g., SacB or PheS) and an origin of replication (e.g., R6K).

[0288] In certain embodiments, any method disclosed herein (for example, transformed by electroporation, joining, etc.) is used to introduce a recombinant polynucleotide comprising a homology arm and a sequence for gene editing into a microorganism. In certain embodiments, after transformation, the resulting transformant can be inoculated on a culture medium to select a transformant expressing a selective genetic marker. The recombination of the target locus in a plasmid comprising homology arms and nucleic acid (for example, genome, plasmid, etc.) can occur at one of two homology sites targeted by the homology arms present on the plasmid, and the two homology sites are flanked by the gene editing of the design. In certain embodiments, the resulting transformant is grown as a colony on a selective culture medium, and can be selected and inoculated on a second selective culture medium (for example, a reverse selective culture medium). In certain embodiments, the second selective culture medium allows selection of cells comprising desired genetic editing.

[0289] In some embodiments, method disclosed herein includes using the protein from one or more recombination systems.Described recombination system can be microorganism endogenous or microorganism exogenous.In some embodiments, protein from one or more recombination systems can be introduced as nucleic acid (for example, as plasmid, linear DNA or RNA or integron), and be integrated into the genome of host cell or stably expressed from extrachromosomal element.In some embodiments, protein from one or more recombination systems can be introduced as RNA and translated by host cell.In some embodiments, protein from one or more recombination systems can be introduced into host cell as protein.The limiting examples of recombination system includes λ red recombination system, RecET recombination system, Red / ET recombination system, any homologue, orthologue or paralogue of recombination system from λ red recombination system, RecET recombination system, Red / ET recombination system, λ red mediation, or its any combination.The details of the recombination system from the RecET recombination system may be any of the recombination systems described in: Zhang Y., Buchholz F., Muyrers JPP and Stewart AF "A new logic for DNA engineering using recombination in E. coli." Nature Genetics 20 (1998) 123-128; Muyrers, JPP, Zhang, Y., Testa, G., Stewart, AF "Rapid modification of bacterial artificial chromosomes by ET-recombination." Nucleic Acids Res. 27 (1999), 1555-1557; Zhang Y., Muyrers JPP, Testa G. and Stewart AF "DNA cloning by homologous recombination in E. coli." Nature Biotechnology 18 (2000), 1314-1317 and Muyrers JP et al., "Techniques: Recombinogenic engineering--new options for cloning and manipulating DNA," Trends Biochem Sci. 2001 May; 26(5): 325-31, incorporated herein by reference in their entirety.

[0290] 4. Method for producing allulose

[0291] The present disclosure also provides methods for producing psicose. Predicted ΔG for cell-free methods (e.g., in vitro synthesis) o The value is +5 kJ / mol, and therefore thermodynamically unfavorable. In certain embodiments, the method for producing psicose disclosed herein includes culturing a microorganism (eg, a microorganism disclosed in Section 2) and purifying psicose.

[0292] Cell culture

[0293] The present disclosure provides methods for cultivating the microorganisms disclosed herein. As used herein, "culturing" cells refers to introducing an appropriate culture medium under appropriate conditions to promote the growth of the cells. In certain embodiments, liquid or solid growth medium is used for cultivation. In certain embodiments, cultivation is carried out under aerobic or anaerobic conditions, depending on the requirements of the microorganism and its desired metabolic state. In certain embodiments, cultivation includes specific conditions such as temperature, pressure, light, pH and cell density.

[0294] In certain embodiments, the method for producing psicose includes a culture medium for culturing recombinant bacteria. As used herein, "culture medium" refers to any composition or broth that supports the growth of the microorganisms disclosed herein. The culture medium can be liquid or solid. In certain embodiments, the culture medium includes nutrients, salts, buffers, elements, and other compounds that support cell growth and vitality. In addition, the culture medium can include a nitrogen source, a carbon source, amino acids, carbohydrates, trace elements, vitamins, and minerals. In certain embodiments, the culture medium includes a complex extract (e.g., yeast extract). In certain embodiments, the culture medium is enriched to support rapid growth. In certain embodiments, the culture medium is modified to support slower growth. In certain embodiments, the culture medium includes an agent (e.g., antibiotic) that can inhibit the growth of contaminating organisms or kill contaminating organisms. In certain embodiments, the culture medium includes an agent (e.g., IPTG) that can activate an inducible promoter or enzyme. Non-limiting examples of culture media encompassed by the present disclosure include M9 culture medium, Lysogeny Broth (LB), Terrific Broth (TB), and YT broth (YT broth). In certain embodiments, the culture medium contains a substrate that is converted to psicose by the recombinant microorganism.

[0295] In certain embodiments, substrate is sugar (such as glucose or fructose), which can be phosphorylated by bacteria and converted into fructose-6-phosphate by kinase (such as hexokinase). In certain embodiments, substrate is glucose. In certain embodiments, glucose can be derived from cellulose, C5 sugar, hemicellulose and / or xylose. In certain embodiments, substrate is a component of culture medium. In certain embodiments, substrate culture medium is supplemented. In certain embodiments, substrate persists in culture medium. In certain embodiments, substrate is supplemented in the growth phase. In certain embodiments, substrate is supplemented in the stationary phase.

[0296] Purification of Psicose

[0297] In certain embodiments, the methods of the present disclosure further include purifying the psicose produced by the microorganisms of the present disclosure, for example, from a cell culture or cell culture medium. Various methods known in the art can be used to purify products from microorganisms or microbial cultures. In certain embodiments, one or more products can be purified continuously, for example, from a continuous culture. In certain embodiments, one or more products can be purified independently of fermentation, for example, from a batch or fed-batch culture. Those skilled in the art will appreciate that the specific purification method used may depend on the microorganism, culture conditions, and / or the specific product, among other things.

[0298] In certain embodiments, purifying psicose includes separating or filtering the microorganism from the cell culture medium, separating psicose from the culture medium (e.g., by chromatography), concentrating the water (e.g., by evaporation), and lyophilizing the psicose.

[0299] Purity

[0300] In certain embodiments, the methods of the present disclosure allow for obtaining high purity values ​​of psicose. As used herein, the term "psicose purity" refers to the percentage value of the psicose concentration to the sum of the psicose, mannose, and glucose concentrations. In other words, the term psicose purity refers to the relative value of psicose that does not contain mannose and / or glucose (e.g., foreign or contaminating sugars). In certain embodiments, the psicose purity is calculated using the sugar concentration and the following equation:

[0301]

[0302] In order to determine the purity (e.g., psicose purity) of a sample (e.g., culture medium sample after production, sample after purification), high performance liquid chromatography (HPLC) can be used to analyze the concentration of glucose, psicose, and mannose. Glucose, psicose, and mannose standards of known concentration can be run on HPLC, and the area under each corresponding peak can be integrated. For each sugar standard, peak integral can be drawn for concentration and fitted with a best fit line. While using standard, production samples can be run on HPLC. Standard can be used to identify the corresponding sugar peak in each production sample (e.g., culture medium sample after production, sample after purification). Each sample peak can be integrated, and their area recorded. Using a best fit line, peak integral can be used to find the sugar concentration in each sample.

[0303] In certain embodiments, the psicose purity is determined after culturing the microorganisms disclosed herein. In certain embodiments, the psicose purity has a percentage value (%) between about 50% and about 100%. In certain embodiments, the psicose purity has a percentage value of at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%. In certain embodiments, the psicose purity has a percentage value of at least about 80%. In certain embodiments, the psicose purity has a percentage value of at least about 90%. In certain embodiments, the psicose purity has a percentage value of at least about 95%. In certain embodiments, the psicose purity has a percentage value of at least about 100%.

[0304] In certain embodiments, the purity of psicose is measured after purification. In certain embodiments, the percentage value (%) of psicose purity is between about 50% and about 100%. In certain embodiments, the percentage value of psicose purity is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%. In certain embodiments, the percentage value of psicose purity is at least about 80%. In certain embodiments, the percentage value of psicose purity is at least about 90%. In certain embodiments, the percentage value of psicose purity is at least about 95%. In certain embodiments, the percentage value of psicose purity is at least about 100%.

[0305] In certain embodiments, the purity of psicose meets or exceeds the standards set by the American Chemical Society (ACS) or the standards defined in the United States Pharmacopoeia (USP).

[0306] 5. Food

[0307] The present disclosure also provides delivery systems and methods for food products, which include psicose produced and / or generated by any of the microorganisms disclosed herein.

[0308] As used herein, the term "food" includes any food, such as those listed in 21 CFR 101.12. Non-limiting examples of such food include frozen desserts, baked goods, fillings, nutritional drinks, beverages, salad dressings or similar sauces, sauces, frostings, puddings and custards, batters, and the like. U.S. Patent No. 6,536,599 discloses various baked goods, the disclosure of which is incorporated herein by reference in its entirety. Non-limiting examples of baked goods include cookies, cakes, rolls, pastries, pie dough, brownies, bread, bagels, and the like. Allulose prepared and / or produced by any of the microorganisms disclosed herein is also suitable as an ingredient in frozen foods.

[0309] In certain embodiments, a food product is prepared by mixing psicose with any optional ingredients in an ingestible carrier to form a homogeneous mixture. The final composition can be readily prepared using standard methods and equipment commonly known to those skilled in the art (such as the confectionery field). Equipment useful in accordance with the present disclosure includes mixing equipment well known in the art, and thus the selection of a specific equipment will be readily apparent to those skilled in the art.

[0310] In certain embodiments, the present application relates to improved edible food products produced by the methods disclosed herein.In certain embodiments, the food product can be produced by processes well known to those of ordinary skill in the art for producing edible products.

[0311] In certain embodiments, psicose prepared and / or produced by any of the microorganisms disclosed herein can be dissolved or dispersed in many known edible liquids, solids, or other carriers, such as water at a neutral, acidic, or alkaline pH, fruit or vegetable juice, vinegar, marinade, beer, wine, natural water / fat emulsions (such as milk or condensed milk), whey or whey products, edible oils and shortenings, fatty acids, certain low molecular weight oligomers of propylene glycol, glycerides of fatty acids, and dispersions or emulsions of such hydrophobic substances in aqueous media, salts (such as sodium chloride), plant powders, solvents (such as ethanol), solid edible diluents (such as vegetable powder or flour), etc., and then combined with a precursor of an edible product or pharmaceutical product, or directly applied to an edible product or pharmaceutical product.

[0312] Those skilled in the art of food preparation and marketing are well aware of the numerous types, subtypes, and categories of edible compositions and use well-known and recognized terminology to refer to these edible compositions in their efforts to prepare and market various edible compositions. A list of such terminology is provided below. It is specifically contemplated that psicose produced and / or generated by any of the microorganisms disclosed herein can be used to modify or enhance the taste of the following edible compositions, either alone or in all reasonable combinations or mixtures thereof.

[0313] In certain embodiments, food products mixed with psicose prepared and / or produced by any of the microorganisms disclosed herein include, for example, the wet soup category, the dehydrated and cooked food category, the beverage category, the frozen food category, the snack food category, and the condiments or condiment mixes described herein.

[0314] In certain embodiments, allulose produced and / or generated by any of the microorganisms disclosed herein is combined with one or more of candies, chocolate candies, tablets, chocolate countlines, bagged selfies / softlines, boxed assortments, standard boxed assortments, twistwrapped miniatures, seasonal chocolates, chocolates with toys, assortments, other chocolate candies, mints, standard mints, strong mints, hard candies, lozenges, chewing gum, jellies and chews, toffees, caramels and nougats, medicinal candies, lollipops, licorice, other candies, chewing gum, chewing gum, sugary chewing gum, sugar-free chewing gum, functional chewing gum, bubble gum, bread, packaged / industrial bread, unpackaged / artisanal bread, pastries, cakes, packaged / industrial cakes, unpackaged / artisanal cakes, cookies, chocolate-coated cookies, sandwich cookies, filled cookies, savory cookies and crackers, bread substitutes, breakfast cereals cereals, ready-to-eat cereals, breakfast cereals for families, flakes, oatmeal, other ready-to-eat cereals, breakfast cereals for children, hot cereals, ice cream, ready-to-eat ice cream, single-serve dairy ice cream, single-serve water-based ice cream, multi-pack dairy ice cream, multi-pack water-based ice cream, family-sized ice cream, family-sized dairy ice cream, ice cream desserts, bulk ice cream, family-sized water-based ice cream, frozen yogurt, artisanal ice cream, dairy products, milk, fresh / pasteurized milk, whole fresh / pasteurized milk, semi-skimmed fresh / pasteurized milk, shelf-stable / UHT milk, whole shelf-stable / UHT milk, semi-skimmed shelf-stable / UHT milk, skimmed shelf-stable / UHT milk Milk, goat's milk, condensed milk / evaporated milk, pure condensed milk / evaporated milk, flavored milk, functional and other condensed milk, flavored milk beverages, pure dairy flavored milk beverages, fruit juice flavored milk beverages, soy milk, yogurt beverages, fermented dairy beverages, coffee whiteners, milk powder, flavored milk powder beverages, cream, cheese, processed cheese, spreadable processed cheese, non-spreadable processed cheese, unprocessed cheese, spreadable unprocessed cheese, hard cheese, packaged hard cheese, unpackaged hard cheese, yogurt, plain / natural yogurt, flavored yogurt, fruit yogurt, probiotic yogurt, drinkable yogurt, plain drinkable yogurt, probiotic drinkable yogurt, refrigerated and shelf-stable desserts, dairy desserts, soy sweeteners snacks, refrigerated snacks, fresh cheese and quark, plain fresh cheese and quark, flavored fresh cheese and quark, salted fresh cheese and quark, sweet and savory snacks, fruit snacks, potato chips / crisps, extruded snacks, tortillas / corn chips, popcorn, pretzels, nuts, other sweet and savory snacks, snack bars, granola bars, breakfast bars, energy bars, fruit bars, other snack bars, meal replacement products, weight loss products, recovery drinks, ready meals, canned ready meals, frozen ready meals, dried ready meals, refrigerated ready meals, dinner mixes, frozen pizza, refrigerated pizza, soup, canned soup, dehydrated soup,Instant soup, refrigerated soup, UHT soup, frozen soup, pasta, canned pasta, dried pasta, refrigerated / fresh pasta, noodles, white noodles, instant noodles, cup / bowl instant noodles, packaged instant noodles, refrigerated noodles, snack noodles, canned food, canned meat and meat products, canned fish / seafood, canned vegetables, canned tomatoes, canned beans, canned fruit, canned ready meals, canned soup, canned pasta, other canned food, frozen food, frozen processed red meat, frozen processed Poultry, Frozen Processed Fish / Seafood, Frozen Processed Vegetables, Frozen Meat Substitutes, Frozen Potatoes, Baked Potato Chips, Other Baked Potato Products, Non-Oven Frozen Potatoes, Frozen Baked Products, Frozen Desserts, Frozen Ready-to-Eat Meals, Frozen Pizza, Frozen Soup, Frozen Noodles, Other Frozen Foods, Dried Foods, Dessert Mixes, Dried Ready-to-Eat Meals, Dehydrated Soup, Instant Soup, Dried Noodles, White Noodles, Instant Noodles, Instant Noodles in Cups / Bowls, Instant Noodles in Packages, Refrigerated Foods, Refrigerated Processed Meats, Refrigerated Fish / Seafood products, refrigerated processed fish, refrigerated breaded fish, refrigerated smoked fish, refrigerated lunch kits, refrigerated ready meals, refrigerated pizzas, refrigerated soups, refrigerated / fresh noodles, refrigerated noodles, oils and fats, olive oil, vegetable and seed oils, edible fats and fats, butter, margarine, spreadable oils, functional spreadable oils and fats, sauces, condiments and dressings, tomato paste and tomato puree, bouillon / stock cubes, stock cubes, gravy granules, liquid stocks and soup bases, herbs and spices, fermented sauces, soy sauce-based sauces, Pasta sauces, wet sauces, dry sauces / powder mixes, ketchup, mayonnaise, regular mayonnaise, mustard, salad dressings, regular salad dressings, reduced-fat salad dressings, vinaigrettes, dips, pickles, other sauces, dressings and condiments, baby food, milk powder, standard milk powder, follow-on milk powder, toddler milk powder, hypoallergenic milk powder, prepared baby food, dry baby food, other baby food, spreads, jams and preserves, honey, chocolate spreads, nut spreads and yeast spread mixes.

[0315] 5.1. Chewing gum

[0316] In certain embodiments, the psicose prepared and / or generated by any microorganism disclosed herein can be used in low-calorie chewing gum formulations, and can also be used in sugar-containing chewing gums. U.S. Patent No. 6,899,911 discloses various details of chewing gum compositions, and the disclosure of this patent is incorporated herein by reference in its entirety. The chewing gum compositions of the current disclosed subject matter follow the general pattern outlined below. Typically, the chewing gum composition typically contains a chewable gum base portion that is substantially free of water and insoluble in water, a water-soluble bulk portion, and a flavoring that is typically insoluble in water. The water-soluble portion dissipates along with the flavoring during chewing. The gum base portion remains in the mouth throughout the chewing process. The insoluble gum (gum) base typically includes an elastomer, an elastomer solvent, a plasticizer, a wax, an emulsifier, and an inorganic filler. Plastic polymers (such as polyvinyl acetate) are also often included, which are also plasticizers to some extent. Other available plastic polymers include polyvinyl laurate, polyvinyl alcohol, and polyvinyl pyrrolidone. Elastomers can include polyisobutylene, butyl rubber (isobutylene-isoprene copolymer) and styrene butadiene rubber, as well as natural latex (such as chicle). Elastomer solvents are typically resins (such as terpene resins). Plasticizers, sometimes called softeners, are typically fats and oils, including tallow, hydrogenated and partially hydrogenated vegetable oils, and cocoa butter. Commonly used waxes include paraffin wax, microcrystalline wax, and natural waxes (such as beeswax and carnauba wax). Microcrystalline waxes, especially those with high crystallinity, can be considered thickeners or texture modifiers.

[0317] In certain embodiments, the insoluble chewing gum matrix accounts for about 5% to about 95% by weight of the chewing gum. More preferably, the insoluble chewing gum matrix is ​​10% to 50% by weight of the chewing gum, and most preferably is about 20% to 35% by weight of the chewing gum. The chewing gum matrix also typically includes a filler component. The filler component can be calcium carbonate, magnesium carbonate, talc, dicalcium phosphate, etc. The filler can account for about 5% to about 60% by weight of the chewing gum matrix. Preferably, the filler is about 5% to 50% by weight of the chewing gum matrix.

[0318] The chewing gum base also typically contains softeners, which include glyceryl monostearate and glyceryl triacetate. The chewing gum base may also contain optional ingredients such as antioxidants, pigments, and emulsifiers. The subject matter disclosed herein contemplates the use of any commercially acceptable chewing gum base.

[0319] The water-soluble portion of chewing gum may also contain softeners, sweeteners, flavorings, physiological cooling agents, and combinations thereof. Sweeteners typically act as bulking agents in chewing gum. Bulking agents typically comprise from about 5% to about 95% of the chewing gum composition.

[0320] In order to optimize the chewability and mouthfeel of chewing gum, softeners are added to chewing gum. Softeners, also known in the art as plasticizers or plasticizers, typically comprise from about 0.5% to about 15% of the chewing gum. Softeners contemplated by the present disclosure include glycerin, lecithin, and combinations thereof. In addition, aqueous sweetener solutions (such as solutions containing sorbitol, hydrogenated starch hydrolysates, corn syrup, and combinations thereof) can be used as softeners and binders in chewing gum.

[0321] As mentioned above, the psicose prepared and / or generated by any microorganism disclosed herein can be used in low-calorie chewing gum formulations. However, sugar-containing formulations are also within the scope of the present invention. Sugar sweeteners generally include sugar-containing components known in the chewing gum field, including but not limited to sucrose, glucose, maltose, dextrin, dry invert sugar, fructose, galactose, corn syrup solids, etc., alone or in any combination. The psicose prepared and / or generated by any microorganism disclosed herein can also be used in combination with sugar-free sweeteners. Generally, sugar-free sweeteners include components with sweetening properties but do not contain commonly known sugars, including but not limited to sugar alcohols, such as sorbitol, hydrogenated isomaltulose, mannitol, xylitol, lactitol, erythritol, hydrogenated starch hydrolysate, maltitol, etc., alone or in any combination.

[0322] Depending on the specific sweetness release profile and shelf life desired, coated or uncoated high-intensity sweeteners can be used in chewing gum compositions, or can be used in center coatings made from these chewing gum compositions. High-intensity sweeteners, preferably aspartame, can be used in amounts of about 0.01% to about 3.0%. Encapsulated aspartame is a high-intensity sweetener with improved stability and release characteristics compared to free aspartame. Free aspartame can also be added, and when aspartame is used, a combination of some free aspartame and encapsulated aspartame is preferred. Other high-intensity sweeteners that can be used in chewing gum centers are: saccharin, samatin, alitame, saccharin salts, sucralose, stevia, and acesulfame potassium. Overall, chewing gum compositions preferably contain about 0.5% to about 90% sweeteners. The most typical sweeteners will include at least one bulk sweetener and at least one high-intensity sweetener. Optional ingredients such as colors, emulsifiers, and pharmaceutical agents may also be added as separate components of the chewing gum composition, or as part of the chewing gum base.

[0323] Aqueous syrups such as corn syrup and hydrogenated corn syrup can be used, especially when their water content is reduced. This is preferably achieved by co-evaporating the aqueous syrup with a plasticizer such as glycerol or propylene glycol to a water content of less than 10%. A preferred composition includes hydrogenated starch hydrolysate solids and glycerol. Such syrups and methods for their preparation are discussed in detail in U.S. Patent No. 4,671,967.

[0324] The method for making chewing gum according to the disclosed subject matter comprises sequentially adding the various chewing gum ingredients to any commercially available mixer known in the art. After the ingredients are thoroughly mixed, the chewing gum is discharged from the mixer and formed into the desired shape, such as rolled into sheets and cut into strips, extruded into blocks, or cast into pellets. Typically, the ingredients are first mixed by melting the chewing gum base and adding it to the running mixer. The base can also be melted in the mixer itself. At this point, color or emulsifiers can also be added, as well as syrup and some leavening agent. Further portions of leavening agent can then be added to the mixer. The flavoring system is usually added with the last portion of leavening agent. If the flavoring system is coated or otherwise modified to change its release rate when incorporated into the delivery system, it is preferably added after the last portion of leavening agent is added. The entire mixing process typically takes five to twenty minutes, but longer mixing times may sometimes be required. Those skilled in the art will recognize that many variations of the above procedure can be followed.

[0325] If the chewing gum composition is in pellet or ball form, it can be coated. The coating is initially in the form of a liquid syrup containing from about 30% to about 80% or 85% sugar or sugar alcohol and from about 15% or 20% to about 70% solvent (such as water). Typically, the coating process is performed in conventional panning equipment. The gum center tablets to be coated are placed in the panning equipment to form a moving mass.

[0326] The material or syrup that will ultimately form the coating is applied or distributed over the gum center tablet. Psicose can be added before, during, or after the syrup is applied to the gum center. Once the coating has dried to form a hard surface, additional syrup can be added to create multiple coatings or layers. Psicose may or may not be added to any coating and / or layer.

[0327] During the pan coating process, the syrup is added to the gum center tablets at a temperature ranging from about 100°F to about 240°F. Preferably, the syrup temperature is about 140°F to about 200°F. Most preferably, the syrup temperature should be kept constant throughout the process to prevent crystallization of the polyols in the syrup. The syrup can be mixed with, sprayed on, poured onto, or added to the gum center tablets in any manner known to those skilled in the art.

[0328] In certain embodiments, the soft coating is formed by adding a powder coating after the liquid coating. The powder coating may include natural sugar gum hydrolysates, maltodextrin, gelatin, cellulose derivatives, starch, modified starch, sugar, sugar alcohol, natural sugar gum, and fillers such as talc and calcium carbonate.

[0329] The various components of the coating on the gum center can be applied in a single layer or multiple layers. Typically, multiple layers are obtained by applying a single layer of coating, allowing each layer to dry, and then repeating the process. The amount of solids added for each coating step depends primarily on the concentration of the coating syrup. Any number of coatings can be applied to the gum center tablet. Preferably, no more than about 75 coatings are applied to the gum center. More preferably, less than about 60 coatings are applied, and most preferably, from 30 to about 60 coatings are applied. Regardless, the subject matter of the present disclosure contemplates applying an amount of syrup sufficient to produce a coated gum product containing from about 10% to about 65% coating. Preferably, the finished product will contain from about 20% to about 50% coating.

[0330] Those skilled in the art will recognize that to achieve multiple coatings, multiple pre-weighed aliquots of coating syrup can be applied to the gum center. However, it is contemplated that the volume of the syrup aliquots applied to the gum center can vary throughout the coating process.

[0331] Once the syrup coating is applied to the gum center, the syrup is dried in an inert medium. A preferred drying medium comprises air. Preferably, the forced drying air contacts the wet syrup coating at a temperature in the range of about 70°F to about 110°F. More preferably, the drying air has a temperature in the range of about 80°F to about 100°F. The present invention also contemplates that the drying air has a relative humidity of less than about 15%. Preferably, the relative humidity of the drying air is less than about 8%.

[0332] Dry air can be passed and mixed onto the syrup-coated gum centers by any means known in the art. Preferably, the dry air is blown over and around the syrup-coated gum centers at a flow rate of approximately 2,800 cubic feet per minute (for large-scale operations). If the amount of material to be processed is small or if smaller equipment is used, a lower flow rate should be used. If flavoring is applied after the syrup coating has dried, the presently disclosed subject matter contemplates drying the flavor with or without a drying medium.

[0333] The amount of psicose used herein generally depends on preferences based on factors such as the type of final chewing gum composition, the individual flavors, the chewing gum base used, and the desired flavor concentration. Thus, the amount of psicose can be varied to achieve the desired results in the final product, and such variations are within the capabilities of those skilled in the art without undue experimentation. In a chewing gum composition, the amount of psicose produced and / or generated by any of the microorganisms disclosed herein is generally from about 0.02% to about 5% by weight, preferably from about 0.1% to about 2% by weight, and more preferably from about 0.8% to about 1.8% by weight of the chewing gum composition.

[0334] 5.2. Candy

[0335] Another important aspect of the presently disclosed subject matter includes candy compositions incorporating psicose produced and / or generated by any of the microorganisms disclosed herein and methods for preparing the candy compositions. The preparation of candy formulations is well known in the art. Candy items are categorized as either "hard" candies or "soft" candies. Psicose produced and / or generated by any of the microorganisms disclosed herein can be incorporated into candies by mixing the compositions of the presently disclosed subject matter into conventional hard and soft candies.

[0336] Hard candies can be processed and formulated by conventional means. Typically, hard candies have a matrix comprising a mixture of sugar and other carbohydrate leavening agents, which is maintained in an amorphous or glassy state. Hard candies can also be sugar-free. Hard candies can also be low-calorie. This form is considered a solid syrup of sugar, which typically has about 0.5% to about 1.5% moisture. Such materials typically contain up to about 92% sugar, up to about 55% corn syrup, and about 0.1% to about 5% water, based on the weight of the final composition. The syrup component is typically made from sucrose and corn syrup, but may include other materials. In certain embodiments, the syrup component includes psicose prepared and / or generated by any microorganism disclosed herein. Other ingredients, such as flavorings, sweeteners, acidulants, coloring agents, etc., may also be added.

[0337] Such confectionery can generally be prepared by conventional methods, including but not limited to methods involving open flame cookers, vacuum cookers, and scraped surface cookers (also known as high-speed atmospheric cookers). Equipment useful in accordance with the presently disclosed subject matter includes cooking and mixing equipment well known in the art of confectionery manufacturing, and thus the selection of specific equipment will be readily apparent to the skilled artisan.

[0338] The open flame cooking method involves a traditional method of making a candy base. In this method, the desired amount of carbohydrate bulking agent is dissolved in water by placing the leavening agent in a pot and heating it until it dissolves. Additional leavening agent can then be added and cooked until the final temperature reaches 145°C to 156°C. The batch is then cooled and processed into a plastic-like mass to incorporate additives (such as flavorings, colorings, etc.).

[0339] High-speed, atmospheric cookers use a heat exchanger surface onto which a thin film of candy is laid, which is heated to 165°C to 170°C in a matter of seconds. The candy is then rapidly cooled to 100°C to 120°C and processed into a plastic-like mass for the incorporation of additives such as flavorings, colorings, etc. In a vacuum cooker, a carbohydrate leavening agent is boiled to 125°C to 132°C, a vacuum is applied, and additional water is boiled without additional heating. When cooking is complete, the mass is semi-solid and has a plastic-like consistency. At this point, flavorings, colorings, and other additives are mixed into the mass through conventional mechanical mixing operations.

[0340] In the traditional hard candy production process, the optimal mixing time required to uniformly mix flavorings, coloring agents and other additives depends on the time required to obtain a uniform distribution of materials. Generally speaking, a mixing time of 2 to 10 minutes is acceptable.

[0341] Once the candy mass has been properly tempered, it can be cut into workable portions or formed into the desired shape. A variety of forming techniques can be used depending on the shape and size of the desired final product. A general discussion of the composition and preparation of hard candies can be found in H.A. Lieberman, Pharmaceutical Dosage Forms: Tablets, Vol. 1 (1989) (Marcel Dekker, Inc., New York, NY), pp. 419-582, the disclosure of which is incorporated herein by reference.

[0342] Compressed tablet candies contain specialized materials that form a structure under pressure. These candies typically contain up to about 95% sugar by weight of the composition, along with typical tablet excipients (such as binders and lubricants, as well as flavorings, colorings, etc.). These candies can also be sugar-free.

[0343] Similar to hard candies, soft candies can be used in embodiments of the disclosed subject matter. The preparation of soft candies, such as nougat, involves conventional methods, such as a combination of two main ingredients, namely (1) a high-boiling point syrup (such as corn syrup) and (2) a relatively light-textured smoothie, typically prepared from egg whites, gum arabic, gelatin, vegetable proteins (such as soy-derived compounds), unsweetened milk-derived compounds (such as milk protein), and mixtures thereof. Smoothies are generally relatively light and, for example, may have a density ranging from about 0.5 to about 0.7 g / cm3.

[0344] The high-boiling point syrup or "bob syrup" of soft candies is relatively viscous, has a higher density than smoothie ingredients, and typically contains a large amount of carbohydrate leavening agents. Traditionally, the final nougat composition is prepared by adding the "bob syrup" to the smoothie while stirring to form a basic nougat mixture. Subsequently, other ingredients such as flavorings, additional carbohydrate leavening agents, coloring agents, preservatives, medicaments, mixtures thereof, etc. may also be added while stirring. Soft candies can also be made sugar-free. A general discussion of the composition and preparation of nougat can be found in BW Minifie, Chocolate, Cocoa and Confectionery: Science and Technology, 2nd ed., AVI Publishing Company, Westport, Connecticut (1983), pp. 576-580, the disclosure of which is incorporated herein by reference.

[0345] Typically, the smoothie component is prepared first, and the syrup component is then slowly added with stirring at a temperature of at least about 65° C., preferably at least about 100° C. The components are continued to mix to form a homogeneous mixture, and then the mixture is cooled to a temperature below 80° C., at which time flavoring can be added. The mixture is continued to mix for a period of time until it is ready to be removed and formed into suitable candy shapes.

[0346] According to the present disclosure, the amount of psicose prepared and / or generated by any microorganism disclosed herein can be mixed into hard candies and soft candies. The precise amount of psicose adopted depends on preference usually, depends on factors such as the specific type of prepared candy, the type of leavening agent or carrier adopted, the type of flavorings adopted, and the intensity of required breath freshening sensation. Therefore, the amount of psicose can be changed to obtain the result desired in the finished product, and this variation is within the capabilities of those skilled in the art without excessive experiments. Generally speaking, the amount of psicose normally present in hard candies or soft candies is about 0.001 % by weight to about 20 % by weight of sugar, preferably about 0.01 % by weight to about 15 % by weight, more preferably about 0.01 % by weight to about 10 % by weight, more preferably about 0.01 % by weight to about 5 % by weight, more preferably about 0.01 % by weight to about 0.5 % by weight.

[0347] The presently disclosed subject matter also extends to methods for making improved candies. Using standard techniques and equipment known to those skilled in the art, any microbially produced and / or generated psicose disclosed herein can be incorporated into otherwise conventional hard or soft candy compositions. Equipment useful in accordance with the presently disclosed subject matter includes mixing and heating equipment well known in the candy making art, and thus the selection of specific equipment will be readily apparent to those skilled in the art.

[0348] In this method, a candy composition is prepared by incorporating psicose and the other components of the final desired composition into a candy composition. The other ingredients are typically incorporated into the composition based on the properties of the desired composition, as is well known to those skilled in the art. The final candy composition can be easily prepared using methods commonly known in the fields of food technology and pharmacology. The candy mixture can then be formed into the desired candy shape.

[0349] Allulose prepared and / or produced by any of the microorganisms disclosed herein can be formulated with conventional ingredients that provide a variety of textures suitable for specific applications. Such ingredients can be hard and soft candies, tablets, toffees, nougat, chewy candies, chewing gum, etc., candies with fillings, and candies containing sugar and sugar-free. Acceptable ingredients can be selected from a variety of materials. However, such materials include, but are not limited to, diluents, binders and adhesives, lubricants, disintegrants, leavening agents, humectants, buffers, and adsorbents. The preparation of such candy and chewing gum products is well known.

[0350] 5.3. Chocolate and fillings

[0351] The subject matter of the present disclosure is also useful in chocolate products, chocolate-flavored candies, and chocolate-flavored compositions. Chocolates also include those containing granulated solids or made entirely or partially from a granulated process. For example, U.S. Patent Nos. 7,968,140 and 8,263,168 disclose various chocolates, the disclosures of which are incorporated herein by reference in their entireties. A general discussion of the composition and preparation of chocolate can be found in BW Minifi e , Chocolate, Cocoa and Confectionery: Science and Technology, 2nd ed., AVI Publishing Company, Westport, Connecticut (1982), pp. 576-580, the disclosure of which is incorporated herein by reference.

[0352] As used herein, the term "chocolate" refers to a solid or semi-plastic food, meaning all chocolates or chocolate-like compositions containing a fat-based component phase or a fat-like composition. The term is intended to include standardized or non-standardized compositions that meet U.S. Standards of Identity (SOI), CODEX Alimentarius, and / or other international standards, as well as compositions that do not meet U.S. Standards of Identity or other international standards. Unless otherwise indicated, the term includes dark chocolate, baking chocolate, sweet chocolate, bittersweet or semisweet chocolate, milk chocolate, buttermilk chocolate, skim milk chocolate, mixed dairy chocolate, white chocolate, sweet cocoa and vegetable fat coatings, sweet chocolate and vegetable fat coatings, milk chocolate and vegetable fat coatings, vegetable fat-based coatings, chocolates comprising white chocolate or a coating made with cocoa butter or vegetable fat, or combinations of these, nutritionally improved chocolate-like compositions (chocolate or coatings made with low-calorie ingredients), and low-fat chocolate, aerated chocolate, compound coatings, non-standardized chocolate, and chocolate-like compositions.

[0353] For example, when a nutritive carbohydrate sweetener is partially or fully substituted; or when cocoa butter, cocoa butter substitutes, cocoa butter equivalents, cocoa butter extenders, cocoa butter substitutes, cocoa butter substitutes, or milk fat is partially or fully substituted; or when ingredients with flavors that mimic milk, butter, or chocolate are added or other additions or omissions are made to the recipe that exceed the FDA chocolate identity standards, or a combination thereof. Chocolate-like compositions are fat-based compositions that can be used as a substitute for chocolate in applications such as pan enrobing, molding, or enrobing; for example, carob.

[0354] In the United States, chocolate is subject to labeling standards established by the U.S. Food and Drug Administration (FDA) under the Federal Food, Drug, and Cosmetic Act. The United States has clearly defined definitions and standards for various types of chocolate. Non-standardized chocolate refers to chocolate whose composition falls outside the specified range for standardized chocolate.

[0355] In certain embodiments, chocolate may contain psicose prepared and / or generated by any microorganism disclosed herein. In addition, chocolate may contain syrup / solids, invert sugar, hydrolyzed lactose, maple syrup, brown sugar, molasses, honey, sugar substitutes, etc. Nutritive carbohydrate sweeteners with different sweetness intensities can be those commonly used in any of the art, including but not limited to sucrose (e.g., from sugar cane or beets), glucose, fructose, lactose, maltose, glucose syrup solids, corn syrup solids, invert sugar, hydrolyzed lactose, honey, maple syrup, brown sugar, molasses, etc. Sugar substitutes can partially replace nutritive carbohydrate sweeteners. High-potency sweeteners include aspartame, cyclamate, saccharin, acesulfame potassium, neohesperidin dihydrochalcone, sucralose, alitame, stevia sweetener, glycyrrhizin, thaumatin, etc., and mixtures thereof. Preferred high-potency sweeteners are aspartame, cyclamate, saccharin, and acesulfame potassium. Examples of sugar alcohol may be any of those generally used in the art, including sorbitol, mannitol, xylitol, maltitol, isomalt, lactitol, and the like.

[0356] The chocolate may also contain a leavening agent. The term "leaving agent" as defined herein may be any of those commonly used in the art, including polydextrose, cellulose and its derivatives, maltodextrin, gum arabic, and the like.

[0357] The chocolate product may contain an emulsifier. Examples of safe and suitable emulsifiers may be any of those commonly used in the art, including lecithins derived from plant sources (such as soy, safflower, corn, etc.), fractionated lecithins enriched with phosphatidylcholine or phosphatidylethanolamine, or both, mono- and diglycerides, diacetyltartaric acid esters of mono- and diglycerides (also known as DATEM), monosodium phosphate derivatives of mono- and diglycerides of edible fats or oils, sorbitan monostearate, hydroxylated lecithins, lactylated fatty acid esters of glycerol and propylene glycol, polyglycerol esters of fatty acids, propylene glycol mono- and diesters of fats and fatty acids, or emulsifiers approved for use in the soft candy category defined by the US FDA. Other emulsifiers that may be used include polyglycerol polyricinoleate (PGPR), ammonium salts of phosphatidic acid, (e.g., YN) sucrose esters, oat extract, and the like, any emulsifier found to be suitable for chocolate or similar fat / solid systems or any mixtures thereof.

[0358] The term "chocolate-flavored candy" refers to food products that, in addition to being "chocolate," have a chocolate flavor / aroma and contain a cocoa portion. These products are stable for extended periods of time (e.g., greater than one week) at ambient temperature and have a microbiological shelf life at 18-30°C under normal atmospheric conditions. Examples include chocolate-flavored hard candies, chews, and gum.

[0359] The term "chocolate-flavored composition" refers to a chocolate-flavored composition that does not contain "chocolate," contains a cocoa portion, and has a chocolate flavor / aroma. Examples include chocolate-flavored cake mixes, ice cream, syrups, baked goods, and the like. The term encompasses chocolate-flavored compositions (e.g., cake, nougat, pudding, etc.), as well as compositions that do not have a chocolate flavor (e.g., caramel, etc.).

[0360] 5.4. Salty foods and other foods

[0361] In certain embodiments, psicose produced and / or generated by any of the microorganisms disclosed herein is incorporated into a savory food. In certain embodiments, the savory food is a food having a savory flavor, including, but not limited to, spicy, peppery, dairy, vegetable, tomato, dill, meat, poultry, chicken, and reaction flavors added or generated when the food is heated.

[0362] In certain embodiments, allulose prepared and / or generated by any microorganism disclosed herein is incorporated into wet soup foods, which include wet / liquid soups, including frozen soups, regardless of concentration or container. In certain embodiments, soup foods refer to foods prepared from meat, poultry, fish, vegetables, grains, fruits, and / or other ingredients, cooked in a liquid that may include some or all of the visible fragments of these ingredients. It can be clear (as broth) or thick (as chowder), smooth, thick or chunky, ready-to-eat, semi-concentrated, or concentrated, and can be eaten hot or cold, as a first course or as the main course of a meal, or as a snack between meals (sipping like a beverage). Soup can be used as an ingredient to prepare other dietary components, ranging from broth (clear soup) to sauce (cream or cheese soup).

[0363] In certain embodiments, allulose produced and / or generated by any of the microorganisms disclosed herein is incorporated into foods in the category of dehydrated and culinary foods, which include (i) culinary aid products such as powders, granules, pastes, concentrated liquid products, including concentrated bouillon, bouillon, and bouillon-like products in pressed cube, tablet, or powder or granule form, sold separately as a finished product or as an ingredient within a product, sauces, and recipe mixes (regardless of the technology used); (ii) dietary liquid products, such as dehydrated and freeze-dried soups, including dehydrated soup mixes, dehydrated instant soups, dehydrated instant soups, ready-to-eat dishes that are dehydrated or prepared at room temperature, meals, and single-serving entrees, including pasta, potatoes, and rice; and (iii) meal bellishment products. product), such as: dressings, marinades, salad dressings, salad toppings, dips, breadings, batter mixes, shelf-stable spreads, barbecue sauces, liquid recipe mixes, concentrates, sauces or sauce mixes, including salad recipe mixes sold as a finished product or as an ingredient in a product, whether dehydrated, liquid or frozen.

[0364] In certain embodiments, the psicose prepared and / or generated by any microorganism disclosed herein is incorporated into meat foods. In certain embodiments, meat foods include foods made from the edible residues of any dead animal (including birds, fish, crustaceans, shellfish and mammals) processed. Meat foods include, but are not limited to, for example, processed beef, mutton, pork, poultry or seafood products. Examples of such meat foods include, for example, bologna sausage, frankfurters, sausages, luncheon meat, deli slices, bread, bacon, meatballs, fish sticks, chicken fillet and minced meat, for example, meatloaf, meatballs and hamburgers. Meat foods can be combined with simulated meat foods. Simulated meat foods include, but are not limited to, for example, meat substitutes, meat analogs, soy burgers, soy bologna sausages, soy frankfurters, soy sausages, soy luncheon bread, soy bacon and soy meatballs. Simulated meat foods can be combined with meat foods.

[0365] In certain embodiments, allulose prepared and / or produced by any of the microorganisms disclosed herein is incorporated into snack foods. In certain embodiments, snack products include any food that can be consumed as a light meal, including but not limited to sweet and salty snacks and snack bars. Examples of snacks include but are not limited to fruit snacks, potato chips / crisps, extruded snacks, tortillas / corn flakes, popcorn, pretzels, nuts, and other sweet and salty snacks. Examples of snack bars include but are not limited to granola / cereal bars, breakfast bars, energy bars, fruit bars, and other snack bars.

[0366] In certain embodiments, allulose produced and / or generated by any of the microorganisms disclosed herein is incorporated into frozen foods, including refrigerated or frozen foods, such as, but not limited to, ice cream, ready-to-eat ice cream, single-serving dairy ice cream, single-serving water-based ice cream, multi-pack dairy ice cream, multi-pack water-based ice cream, family-sized ice cream, family-sized dairy ice cream, ice cream desserts, bulk ice cream, family-sized water-based ice cream, frozen yogurt, artisanal ice cream, frozen ready-to-eat foods, frozen pizza, refrigerated pizza, frozen soup, frozen pasta, frozen processed red meat, frozen processed poultry, frozen processed fish / seafood, frozen processed vegetables, frozen meat substitutes, frozen potatoes, frozen baked products, and frozen desserts.

[0367] 5.4.Medications

[0368] D-psicose prepared and / or produced by any of the microorganisms disclosed herein can also be in pharmaceutical form. A non-limiting example of a pharmaceutical form is a suspension. Pharmaceutical suspensions can be prepared by conventional formulation methods. The suspension may contain auxiliary materials used to prepare suspensions in the art. The suspensions of the presently disclosed subject matter may include preservatives, buffers, suspending agents, defoaming agents, sweeteners, flavorings, colorants or decolorizing agents, solubilizers, and combinations thereof.

[0369] Flavoring agents such as those well known to those skilled in the art, such as natural and artificial flavorings and mints (such as peppermint), menthol, citrus flavorings (such as orange and lemon), artificial vanilla, cinnamon and various fruit flavorings, individual and mixed flavorings, etc., are used in an amount of about 0.01% to about 5% by weight of the suspension, more preferably 0.01% to about 0.5% by weight.

[0370] The pharmaceutical suspension of the presently disclosed subject matter can be prepared as follows: (i) mixing a thickener with water heated to about 40° C. to about 95° C., preferably about 40° C. to about 70° C., to form a dispersion (if the thickener is not water-soluble) or a solution (if the thickener is water-soluble); (ii) mixing psicose produced and / or generated by any of the microorganisms disclosed herein with water to form a solution; (iii) if desired, mixing a flavoring with the thickener-water mixture to form a uniform thickener-flavoring; (i v ) combining the sweetener solution with the thickener-flavoring agent and mixing until homogeneous; and (v) mixing optional auxiliary materials (such as colorants, flavorings, decolorizing agents, solubilizers, antifoaming agents, buffers and additional water) with the mixture of step (iv) to form a suspension.

[0371] Allulose prepared and / or produced by any microorganism disclosed herein can also be in a chewable form. In order to achieve acceptable stability and quality as well as good taste and mouthfeel in a chewable formulation, several important factors need to be considered. These factors include the amount of active substance per tablet, the flavoring agent used, the compressibility of the tablet, and the additional properties of the composition. The preparation method of chewable medicinal candies is similar to that of soft candies. A general discussion of lozenges and chewable tablet candies can be found in HA Lieberman and L Lachman, Pharmaceutical Dosage Forms: Tablets, Vol. 1 (1989) (Marcel Dekker, Inc., New York, NY), pp. 367-418, the disclosure of which is incorporated herein by reference. In a typical method, a boiled sugar-corn syrup mixture is formed, to which a smoothie mixture is added. A boiled sugar-corn syrup mixture can be prepared from sugar and corn syrup mixed in a weight ratio of about 90:10 to about 10:90. The sugar-corn syrup mixture is heated to a temperature above about 120°C to remove water and form a melt. Smoothies are typically prepared from gelatin, egg whites, milk proteins (such as casein), and plant proteins (such as soy protein), which are added to the gelatin solution and quickly mixed at ambient temperature to form an aerated sponge-like substance. The smoothie is then added to the molten candy mass and mixed at a temperature of about 65°C to about 120°C until uniform. Any microbial preparation and / or production of allulose disclosed herein can then be added to the homogenous mixture, and additional ingredients, such as flavorings and coloring agents, can then be added when the temperature is reduced to about 65°C-95°C. The preparation is further cooled and formed into pieces of the desired size.

[0372] In other pharmaceutical embodiments, the flavoring agent is incorporated into an ingestible topical vehicle, which may be in the form of a mouthwash, rinse, ingestible spray, suspension, tooth gel, or the like. Typical non-toxic ingestible vehicles known in the pharmaceutical art may be used in the subject matter of the present disclosure. Preferred ingestible vehicles are water, ethanol, and water-ethanol mixtures. Water-ethanol mixtures are typically employed in weight ratios of about 1:1 to about 20:1, preferably about 3:1 to about 20:1, and most preferably about 3:1 to about 10:1, respectively. The pH of the ingestible vehicle is typically from about 4 to about 7, preferably from about 5 to about 6.5. Ingestible topical vehicles having a pH below about 4 will typically irritate the ingestible cavity, while ingestible vehicles having a pH greater than about 7 will typically result in an unpleasant taste.

[0373] Ingestible topical flavorings can also contain conventional additives that are generally used for those products. Conventional additives include fluorine compounds, sweeteners, flavorings, coloring agents, wetting agents, buffers and emulsifiers, as long as these additives do not interfere with the flavoring properties of the composition. The coloring agents and wetting agents described above and the consumption of these additives can be used for ingestible topical compositions. Operable flavorings (spices, seasonings) include those well known to those skilled in the art, such as natural flavors and artificial flavors. Suitable flavorings include mint (such as peppermint), citrus flavors (such as oranges and lemons), artificial vanilla, cinnamon, various fruit flavors, etc., which can be used alone and in combination. The amount of the flavorings used in the ingestible topical compositions generally depends on factors such as the type of the final ingestible composition, the concentration of a separate flavoring and the required flavoring. Therefore, the amount of flavorings can be changed to obtain the desired result in the final product, and this variation is within the capabilities of those skilled in the art, without excessive experimentation. During use, the amount of flavorings can generally vary, for example, and the scope of amount is approximately 0.05% by weight to approximately 6% by weight of the ingestible topical compositions.

[0374] 5.5. Pet food

[0375] D-psicose produced and / or generated by any of the microorganisms disclosed herein can be used in a variety of pet foods.

[0376] As used herein, the term "pet food" or "pet food product" refers to a product or composition for consumption by companion animals (e.g., cats, dogs, guinea pigs, rabbits, birds, and horses). For example, but not limited to, the companion animal can be a "domestic" dog, such as the Chinese garden dog (Canis lupus familiaris). "Pet food" or "pet food product" includes any food, feed, treat, food supplement, liquid, beverage, snack, toy (chewable and / or edible toys), meal substitute, or meal replacement.

[0377] In certain embodiments, psicose produced and / or generated by any of the microorganisms disclosed herein is added directly to pet food. In certain embodiments, psicose produced and / or generated by any of the microorganisms disclosed herein can be added before, during, or after the formulation or packaging of the pet food.

[0378] Non-limiting examples of suitable pet foods include wet foods, dry foods, moist foods, pet food supplements (eg, vitamins), pet beverage products, treats and nibbles, and the pet food categories described herein.

[0379] In some embodiments, the pet food is a dry food. A dry or low-moisture nutritionally complete pet food may contain less than about 15% moisture. In some embodiments, the pet food is a wet food. A wet or high-moisture nutritionally complete pet food may contain more than about 50% moisture. In some embodiments, the pet food is a nutritionally complete wet food. A wet, e.g., semi-moist or semi-dry or soft-dry or soft-moist or medium or medium moisture nutritionally complete pet food may contain from about 15% to about 50% moisture.

[0380] In certain embodiments, the pet food is a pet food treat product. Non-limiting examples of pet food treat products include treat bars, pet chews, crunchy treats, cereal bars, snacks, cookies, and desserts.

[0381] Example

[0382] The subject matter of the present disclosure may be better understood with reference to the following: The following examples are merely illustrative and should not be considered limiting.

[0383] Example 1 - Biosynthesis of D-psicose from Glucose

[0384] E. coli naturally produces trace amounts of D-psicose. In this example, D-psicose production was increased by overexpressing key genes, removing competing pathway genes, and optimizing production conditions.

[0385] Evaluation of the Allulose Production Ability of Escherichia coli

[0386] First, we tested whether E. coli possesses an enzyme capable of producing psicose. The production of psicose was tested in the production strain AL3601 shown in Table 1 below. Cultures were grown at 30°C in M9P medium (M9 minimal medium containing 5g / L yeast extract) containing 10g / L glucose. Supernatant samples were collected 24 hours after inoculation and analyzed using HPLC. Notably, no psicose was detected in AL3601 ( Figure 1). A series of single gene knockouts (KOs) were constructed in AL3601. These KOs included ΔpfkA, ΔpfkB, and Δzwf, resulting in strains AL3694, AL3689, and AL3725, respectively (see Table 1). The gene zwf encodes the enzyme glucose-6-phosphate dehydrogenase (Zwf) (EC 1.1.1.363), which converts glucose-6-phosphate (G6P) to 6-phospho-D-glucono-1,5-lactone as the first step in the pentose phosphate pathway (PPP). pfkA and pfkB encode phosphofructokinase A and phosphofructokinase B (EC 2.7.1.11 and EC 2.7.1.105), which work together to convert fructose-6-phosphate (F6P) to fructose-1,6-bisphosphate (F16BP), the first step in glycolysis. A single KO strain containing ΔpfkA produced 0.15 g / L psicose, indicating that E. coli possesses an enzyme capable of producing psicose ( Figure 1 No psicose was detected in the single KO strains containing Δzwf and ΔpfkB.

[0387] Table 1. List of strains

[0388]

[0389]

[0390] Identification of the allulose-producing enzyme

[0391] Next, we hypothesized that fructose-6-phosphate (F6P) is an intermediate of psicose in E. coli, as deletion of pfkA increases psicose production. The production pathway begins with the natural assimilation of glucose by E. coli via the phosphotransferase system (PTS) or GalP / Glk, converting glucose into glucose-6-phosphate (G6P) ( Figure 2 ). G6P is then isomerized to F6P by glucose-6-phosphate isomerase (Gpi) (EC 5.3.1.9). F6P can be converted to psicose-6-phosphate by epimerase ( Figure 2 Phosphatases can dephosphorylate psicose-6-phosphate to free psicose, which is then excreted from the cell ( Figure 2 The psicose production system using phosphorylation and dephosphorylation steps as the driving force of the system should be more efficient than the pathway currently used in the industrial production of psicose.

[0392] Through literature search and genome mining, one epimerase and two phosphatase candidates were identified. D-psicose-6-phosphate 3-epimerase (AlsE) has shown activity towards psicose, and two phosphatases (HxpB and YbiV) have shown promiscuity towards various hexose sugars.

[0393] Two plasmids, pAL1946 and pAL1947 (Table 2), were constructed, expressing the gene under the inducible promoter P T7 Each plasmid was introduced into AL3601 and tested for psicose production. Cultures were grown in M9P medium supplemented with 10 g / L glucose at 30°C and induced with 1 mM IPTG. After 24 hours, induced AL3601 / pAL1946 produced 1.0 g / L psicose, while induced AL3601 / pAL1947 produced 0.4 g / L, indicating that HxpB is the superior phosphatase for psicose production. Figure 3 ).

[0394] Table 2. Plasmid list

[0395] plasmids pAL1946 <![CDATA[P T7 :alsE-yniC,amp R ,pBR322 ori]]> pAL1947 <![CDATA[P T7 :alsE-ybiV,amp R ,pBR322 ori]]> pAL1952 <h2 style=";text-align:left;direction:ltr"><![CDATA[P <h2 style=";text-align:left;direction:ltr"> tet <h2 style=";text-align:left;direction:ltr"> :dcas9,kan<h2 style=";text-align:left;direction:ltr"> R <h2 style=";text-align:left;direction:ltr"> ,p1 5A ori]]><h2 style=";text-align:left;direction:ltr"> pAL2001 <![CDATA[P LIac01 :alsE-yniC,amp R ColE1 ori]]> pAL2160 <![CDATA[P LIac01 :alsE-yniC,Pj2 3119 : Empty boot, amp R ,ColE1 ori]]> pAL2179 <![CDATA[P LIac01 :alsE-yniC,P j23119 :pfkB boot,amp R ,ColE1 ori]]> pAL2247 <![CDATA[P gadB :alsE-hxpB,amp R ColE1 ori]]> pAL2274 <![CDATA[P LtetO1 :galP-glk,gent R ,Cola ori]]>

[0396] Increasing allulose production by removing competing pathways

[0397] To increase carbon flux through the psicose production pathway, a triple knockout (TKO) strain containing Δzwf, ΔpfkA, and ΔrpiB was constructed (see AL3729 in Table 1) to increase the pool of F6P for psicose production. The gene rpiB encodes the enzyme allose-6-phosphate isomerase (RpiB), which re-assimilates P6P into central carbon metabolism by converting it to aldehyde-D-allose 6-phosphate in the allose degradation pathway ( Figure 2 pAL1946 (Table 2) was introduced into AL3729 (Table 1) and tested for psicose production. Cultures were grown in M9P medium supplemented with 10 g / L glucose at 30°C and induced with 25 μM IPTG. Uninduced AL3729 harboring pAL1946 produced the most psicose after 24 hours, at 1.5 g / L, while induced AL3729 harboring pAL1946 produced 0.2 g / L ( Figure 4 The induced AL3601 containing pAL1946 produced 0.6 g / L of psicose after 24 hours, while the uninduced AL3601 containing pAL1946 produced 0.2 g / L.

[0398] Comparison of expression systems for psicose-producing enzymes

[0399] AL3601 has a P encoding T7 RNA polymerase lacUV5 : T7 RNAP (Table 1). T7 RNAP seems to impair the growth of the strains tested. In order to eliminate this growth burden, a new psicose production plasmid (see pAL2001 in Table 2) was generated to produce psicose under the IPTG inducible promoter P LlacO1Under overexpression of alsE and hxpB, this promoter is more T7 pAL2001 was introduced into strain AL1050 (see Table 1). Strain AL1050 carries the same genotype as AL3601, but lacks P lacUV5 : T7 RNAP. Cultures were grown at 30°C in M9P medium supplemented with 10 g / L glucose and induced with 1 mM IPTG. After 24 hours, induced AL1050 carrying pAL2001 produced 0.5 g / L of psicose, while induced AL3601 carrying pAL1946 produced 0.5 g / L. Growth of induced and uninduced AL1050 cultures carrying pAL2001 appeared comparable. In contrast, AL3601 containing pAL1946 grew less well, with the induced culture reaching a much lower culture density ( Figure 5 Based on the results that pAL2001 produced similar titers of psicose without causing growth burden, in the following experiments, we selected pAL2001-based LlacO1 The expression system of psicose was used as a plasmid for the production of psicose.

[0400] Comparison of expression systems for psicose-producing enzymes in TKO strains

[0401] P was tested in the TKO strain AL3756. LlacO1 The yield of allulose was tested in AL3729. T7 AL3756 carrying pAL2001 and AL3729 carrying pAL1946 were grown in M9P medium containing 10 g / L glucose at 30°C and induced with 1 mM IPTG. After 24 hours, AL3756 carrying pAL2001 produced 1.4 g / L psicose, while the uninduced culture produced 0.6 g / L psicose. AL3729 carrying pAL1946 produced 0.6 g / L psicose when induced, while the uninduced culture produced 1.8 g / L psicose. Figure 6 ).

[0402] Identify and reduce by-products

[0403] When analyzing samples from the psicose-producing strain, a prominent peak was consistently observed on the HPLC chromatogram that did not match any of the culture medium components or standards of typical metabolites. It was found that the retention time and mass spectrum of the unknown peak matched those of the mannose standard ( Figure 7To further test the hypothesis that the byproduct is mannose, manA was knocked out in AL3756 to generate the quadruple knockout (QKO) strain AL3990 (see Table 1). The manA gene encodes the enzyme mannose-6-phosphate isomerase (ManA), which catalyzes the reversible isomerization of mannose-6-phosphate and F6P. pAL2001 (Table 2) was introduced into AL3990 and AL3756 (Table 1) and tested for the production of psicose ( Figure 8 and Figure 9 Cultures were grown at 30°C in M9P medium supplemented with 10 g / L glucose and induced with 1 mM IPTG. After 24 hours, the QKO strain produced 0.7 g / L mannose but 3.5 g / L psicose, a yield of 34%. The TKO strain produced 2.5 g / L mannose and 2.3 g / L psicose.

[0404] AL3990 carrying pAL2001 was cultured in M9P medium containing 15 g / L glucose (instead of the usual 10 g / L). The culture was grown at 30°C and induced with 1 mM IPTG. After 24 hours, the induced strain produced 2.3 g / L psicose, a yield of 40.3% ( Figure 10 ).

[0405] Dynamic regulation

[0406] Balancing carbon flux between glycolysis and production is crucial for maximizing culture health and allulose production. During the logarithmic growth phase, cells require more energy to maintain rigorous growth and division. Genes associated with glycolysis should be expressed, while genes associated with allulose production should be suppressed. When cells enter a stationary phase and are not actively dividing, carbon flux can shift from glycolysis to allulose production.

[0407] pfkB was downregulated in the QKO strain using CRISPR interference (CRISPRi). CRISPRi involves an inactivated Cas9 enzyme, dCas9, which, when recruited by a single guide RNA scaffold (sgRNA), can target and block transcription initiation by RNA polymerase. Regulation of pfkB was tested using the dCas9 plasmid pAL1952 (see Table 2), which contains an anhydrotetracycline (aTC)-inducible promoter, P tet Separate production / guide plasmids were constructed, containing P LlacO1 :alsE-hxpB and P J23119 : guide (pAL2179) or P J23119: Empty guide (pAL2160). The sgRNA encoded by pAL2179 guides dCas9 to the promoter region of pfkB. Strain AL3990 was transformed with pAL1952 and pAL2160 or pAL2179 and tested for the production of psicose. Cultures were grown in M9P medium (10 mL) supplemented with 10 g / L glucose at 37°C until the OD 600 The cells were then centrifuged and resuspended in 3.0 mL of fresh M9P medium supplemented with 10 g / L glucose, 1 mM IPTG, and 100 ng / mL aTC. After 24 hours of growth at 30°C, the aTC- and IPTG-induced AL3990 culture containing pAL1952 and pAL2179 produced 1.7 g / L psicose (yield = 46%), while the IPTG-induced culture without aTC induction produced 2.0 g / L psicose (yield = 42%). The aTC-induced AL3990 culture containing pAL1952 and pAL2160 produced 0.7 g / L psicose (yield = 8%), while the uninduced culture produced 0.7 g / L psicose (yield = 8%). Figure 11 ).

[0408] Next, the use of the stationary phase promoter P was evaluated. gadB Inducer-free production system. gadB Controls the expression of the gene glutamate decarboxylase B, which is mainly active during the resting phase. gadB Controlling the expression of alsE and hxpB genes can shift carbon flux to production during the stationary phase. LlacO1 : plasmid pAL2001 containing alsE-hxpB and P gadB pAL2247 (Table 2) containing alsE-hxpB was introduced into QKO strain AL3990 (Table 1). Cultures were grown at 30°C in M9P medium supplemented with 30 g / L glucose and 1 mM IPTG (only for strains with P LlacO1 The induced culture containing pAL2001 produced 6.8 g / L psicose, with a yield of 56.5%, while the culture containing P gadB : The culture of alsE-hxpB produced 8.8 g / L psicose, with a yield of 63% ( Figure 12 ). P gadB Allowing for increased expression of the enzymes producing it and achieving greater flux through the allulose pathway ( Figure 12 Together, these data suggest that inducer-independent promoters favor control of alsE and hpxB gene expression and facilitate shutting off carbon flux through glycolysis.

[0409] Increased glucose uptake using the sugar symporter GalP

[0410] In Escherichia coli, the preferred method of glucose uptake is the phosphotransferase system (PTS). The PTS relies on the activation of phosphoenolpyruvate (PEP), a downstream product of glycolysis. By inhibiting glycolysis through gene knockout or CRISPRi, we hypothesized that the cellular PEP reserve might be depleted, leading to reduced glucose consumption. To increase glucose consumption, we introduced additional copies of the genes galP and glk, which encode the galactose:H + The symporter (GalP) and glucokinase (Glk) are involved. GalP transports glucose into the cell where it is phosphorylated by Glk to glucose-6-phosphate and assimilated into the central carbon metabolism. Plasmid pAL2274 (Table 2) was constructed to LtetO1 pAL2274 was expressed under the galP and glk promoters. Llaco1 : alsE-hxpB) or pAL2247 (P gadB : alsE-hxpB) were introduced into QKO strain AL3990 (Table 1) and tested for psicose production. Cultures were grown at 30°C in M9P medium supplemented with 30 g / L glucose and induced with 1 mM IPTG for 24 hours. The strain containing pAL2247 and pAL2274 produced the most psicose, with a titer of 10.7 g / L and a yield of 61%. The strain containing only pAL2247 produced 8.3 g / L psicose, with a yield of 46%. In contrast, the IPTG-induced culture containing pAL2001 and p2274 produced 6.6 g / L psicose, with a yield of 45%, while the induced culture containing only pAL2001 produced 5.8 g / L psicose, with a yield of 46% ( Figure 13 ). It appears that overexpression of GalP-Glk can complement glucose input when paired with the production plasmid pAL2247.

[0411] Table 3

[0412]

[0413]

[0414]

[0415]

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447] Example 2 - Biosynthesis of D-psicose from Glucose

[0448] Sedentary lifestyles and a dramatic increase in the intake of high-calorie foods have led to a tripling of global obesity rates from 1975 to 2016 (Blüher, Nat. Rev. Endocrinol. 2019 15515, 288-298 (2019)). Nearly 40% of adults worldwide are overweight, a major risk factor for cardiovascular disease, diabetes, musculoskeletal diseases, and some cancers. In response to this public health crisis, more and more people are looking to treat or prevent disease by adopting a health-conscious, low-calorie diet. The food industry can help people make better choices by replacing sucrose and high-fructose corn syrup with zero-calorie and low-calorie sugar substitutes. As a result, the sugar substitutes market size is expected to reach USD 20.6 billion by 2025 (MarketandMarket. Sugar Substitutes Market by Type (High Fructose Syrup, High-Intensity Sweetener, Low-Intensity Sweetener), Composition, Application (Beverages, Food Products, and Health & Personal Care Products), and Region - Global Forecast t02025) (2020)).

[0449] Rare sugars are monosaccharides that are not commonly found in nature and have slightly different structures from common sugars such as glucose and fructose. Many rare sugars have no nutritional value but have potential health benefits, making them attractive targets for sugar substitutes. The GRAS (zero-calorie) rare sugar D-psicose is the 3' diastereomer of fructose and has 70% the sweetness of sucrose. D-psicose, marketed to consumers as "allulose," has a desirable flavor profile as well as good browning, hygroscopic, and solubility properties. Furthermore, studies have linked the consumption of D-psicose to anti-hyperglycemic, anti-hyperlipidemic, anti-parasitic, and antioxidant health benefits. 3 Like many rare sugars, research on D-psicose and its application in industry are limited by the lack of economical, large-scale production systems. D-psicose exists naturally in some fruits and grains, but the concentration is extremely low and cannot be extracted (Oshima et al., Food Sci. Technol. Res. 12, 137-143 (2006)).

[0450] Although synthetic methods for producing D-psicose have been proposed, these methods have poor stereoselectivity, yield, and purification capabilities (Wang et al., Nature 578, 403-408 (2020)). Therefore, biosynthesis using the enzymes D-tagatose-3-epimerase (DTEase) and D-psicose-3-epimerase (DPEase) has been the main focus of D-psicose production (Itoh et al., OUP 58, 2168-2171 (2014); Jiang et al., Front. Bioeng. Biotechnol. 8, 26 (2020); Armetta et al., Synth. Biol. 4, ysz028 (2019)). Both enzymes operate at high temperatures and alkaline pH, epimerizing the C3 carbon of D-fructose to form D-psicose. Attempts to increase yields have focused on modifying these enzymes to improve catalytic efficiency, thermal stability, and the ability to function at lower pH and temperature (Hu et al., Compr. Rev. Food Sci. Food Saf. 20, 6012-6026 (2021)). Despite concerted efforts to improve D-psicose-3-epimerase and D-tagatose-3-epimerase, the method is inherently yield-limited due to the lack of a thermodynamic driving force, so conversion rates do not reach above 50%. The epimerization of D-fructose to D-psicose is reversible, with a predicted ΔG of 1. o +5 kJ mol -1 , which is thermodynamically unfavorable and more likely to favor D-fructose ( Figure 14ALow conversion yields a mixed solution of D-fructose and D-psicose, making separation and purification a significant challenge. Methods that achieve conversions exceeding 50% rely on the addition of toxic or expensive cofactors.

[0451] To overcome this obstacle, this example proposes to use phosphorylation and dephosphorylation to provide thermodynamic incentives and driving forces for D-psicose production ( Figure 14B ). At a typical physiological concentration of 1 mM, the predicted ΔG′ m -31.1 kJmol -1 , the dephosphorylation of D-psicose-6-phosphate (P6P) to D-psicose is a very favorable reaction. The enzymatic machinery and cofactors required for sugar phosphorylation / dephosphorylation are readily available in living cells. In addition, most organisms, including the model organism Escherichia coli, utilize sugar phosphorylation / dephosphorylation as part of sugar consumption and central carbon metabolism. In E. coli, carbon metabolism begins with the phosphotransferase system (PTS), in which D-glucose is simultaneously phosphorylated and transferred across the cell membrane. Alternatively, glucose can be transported across the cell membrane by the galactose proton symporter GalP, followed by phosphorylation by the glucokinase Glk13,14. D-glucose-6-phosphate (G6P) can then be isomerized to D-fructose-6-phosphate (F6P) and used for glycolysis. In theory, some F6P can be transferred from glycolysis and epimerized to P6P, which can then be dephosphorylated to D-psicose and excreted from the cell.

[0452] In this example, it was found that E. coli naturally possesses an enzyme capable of completing the above-mentioned D-psicose biosynthesis pathway ( Figure 14B Epimerization of F6P to P6P can be accomplished using D-psicose-6-phosphate 3-epimerase (AlsE), while dephosphorylation of P6P can be accomplished using the phosphatase hexitol-phosphatase B (HxpB). By utilizing native genes of E. coli, this example successfully produced D-psicose from D-glucose with a yield exceeding 50%, without the need for heterologous enzyme expression.

[0453] The ability of Escherichia coli to produce D-psicose was enhanced by additionally expressing alsE and hxpB and depleting or regulating competing metabolic pathways, including the pentose phosphate pathway (PPP), glycogen biosynthesis, glycolysis, the D-allose degradation pathway, and the D-mannose degradation pathway. D-glucose input was supplemented by additionally expressing the native galactose proton symporter gene galP and the glucokinase gene glk. To further improve production while maintaining cell viability, we explored various dynamic gene regulation and carbon flux allocation strategies. During the growth phase, cells require more energy for strict growth and should express genes related to glycolysis. When cells enter the stationary phase and are not actively growing, carbon flux can be shifted from glycolysis to D-psicose production. To balance this growth-phase-dependent carbon allocation, we explored two strategies for dynamically regulating the expression of key metabolic and D-psicose production genes: an inducer-free stationary promoter and clustered regularly interspaced short palindromic repeat interference (CRISPRi).

[0454] Design a thermodynamically favorable D-psicose production pathway.

[0455] Currently, the main method for producing D-psicose involves in vitro enzymatic isomerization of D-fructose to D-psicose, a thermodynamically unfavorable process that results in incomplete product formation (approximately 50%) and requires expensive purification ( Figure 14A To overcome the thermodynamic limitations of this approach, this example proposes a pathway driven by sugar phosphorylation and dephosphorylation, where the cofactors and enzymes required are readily available in living organisms ( Figure 14B and 14C Therefore, the model organism Escherichia coli was selected as the host of the D-psicose biosynthesis pathway.

[0456] The proposed pathway begins with the assimilation of D-glucose by E. coli through the PTS, which in turn converts D-glucose into G6P ( Figure 14C Alternatively, D-glucose can be taken up by the galactose proton symporter GalP and then phosphorylated to G6P by the glucokinase Glk ( Figure 14C ). G6P is then isomerized to F6P by glucose 6-phosphate isomerase (Gpi). Here, the proposed pathway diverges from native carbon metabolism. Many enzymes are promiscuous, utilizing a variety of substrates. Therefore, it has been speculated that under the right conditions and altered sugar metabolism, E. coli may naturally possess enzymes capable of producing D-psicose. F6P can be epimerized to P6P and then dephosphorylated to D-psicose in the final thermodynamically favorable step ( Figure 14CIn theory, placing this favorable reaction at the end of the biosynthetic pathway could boost the pathway's flux by restoring equilibrium. Because D-psicose 6-phosphate is favorably dephosphorylated to D-psicose and excreted from the cell, the equilibrium is restored by generating more P6P from F6P.

[0457] Evaluation of the native D-psicose production capacity of Escherichia coli.

[0458] To evaluate the native ability of E. coli to produce D-psicose, E. coli MG1655 and the MG1655 derivative strain AL3601 (carrying the gene encoding T7 RNAP) were tested for production in M9P medium (Table 5, Methods). In the absence of any genetic manipulation, neither strain produced detectable levels of D-psicose ( Figure 15A ).

[0459] It has been hypothesized that carbon flux must be purposefully directed towards the production of D-psicose by accumulating the upstream metabolite F6P. The major metabolic pathway competing for F6P is glycolysis, as F6P is preferentially catalyzed by phosphofructokinase A and B (PfkA and PfkB, Figure 14C ) is converted to D-fructose 1,6-bisphosphate. To establish an intracellular F6P pool, the gene encoding PfkA (which accounts for approximately 90% of phosphofructokinase activity) was deleted in MG1655 and AL3601, generating AL4058 and AL3694, respectively (Table 5). The ΔpfkA strain produced 0.24 g L in AL4058. -1 of D-psicose, producing 0.15 g L -1 This indicates that E. coli contains the enzymes necessary for producing D-psicose, which is likely derived from F6P ( Figure 15A ). The production of D-psicose was not detected in the culture grown in M9P medium without glucose.

[0460] To elucidate the enzymes involved in D-psicose production.

[0461] D-psicose 6-phosphate 3-epimerase (AlsE) was identified as a potential candidate enzyme for converting F6P to P6P. AlsE assimilates D-psicose into central carbon metabolism by converting P6P to F6P. At high concentrations of F6P, AlsE exhibited reverse activity, capable of converting F6P to P6P. To confirm whether AlsE is involved in the production of D-psicose, alsE was deleted in AL3694 and AL4058, resulting in strains AL4063 and AL4082 (Table 5). Neither strain produced detectable levels of D-psicose, indicating that AlsE is the epimerase involved in the production of D-psicose ( Figure 15A).

[0462] Escherichia coli has a variety of phosphatases with potential activity against P6P. Candidate phosphatases were selected based on their broad activity against various sugar substrates, and hexitol phosphatase B (HxpB), sugar phosphatase YbiV, sugar phosphatase YidA, hexitol phosphatase A (HxpA), α-D-glucose-1-phosphate phosphatase YihX, and phosphosugar phosphatase YigL were tested. To test the activity of various phosphatases in converting P6P to D-psicose, genes for various phosphatases were expressed from pT721 and alsE on an expression plasmid (Table 6). The plasmid was introduced into AL3601 (pfkA+). After 24 hours, the strain containing pAL1946 (P T7 alsE-hxpB) produced the most D-psicose, which was 0.55 g L -1 , indicating that HxpB is an excellent phosphatase candidate for D-psicose production ( Figure 15B ). Contains pAL1947 (P T7 : alsE-ybiV) or pAL2351 (P T7 : alsE-yidA) strains produced 0.21 g L -1 and 0.20 g L -1 D-psicose, and containing pAL2348 (P T7 : alsE-hxpA), pAL2352 (P T7 : alsE-yihX) and pAL2349 (P T7 Cultures of α-psicose (α-psicose) did not produce detectable D-psicose ( Figure 15B ).

[0463] Identification of key P6P-binding motifs.

[0464] A combination of AlphaFold and Rosetta Molecular Suite was used to evaluate the predicted binding modes between each of the six phosphatases and P6P. Phosphatases that were predicted to be active on P6P (HxpB, YbiV, and YidA) would form at least two internal hydrogen bonds and one additional hydrogen bond with the terminal hydroxyl group of P6P ( Figure 26A and 26B In contrast, the phosphatases predicted to be inactive on P6P (HxpA, YihX, and YigL) did not form hydrogen bonds with the terminal hydroxyl group of P6P. The phosphatase HxpB, which produced the highest D-psicose titer, was predicted to form hydrogen bonds between the active site residues and the four hydroxyl groups of P6P ( Figure 26B These predictions suggest that a minimum of three hydrogen bonds, including one to the terminal hydroxyl group, is critical for binding P6P in a catalytically competent orientation.

[0465] Comparison of expression systems for D-psicose production.

[0466] In AL3601, a T7 RNA polymerase expression system was used, which includes the T7 RNA polymerase (RNAP) gene under the IPTG-inducible PlacUV5 promoter. However, when IPTG was added to induce T7 RNAP expression, a decrease in growth was observed ( Figure 15C Therefore, an alternative expression system was tested in which the operons of alsE and hxpB were expressed from the IPTG-inducible promoter PLlacO1. LlacO1 : alsE-hxpB strains produced with P T7 : alsE-hxpB strains similar to D-psicose ( Figure 15C ). Importantly, with P T7 : Compared with the alsE-hxpB strain, P LlacO1 : The alsE-hxpB strain obtained better inhibition under non-inducing conditions (without IPTG) and did not produce growth burden under inducing conditions ( Figure 15C ). Therefore, P LlacO1 The expression system can be used for further studies.

[0467] Increasing D-psicose production by removing competing pathways

[0468] E. coli relies on two main glycolytic pathways to metabolize glucose: the PPP and the glycolytic (also known as the Embdem Meyerhof Parnas (EMP) pathway). A branch point between D-psicose production and the PPP occurs when glucose-6-phosphate dehydrogenase (Zwf) converts G6P to 6-phospho-D-glucono-1,5-lactone. Figure 15C The branch point between D-psicose production and glycolysis occurs when one of two phosphofructokinases, PfkA or PfkB, converts F6P to D-fructose 1,6-bisphosphate ( Figure 15C This example determined that the deletion of pfkA resulted in an improvement in D-psicose production compared to the unmodified base strain ( Figure 15A ).

[0469] In addition to PPP and glycolysis, the allulose degradation pathway has the potential to divert carbon flux away from D-psicose production by re-assimilating P6P back into central carbon metabolism. The rpiB gene encodes allose-6-phosphate isomerase (RpiB), which may be able to convert P6P into aldehyde-D-allose 6-phosphate ( Figure 14C ).

[0470] To redirect carbon flux from central carbon metabolism toward D-psicose production, we constructed knockouts of ΔpfkA, Δzwf, and ΔrpiB in AL1050, generating strain 1 (Table 4). These three gene deletions resulted in a fourfold increase in D-psicose production, reaching 2.31 g L -1 D-psicose ( Figure 15D ).

[0471] Table 4. List of key strains used in this example.

[0472]

[0473] All strains and plasmids used in this study are listed in Tables 5 and 6, respectively.

[0474] Table 5. Strains used in this example.

[0475] strains genotype source MG1655 F-λ-ilvG-rfb-50rph-1 Baba et al. 2016 AL1050 <![CDATA[MG1655, but with attB::lacI q tetR spec R > Yoneda et al. 2014 AL3601 <![CDATA[AL1050, but with ss9::P lacUV5 : T7rnap]]> Zhang et al. 2021 AL4058 MG1655, but with ΔpfkA This embodiment AL3694 AL3601, but with ΔpfkA This embodiment AL4063 AL3694, but with ΔalsE This embodiment AL4082 AL4058, but with ΔalsE This embodiment AL3756 AL1050, but with ΔpfkAΔzwfΔrpiB This embodiment AL3990 AL3756, but with ΔmanA This embodiment AL4121 AL3990, but with ΔptsG This embodiment AL4188 AL3990, but with ΔptsH This embodiment AL4189 AL4121, but with ΔptsH This embodiment AL4186 AL3990, but with Δpgm This embodiment AL4187 AL4121, but with Δpgm This embodiment AL4200 AL4188, but with Δpgm This embodiment AL4201 AL4189, but with Δpgm This embodiment

[0476] Table 6. Strains used in this example.

[0477]

[0478]

[0479]

[0480] *See Figure 21A and 21B .

[0481] By knocking out pfkA, zwf, and rpiB, carbon flux was directed toward the D-psicose production pathway. Specifically, knocking out pfkA and zwf resulted in increased intracellular F6P availability. These results reaffirm the hypothesis that F6P plays a key role as a precursor of P6P and that its accumulation is necessary to drive carbon flux through the production pathway.

[0482] Identification of by-products.

[0483] When analyzing a sample of strain 1, a significant peak was observed on the high-performance liquid chromatography (HPLC) chromatogram that was inconsistent with the D-glucose, D-fructose, or D-psicose standards or any of the culture medium components. Analysis using gas chromatography-mass spectrometry (GC-MS) revealed that the retention time and mass spectrum of the unknown peak matched those of D-mannose ( Figure 19 ).

[0484] Under standard conditions, the D-mannose pathway involves the assimilation of D-mannose 6-phosphate (M6P) into central carbon metabolism, where M6P is reversibly isomerized to F6P by mannose-6-phosphate isomerase (ManA). Accumulation of F6P may cause this reaction to reverse, generating M6P and D-mannose. To test the hypothesis that this byproduct is D-mannose, manA was deleted in strain 1 to generate strain 2 (Table 4).

[0485] The deletion of manA resulted in a significant decrease in D-mannose production, with strain 2 producing only 0.69 g L using IPTG. -1 D-mannose, while strain 1 using IPTG produced 2.49 g L -1 D-mannose ( Figure 19 Complementing the decrease in D-mannose production, the D-psicose production in strain 2 was 1.5 times higher than that in strain 1 ( Figure 15D Although not completely eliminated, the deletion of manA significantly reduced the production of unwanted D-mannose. Therefore, subsequent production was performed in a strain containing ΔpfkA, Δzwf, ΔrpiB, and ΔmanA.

[0486] Despite the elimination of the D-mannose pathway, other D-fructose epimers continue to compete for carbon flux. Eliminating the PPP and limiting glycolysis could lead to a substantial increase in the cellular F6P pool, which, if unable to efficiently feed into D-psicose biosynthesis, could be acted upon by other epimerases or isomerases. In short, increased F6P availability could allow enzymes not normally observed to bind substrates and produce alternative sugar products.

[0487] A stationary phase promoter is used.

[0488] Building production pathways in microorganisms requires careful allocation of carbon between essential metabolic processes and production, especially when working around central carbon metabolism. In the disclosed system, dynamically balancing carbon flux between glycolysis and the D-psicose pathway can help maximize cell viability and D-psicose production.

[0489] The life cycle of an Escherichia coli culture consists of five distinct phases: the lag phase, the logarithmic phase, the stationary phase, the death phase, and the long-term stationary phase. The lag phase occurs when cells are inoculated into a culture medium and adjust their metabolic processes to the new environment. The cells then grow and divide rapidly, entering the logarithmic phase. During this phase, enzymes related to central carbon metabolism are most important, and transcription of the corresponding genes is upregulated. Once the cells experience environmental stress (such as a scarcity of nutrients in the culture medium), their growth and division slow, and the culture enters the stationary phase. At this point, the culture density reaches a plateau, and genes related to stress responses are expressed. Transcription of these genes is regulated in part by the σ38 subunit of RNA polymerase, which recognizes the promoter region of the gene.

[0490] The native gene regulatory system of E. coli was exploited to balance carbon flux by anchoring the D-psicose production genes alsE and hxpB downstream of a stationary-active promoter. This prevented the production pathway from competing with central carbon metabolism for carbon flux during the logarithmic growth phase, when cells require carbon strictly for growth and division.

[0491] Four promoters that have been previously shown to be active during quiescence were selected and tested using green fluorescent protein (GFP) as a reporter gene: gadB 、P cbpA2 、P ihfA4 and P dps Each promoter was cloned upstream of sfgfp on the expression plasmid (Table 6). gadB The expression intensity of the IPTG-induced promoter P LlacO1 100-fold, and its expression time is related to the late logarithmic phase or early stationary phase ( Figure 20A The second strongest promoter P cbpA2 Following the induced P LlacO1 expression time, but the expression intensity is about 2 / 3 ( Figure 20B ). P ihfA4 and P dps Also follow P LlacO1 expression time, but the expression intensity was lower than that of P cbpA2 . ( Figure 20C )

[0492] Because P gadB Expression is robust during stationary phase and was therefore used to express the alsE and hxpB operons (pAL2247, Table 6). To evaluate the effect of initial glucose concentration, 10, 20, and 40 g L -1 The production of D-psicose was tested. -1 ) and strain 2 (P LlacO1 :alsE-hxpB, Figure 16A ) compared with strain 3 (PgadB : alsE-hxpB, Table 4) consistently produced higher D-psicose titers and showed a larger ΔOD 600 Growth, which is expressed as the optical density at 600 nm (OD 600 ) difference. When the glucose concentration is 40g L -1 When the titer of D-psicose was the highest, strain 3 could produce 6.92 g L -1 D-psicose, ΔOD during growth 600 was 5.2, while strain 2 could produce 4.55 g L -1 D-psicose, ΔOD during growth 600 is 4.0. In further studies, we used 40g L -1 The initial glucose concentration.

[0493] Next, the effect of the transition time from 37°C to 30°C on D-psicose was tested. Preliminary tests showed that 37°C was suitable for cell growth, while 30°C was suitable for production. To determine the optimal transition time from 37°C to 30°C, the culture was grown at 37°C until OD 600 The OD value was about 0 (no culture at 37°C), about 0.4, or about 1, and then grown at 30°C and induced as needed. In all strains, the culture was grown at a later OD value of 600 Switching to 30 °C resulted in higher D-psicose titers and a larger ΔOD 600 Growth Figure 3 b) When OD 600 When the growth condition was about 1, strain 3 produced the highest titer of D-psicose, which was 9.13 g L -1 , with ΔOD 600 The growth rate was 5.6. Strain 2 produced 5.42 g L -1 D-psicose, expressed as ΔOD 600 Growth is 4.4.

[0494] In theory, although P gadB It is better than P LlacO1 A stronger promoter is used, but its expression timing prevents the D-psicose-producing enzyme from siphoning carbon away from central metabolism during critical growth periods. This enables the culture to grow more robustly and produce higher titers of D-psicose. The use of an endogenous, growth-phase-dependent promoter eliminates the need for expensive chemical inducers and allows the culture to self-regulate pathway expression based on growth and cell viability. Subsequently, P gadB : alsE-hxpB (pAL2247, Table 6) was used for further production experiments.

[0495] Complementation of glucose input using GalP and Glk

[0496] Continuous glucose input, especially during the stationary phase of growth, is highly beneficial for D-psicose production. One consequence of restricting carbon flux through glycolysis by knocking out pfkA is a reduction in downstream metabolites such as phosphoenolpyruvate (PEP). PEP is particularly concerning because it is used by the PTS to import and phosphorylate glucose. Reduced PEP availability due to reduced glycolytic flux could affect the ability to assimilate glucose and produce D-psicose. Furthermore, it has been shown that increased G6P or F6P pools in ΔpfkA mutants lead to degradation of ptsG mRNA, encoding the membrane receptor IICBGlc of the PTS complex.

[0497] To enhance glucose import without the use of a PTS in the ΔpfkA background, galP and glk were also expressed from plasmids. The galP gene encodes the galactose proton symporter GalP, which enables glucose import. The glk gene encodes the glucokinase Glk, which phosphorylates glucose to G6P ( Figure 14C These genes were cloned downstream of PLlacO1 to generate plasmid pAL2264 (Table 6). In strain 4, galP-glk expression was induced (strain 3 had P LlacO1 :galP-glk, Table 4) significantly increased the yield of D-psicose and enabled the strain to produce 13.81 g L -1 , with a specific titer of 3.2 g L -1 OD 600 -1 and a yield of 55% ( Figure 16C In contrast, strain 4 without IPTG produced 8.67 g L -1 D-psicose, specific titer 1.3 g L -1 OD 600 -1 , with a yield of 49%. The yield was calculated based on the probability that 1 mol of D-psicose can be produced for every mol of D-glucose consumed, so the theoretical maximum yield is 100%. Interestingly, the expression of galP and glk resulted in a lower ΔOD 600 ( Figure 16C Overexpression of membrane proteins such as GalP has been shown to impose a growth burden on cells, possibly due to competition for membrane transport mechanisms.

[0498] Controls glucose utilization and metabolism

[0499] Studies have shown that the PTS utilizes approximately 50% of PEP to transport and phosphorylate glucose. To further balance the cellular PEP supply and promote glucose import using GalP-Glk, we attempted to disable the PTS by knocking out the genes ptsG (encoding the membrane receptor IICBGlc) and ptsH (encoding the phosphate transport protein HPr) in AL3990 (Table 5).

[0500] Another source of glucose drawn from the D-psicose pathway is through glycogen biosynthesis. Glycogen is stored for use during starvation, which is unnecessary for the presently disclosed subject matter. Deletion of pgm (encoding the phosphoglucomutase Pgm) prevents E. coli from producing glycogen. Therefore, pgm was deleted in the production strain (Table 5).

[0501] Strain 4 with ΔptsG appeared to be detrimental to psicose production (ΔptsG, ΔptsGΔptsH, and ΔptsGΔpgm), but strain 4 with ΔptsH had no effect on psicose production ( Figure 16D Δpgm is beneficial for psicose production, especially at a specific titer ( Figure 16D Strain 5 (strain 4 with Δpgm, Table 4) produced 14.66 g L -1 D-psicose, specific titer 5.5 g L-1OD 600 -1 , the yield is 58% ( Figure 16D ).

[0502] Knocking out pgm to inhibit glycogen biosynthesis eliminates the carbon source for central metabolism that normally supports cell growth during starvation. The combined effect of impaired growth and increased production resulted in an increase in specific titers from strain 4 to strain 5 ( Figure 16D ).

[0503] Although some studies have shown that eliminating PTS by gene knockout helps restore intracellular PEP balance and rescue growth, this study found that knocking out ptsG or ptsH had no or no effect on D-psicose production. The phosphorylated and dephosphorylated forms of IICBGlc and HPr participate in signaling cascades not only related to carbon metabolism but also through the expression of RNA polymerase σ subunits (including the above σ 38 subunits and logarithmic phase correlation σ 70 subunits) participate in global gene expression. gadB Expression of alsE and hxpB and elimination of part of the PTS may reduce the expression of genes in the D-psicose production pathway.

[0504] Dynamic regulation of glycolysis using CRISPRi

[0505] While deletion of pfkA successfully redirected carbon flux toward D-psicose production, glycolysis remained active through PfkB. Completely shutting down glycolysis through deletion of pfkB did not allow cells to grow under our culture conditions, so we attempted to dynamically restrict pfkB expression to the required time. During the logarithmic growth phase, cells require carbon flux through glycolysis as they build biomass. During the stationary phase, carbon flux through glycolysis can be reduced and redirected toward D-psicose production. To dynamically regulate pfkB expression, a CRISPRi system targeting pfkB on the genome was implemented.

[0506] The CRISPRi system utilizes an inactivated Cas9, dCas9, which, when recruited by a single guide RNA scaffold (sgRNA), can precisely target and block transcription initiation by RNA polymerase. The dCas9 gene is cloned together with a constitutively expressed sgRNA sequence targeting the gene of interest under the control of an aTc inducible promoter. tet To confirm the functionality of the CRISPRi system and determine the position where sgRNA should be targeted to achieve maximal expression inhibition, three different sgRNA sequences were designed to inhibit P LlacO1 The expression of sfGFP ( Figure 17A ). sgRNA targeting P LlacO1 The upstream, middle and downstream sequences ( Figure 17A Here, it was found that targeting P LlacO1 The middle sgRNA resulted in the largest fluorescence difference. Continuous sgRNAs targeting the pfkB promoter region were designed, sharing homology with the middle of the promoter sequence.

[0507] Further exploration of the CRISPRi system involved expressing dCas9 and sgRNA from the same or separate plasmids ( Figure 22A and 22B ). In a single plasmid system, P tet : dcas9 and constitutively expressed sgRNA sequences targeting the pfkB promoter region or without targeting sequence were cloned into the same plasmid (Table 6). For separate plasmid systems, P tet dCas9 was cloned into one plasmid, and the constitutively expressed sgRNA sequences targeting the pfkB promoter region or without targeting sequences were cloned into different plasmids (Table 6). Each CRISPRi system was introduced into AL4186 (Table 5) and cultured with 100 ng mL -1Growth was measured 24 hours after aTc induction. The separate plasmid system resulted in greater growth inhibition, likely because the sgRNA was expressed from a high-copy number plasmid, rather than from a low-copy number plasmid as in the single-plasmid system. When production was tested using the separate plasmid CRISPRi system in strain AL3990 using pAL2247 (Tables 5 and 6), little D-psicose was produced regardless of aTc induction. Therefore, a single-plasmid CRISPRi system was used for production.

[0508] The single-plasmid CRISPRi system was introduced into strain 5 to generate strains 6 (empty guide) and 7 (sgRNA targeting pfkB, Table 4). After using CRISPRi (strain 7), cell growth was reduced and the specific titer was greatly improved with or without aTC, although the titer of strain 7 was lower than that of strains 5 and 6 ( Figure 17A ). Add 100ng mL -1 Strain 7 of 1aTc produced 11.40 g L -1 D-psicose, specific titer 3.6 g L -1 OD 600 -1 , with a yield of 62%. Strain 7 without aTc produced 13.65 g L -1 Allulose, specific titer 3.8 g L -1 OD 600 -1 , with a yield of 60%.

[0509] D-psicose production under high culture density conditions

[0510] To investigate the D-glucose consumption and D-psicose production rates of strain 7 (Table 4), the D-psicose production rate was determined by culturing the culture medium at glucose concentrations of 3, 5, and 10 g L -1 Monitor substrate concentration for 10 h when glucose is present ( Figures 23A-23C ). It was found that the production rate of D-psicose in different cultures was similar and had nothing to do with the glucose concentration in the culture medium. -1 The cultures on glucose consumed all the glucose during the experiment, which means that D-psicose can be more easily extracted and purified from the culture medium in an industrial setting.

[0511] To separate growth and production and minimize the limitation of glucose availability on production, strain 7 was cultured for a shorter time under high cell density conditions with excess D-glucose ( Figure 17B Strain 7 (Table 4) was grown to OD 600 is about 1, then 100 ng mL -1 aTc and 1 mM IPTG and grown for another 30 min. The cells were then pelleted and washed with 40 g L-1 Glucose, 100 ng mL -1 Resuspend M9P with aTc and 1 mM IPTG to OD 600 The yield of strain 7 was approximately 10. Samples were collected and analyzed at 0, 4, and 8 hours. During the 8-hour period, strain 7 produced 15.3 g L -1 D-psicose, specific titer is 1.4 g L-1OD 600 -1 , with a yield of 43% and a productivity of 1.9 g L -1 hr -1 ( Figure 17B Although the titers of D-psicose produced in 0-4 h and 4-8 h were similar, they were 7.3 and 8.0 g L -1 , but the yield at 4-8 hours (53%) was higher than that at 0-4 hours (35%). The higher yield may be a result of the cells responding to their high-density culture conditions, as the production system of the present disclosure relies on the stationary phase promoter P gadB Overall, despite the unoptimized in vitro culture conditions, this production system achieved industrially relevant production with high yields (>0.5 g 产品 g 底物 -1 ) and high productivity (>1g L -1 hr -1 ), which is comparable to bioethanol.

[0512] in conclusion

[0513] In this disclosure, whole-cell catalysis is used as a strategy to produce the industrially relevant rare sugar D-psicose. Living cells possess the ability to assemble stereo- and regio-selective enzymes, provide necessary cofactors, and secrete readily purified products, all under environmentally friendly production conditions. Whole-cell catalysis technology and infrastructure are already established industrially, and the model organism Escherichia coli can be fed a feedstock that does not compete with commercial food products.

[0514] Eliminating competing pathways, additionally expressing native E. coli genes (alsE, hxpB, galP, and glk), and employing both static and dynamic gene regulation strategies resulted in a strain capable of producing D-psicose from readily available feedstocks via a thermodynamically favorable biosynthetic pathway. Under in vitro conditions, the highest titer of D-psicose produced was 16.59 g L-1. -1 , with a specific titer of 5.0 g L -1 OD 60 0 -1The highest yield was 62%, exceeding the current industry standard. In addition, the strain was able to consume all D-glucose in the culture medium, greatly simplifying downstream purification requirements. Overall, the engineered strain represents an effective step towards producing D-psicose in a cost-effective manner, providing the food industry with a viable source for producing low-glycemic index products that consumers demand.

[0515] Overall, the engineered strains represent a major step forward in producing D-psicose and other rare sugars in an efficient and cost-effective manner. The ability to produce rare sugars in bulk would help address rising global obesity rates by providing low-calorie sugar alternatives to ultra-processed foods. Increased production of rare sugars would also enable sustainable pesticides for agriculture and provide the pharmaceutical industry with pharmaceutically relevant monosaccharides. The strategies developed in this study have the potential to revolutionize our ability to generate, measure, and control our relationship with food, creating a world where metabolic health is easily achieved, enabling everyone to live happier, healthier, and longer lives.

[0516] method

[0517] Reagents. All enzymes involved in the molecular cloning experiments were purchased from New England Biolabs. w All oligonucleotides were synthesized by Integrated DNA Technologies. Sanger sequencing was provided by Genewiz, a subsidiary of Azenta Life Sciences. D-psicose and D-mannose were purchased from Sigma-Aldrich. D-glucose was purchased from Fisher Scientific.

[0518] Strains and Plasmids. All strains and plasmids used in this study are listed in Tables 5 and 6, respectively. All oligonucleotides are listed in Table 7. Plasmids used for D-psicose production were constructed using sequence-independent ligation cloning (SLIC). The constructed plasmids were verified by Sanger sequencing. The plasmid construction guidelines used in this study are detailed in Table 8.

[0519] Table 7. Oligonucleotides used in this example.

[0520]

[0521]

[0522]

[0523]

[0524]

[0525]

[0526]

[0527]

[0528]

[0529]

[0530]

[0531] Table 8. Plasmid construction guidelines.

[0532]

[0533]

[0534] *Q5-directed mutagenesis (NEB)

[0535] CRISPR-Cas9-mediated homologous recombination was used to construct genome modifications such as gene deletion and gene insertion. Genomic or plasmid DNA was amplified by PCR assembly to construct linear DNA repair fragments for gene deletion and insertion. Q5 site-directed mutagenesis (New England Biolabs) was used with the pTargetF plasmid (Addgene #62226) as a template to construct plasmids encoding sgRNA for CRISPR-Cas9-mediated homologous recombination. All genome modifications were verified by Sanger sequencing. The CRISPR-Cas9-mediated gene modification guides used in this study are detailed in Table 9.

[0536] Table 9. Guidelines for CRISPR-Cas9-mediated gene deletion and insertion

[0537]

[0538]

[0539]

[0540] Cultures were grown overnight at 37°C in 3 mL of Luria-Bertani (LB) medium containing appropriate antibiotics. Antibiotic concentrations were as follows: spectinomycin (50 μg mL -1 ), ampicillin (200 μg mL -1 ), kanamycin (50 μg mL -1 ), gentamicin (3.75 μg mL-1 M9 minimal medium consists of 33.7 mM Na2HPO4, 22 mM KH2PO4, 8.6 mM NaCl, 9.4 mM NH4Cl, 2 mM MgSO4, 0.1 mM CaCl2, A5 trace metal mixture (2.86 mg H3BO3, 1.81 mg MnCl2.4H2O, 0.079 mg CuSO4·5H2O, 49.4 μg Co(NO3)2·6H2O), different concentrations of glucose and appropriate antibiotics. The M9P medium for the production of psicose consists of 5 g L -1 M9 minimal medium was composed of yeast extract and appropriate antibiotics. When the culture was grown in M9P medium without glucose, no D-psicose production was detected. The inducer concentrations were as follows: isopropyl-β-D-1-thiogalactopyranoside (IPTG) (1 mM), anhydrotetracycline (aTc) (100 ng mL -1 OD was measured using a Synergy H1 hybrid plate reader (BioTek Instruments, Inc.). 600 .

[0541] Fluorescence assay. Inoculate 1% of the overnight culture into 300 μL of LB medium in a 96-well black-walled fluorescence assay plate. Grow the cells at 37°C and 250 rpm until the OD 600 The culture was then induced with IPTG, if necessary, and grown for 24 hours at 37°C and 250 rpm. Fluorescence was measured using a Synergy H1 hybrid plate reader (BioTek Instruments, Inc.) at an excitation wavelength of 485 nm and an emission wavelength of 510 nm.

[0542] D-psicose production. For conventional cell density production experiments, 1% of overnight culture was inoculated into 3 mL of M9P medium. Cells were grown at 37°C until the OD 600 Then induce with IPTG and aTc (if necessary) and grow at 30°C for 24 hours. For high cell density production experiments in M9P medium, inoculate 50 mL of M9P medium with 2% of the overnight culture. Grow cells at 37°C until OD600 is approximately 1. Then induce the culture with IPTG and aTc (if necessary) and grow for another 30 minutes. Centrifuge the culture at 5,000g for 15 minutes and resuspend in M9P medium and resuspend with IPTG and aTc (if necessary) to the target OD 600 The cultures were grown at 30°C for 24 hours.

[0543] HPLC analysis was performed using a 10A refractive index detector (RID) and a Rezex TM D-psicose, glucose, and mannose concentrations were analyzed by high-performance liquid chromatography (HPLC) (Shimadzu) using an RCU-USP sugar alcohol column (Phenomenex). The mobile phase consisted of 100% MilliQ water. Samples were run with an injection volume of 1 μL and a flow rate of 0.5 mL min -1 , run for 7 minutes, with a column oven at 83°C and a refractive index cell (RID) temperature at 40°C. To prepare samples for HPLC analysis, 300 μL of culture was centrifuged at 17,000 g for 5 minutes. The supernatant was added to a 0.2 μm PVDF hydrophilic membrane 96-well filter plate and centrifuged at 17,000 g for 2 minutes into a polystyrene 96-well plate.

[0544] GC-MS analysis. GC-MS analysis was performed by the UC Davis West Coast Metabolomics Center. Chemical standards (D-psicose, D-mannose, D-glucose, D-galactose, D-erythrose, D-tagatose, and D-threose) were purchased from Sigma-Aldrich. For analysis by GC-MS, 4 μL of centrifuged culture supernatant was dried and precipitated by adding 10 μL of 40 mg mL in pyridine (Sigma-Aldrich). -1 Methoxyamine hydrochloride (Sigma-Aldrich) was added and derivatized with shaking at 30°C for 1.5 hours. Subsequently, 90 μL of N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) (Sigma-Aldrich) was added as a retention index marker along with 13 fatty acid methyl esters (FAMEs) and shaken at 80°C for 30 minutes. The samples were immediately transferred to crimp bottles and injected into each GC-MS instrument. The LECO Pegasus IV TOF MS was coupled to an Agilent 7890GC system equipped with a Restek RTX-5Sil MS column (29.70 m long, 0.25 mm inner diameter, 0.25 μM df, 95% dimethyl / 5% diphenylpolysiloxane membrane) and an additional 10 m guard column. 1 μL of the derivatized sample was injected into the GC in splitless mode with an injection temperature of 275°C and a constant flow rate of 1 mL min -1 The initial oven temperature was maintained at 50°C for 1 min and then at 20°C min -1 The mass spectrometer was used in electron ionization mode at +70 eV. The mass spectra were acquired from 85 to 500 m / z at a scan rate of 17 Hz and a source temperature of 250°C. Binbase was used for metabolite annotation and reporting.

[0545] Example 3 - Biosynthesis of Psicose from Glucose under High Density Conditions

[0546] Another source of glucose removed from the D-psicose pathway is through glycogen biosynthesis, which produces glycogen for use during starvation. Deletion of pgm, encoding the phosphoglucomutase Pgm, prevents glycogen production in Escherichia coli (Eydallin, G. et al. Genome-wide screening of genes affecting glycogen metabolism in Escherichia coli K-12. FEBS Lett. 581, 2947-2953 (2007)). Therefore, pgm was deleted in the production strain AL3990 to generate AL4186 (MG1655ΔpfkAΔzwfΔrpiBΔmanAΔpgm).

[0547] The glucose concentrations in the culture medium were 3, 5, and 10 g L -1 The D-glucose consumption rate and D-psicose production rate of strain 7 (see Table 4) were monitored for 10 hours. It was found that the D-glucose consumption rate and D-psicose production rate in different cultures were similar and had nothing to do with the glucose concentration in the culture medium. -1 Cultures on glucose consumed all glucose during the experiment.

[0548] To separate growth and production and to minimize the limitation of production on glucose availability, plasmid pAL2247 (P gadB : alsE-hxpB), pAL2264 (P LlacO1 :galP-glk) and pAL2188 (P tet Strain AL4186 (also identified as strain 7 in Table 4) transformed with dcas9pTargetF-pfkB was cultured under high cell density conditions for a short time in excess available D-glucose. -1 Grow in M9P medium containing glucose at 37°C until OD 600 is about 1, then 100 ng mL -1 aTc and 1 mM IPTG and grown for another 30 min. The cells were then pelleted and washed with 40 g L -1 Glucose, 100 ng mL -1 aTc and 1 mM IPTG were resuspended in M9P to an OD 600 was approximately 10. Samples were collected and analyzed at 0, 4, and 8 hours.

[0549] Over the course of 8 h, the culture produced 15.3 g L -1 D-psicose, specific titer 1.4 g L -1 OD 600 -1 , with a yield of 43% and a productivity of 1.9 g L -1 hr -1 See also Figure 24 Although the titers of D-psicose produced during 0-4 h and 4-8 h were similar, 7.3 and 8.0 g L -1 , but the yield in 4-8 h (53%) was higher than that in 0-4 h (35%). The productivity in 4-8 h was 2.0 g L -1 h -1 The higher yields may be a result of the cells responding to their high density culture conditions, as the production system disclosed herein relies on activation of the quiescent promoter PgadB. Overall, despite being cultured under non-optimized in vitro conditions, this production system still achieves industrially relevant production with high yields (>0.5 g 产品 g 底物 -1 ) and high productivity (>1gL -1 h -1 ), which is comparable to bioethanol

[0550] Next, to determine the purity of the sample, high performance liquid chromatography (HPLC) was used to analyze the concentrations of glucose, psicose, and mannose. Glucose, psicose, and mannose standards of known concentrations were run on HPLC, and the area under each corresponding peak could be integrated. For each sugar standard, peak integrals were plotted for concentration and fitted with a best fit line. While using the standards, production samples were run on HPLC. Standards were used to identify the corresponding sugar peaks in each production sample. Each sample peak was integrated, and their areas were recorded. Using the best fit line, peak integrals were used to find the sugar concentration in each sample. Psicose purity was calculated using sugar concentration and the following equation:

[0551]

[0552] Strain 7 was able to consume all the glucose in the culture medium within 24 hours ( Figure 25 ), indicating that the purity of D-psicose was approximately 100%. Complete substrate consumption is a desirable characteristic for microbial production, as isolating D-psicose from glucose and / or fructose mixtures is costly. Depleting glucose from the culture medium could make it easier to extract and purify D-psicose in an industrial setting.

[0553] Although the present disclosure and its advantages have been described in detail, it should be understood that various changes, substitutions and modifications may be made herein without departing from the spirit and scope defined by the appended claims of the present invention. Moreover, the scope of this application is not intended to be limited to the specific embodiments of the processes, machines, manufactures, material compositions, means, methods and steps described in the specification. A person of ordinary skill in the art will readily understand from the content of the present disclosure that processes, machines, manufactures, material compositions, means, methods or steps that currently exist or will be developed in the future and that perform substantially the same functions as the corresponding embodiments herein or achieve substantially the same results may be used in accordance with the present disclosure. Therefore, the appended claims are intended to include such processes, machines, manufactures, material compositions, means, methods or steps within their scope.

[0554] Patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

Claims

1. A recombinant microorganism comprising an exogenous epimerase and an exogenous phosphatase, wherein the recombinant microorganism produces an increased amount of allulose as compared to a naturally occurring microorganism.

2. The recombinant microorganism according to claim 1, wherein the epimerase is allulose-6-phosphate 3-epimerase (AlsE).

3. The recombinant microorganism according to claim 1 or 2, wherein the epimerase is Escherichia coli AlsE.

4. The recombinant microorganism according to any one of claims 1-3, wherein the epimerase comprises an amino acid sequence having at least about 80% identity with the amino acid sequence shown in SEQ ID NO:

1.

5. The recombinant microorganism according to any one of claims 1-4, wherein the epimerase comprises the amino acid sequence shown in SEQ ID NO:

1.

6. The recombinant microorganism according to any one of claims 1-5, wherein the epimerase consists of the amino acid sequence shown in SEQ ID NO:

1.

7. The recombinant microorganism according to any one of claims 1-6, wherein the phosphatase is hexitol phosphate phosphatase B (HxpB).

8. The recombinant microorganism according to any one of claims 1-7, wherein the phosphatase is Escherichia coli HxpB.

9. The recombinant microorganism according to any one of claims 1-8, wherein the phosphatase comprises an amino acid sequence having at least about 80% identity with the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO:

4.

10. The recombinant microorganism according to any one of claims 1-9, wherein the phosphatase comprises the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO:

4.

11. The recombinant microorganism according to any one of claims 1-10, wherein the phosphatase consists of the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO:

4.

12. The recombinant microorganism according to any one of claims 1-11, wherein the recombinant microorganism further comprises an exogenous galactose:H + symporter (Galp) and glucokinase (G1k).

13. The recombinant microorganism according to claim 12, wherein the GalP is Escherichia coli GalP and the Glk is Escherichia coli Glk.

14. The recombinant microorganism according to claim 13, wherein the GalP comprises an amino acid sequence having at least about 80% identity with the amino acid sequence shown in SEQ ID NO: 38, and the Glk comprises an amino acid sequence having at least about 80% identity with the amino acid sequence shown in SEQ ID NO:

40.

15. The recombinant microorganism according to claim 13, wherein the GalP comprises the amino acid sequence shown in SEQ ID NO: 38, and the Glk comprises the amino acid sequence shown in SEQ ID NO:

40.

16. The recombinant microorganism according to any one of claims 1-15, further comprising a mutation in a gene encoding an enzyme of the pentose phosphate pathway as compared to a naturally occurring microorganism.

17. The recombinant microorganism according to claim 16, wherein the enzyme of the pentose phosphate pathway is glucose-6-phosphate 1-dehydrogenase (Zwf).

18. The recombinant microorganism according to any one of claims 1-17, compared with a naturally occurring microorganism, further comprises a mutation in a gene encoding an enzyme of glycolysis.

19. The recombinant microorganism according to claim 18, wherein the enzyme of glycolysis is phosphofructokinase-1 (PfkA), phosphofructokinase-2 (PfkB) or pyruvate kinase (PykF).

20. The recombinant microorganism according to claim 18 or 19, wherein the enzyme of glycolysis is phosphofructokinase-1 (PfkA).

21. The recombinant microorganism according to any one of claims 1-20, further comprises a mutation in a gene encoding an enzyme of the allose degradation pathway.

22. The recombinant microorganism according to claim 21, wherein the enzyme of the allose degradation pathway is allose-6-phosphate isomerase (RpiB).

23. The recombinant microorganism according to any one of claims 1-22, further comprises a mutation in a gene encoding an enzyme of the mannose biosynthetic pathway.

24. The recombinant microorganism according to claim 23, wherein the enzyme of the mannose biosynthetic pathway is mannose-6-phosphate isomerase (ManA).

25. The recombinant microorganism according to any one of claims 1-24, further comprises an exogenous nuclease and sgRNA.

26. The recombinant microorganism according to claim 25, wherein the nuclease is dCas9.

27. The recombinant microorganism according to claim 25 or 26, wherein the sgRNA targets a gene encoding an enzyme of glycolysis.

28. The recombinant microorganism according to claim 27, wherein the enzyme of glycolysis is phosphofructokinase-2 (PfkB).

29. The recombinant microorganism according to any one of claims 1-28, wherein the exogenous epimerase and exogenous phosphatase are expressed by a stationary-phase promoter.

30. The recombinant microorganism according to any one of claims 25-29, wherein the exogenous nuclease is expressed by an inducible promoter.

31. The recombinant microorganism according to any one of claims 25-30, further comprises a mutation in a gene of an enzyme of glycogen biosynthesis selected from the group consisting of phosphoglucomutase (Pgm), UDP-glucose pyrophosphorylase, glycogen synthase, glycogen branching enzyme and glycogenin.

32. The recombinant microorganism according to claim 31, wherein the enzyme of glycogen biosynthesis is phosphoglucomutase (Pgm).

33. A microorganism comprising a recombinant polynucleotide encoding an epimerase and a phosphatase, wherein the expression of the epimerase and the phosphatase results in an increased production of tagatose compared with a microorganism lacking the recombinant polynucleotide.

34. The microorganism according to claim 33, wherein the epimerase is allose-6-phosphate 3-epimerase (AlsE).

35. The microorganism according to claim 33 or 34, wherein the epimerase is Escherichia coli AlsE.

36. The microorganism according to any one of claims 33-35, wherein the epimerase comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO:

1.

37. The microorganism according to any one of claims 33-36, wherein the epimerase comprises the amino acid sequence shown in SEQ ID NO:

1.

38. The microorganism according to any one of claims 33-37, wherein the epimerase consists of the amino acid sequence shown in SEQ ID NO:

1.

39. The microorganism according to any one of claims 33-38, wherein the phosphatase is hexitol phosphate phosphatase B (HxpB).

40. The microorganism according to any one of claims 33-39, wherein the phosphatase is Escherichia coli HxpB.

41. The microorganism according to any one of claims 33-40, wherein the phosphatase comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO:

4.

42. The microorganism according to any one of claims 33-41, wherein the phosphatase comprises the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO:

4.

43. The microorganism according to any one of claims 33-42, wherein the phosphatase consists of the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO:

4.

44. The microorganism according to any one of claims 33-43, which further comprises a mutation in a gene encoding an enzyme of the pentose phosphate pathway.

45. The microorganism according to claim 44, wherein the enzyme of the pentose phosphate pathway is glucose-6-phosphate 1-dehydrogenase (Zwf).

46. The microorganism according to any one of claims 33-45, which further comprises a mutation in a gene encoding an enzyme of glycolysis.

47. The microorganism according to claim 46, wherein the enzyme of glycolysis is phosphofructokinase-1 (PfkA), phosphofructokinase-2 (PfkB) or pyruvate kinase (PykF).

48. The microorganism according to any one of claims 46 or 47, wherein the enzyme of glycolysis is phosphofructokinase-1 (PfkA).

49. The microorganism according to any one of claims 33-48, which further comprises a mutation in a gene encoding an enzyme of the allose degradation pathway.

50. The microorganism according to claim 49, wherein the enzyme of the allose degradation pathway is allose-6-phosphate isomerase (RpiB).

51. The microorganism according to any one of claims 33-50, which further comprises a mutation in a gene encoding an enzyme of the mannose biosynthetic pathway.

52. The microorganism according to claim 51, wherein the enzyme of the mannose biosynthetic pathway is mannose-6-phosphate isomerase (ManA).

53. The microorganism according to any one of claims 33-52, which further comprises an exogenous nuclease and sgRNA.

54. The microorganism according to claim 53, wherein the nuclease is dCas9.

55. The microorganism according to claim 53 or 54, wherein the sgRNA targets a gene encoding an enzyme of glycolysis.

56. The microorganism according to claim 55, wherein the enzyme of glycolysis is phosphofructokinase-2 (PfkB).

57. The microorganism according to any one of claims 33-56, wherein the exogenous epimerase and exogenous phosphatase are expressed by a stationary-phase promoter.

58. The microorganism according to any one of claims 33-57, wherein the exogenous nuclease is expressed by an inducible promoter.

59. The microorganism according to any one of claims 33-58, which further comprises a mutation in a gene encoding an enzyme of glycogen biosynthesis selected from the group consisting of phosphoglucomutase (Pgm), UDP-glucose pyrophosphorylase, glycogen synthase, glycogen branching enzyme, and glycogenin.

60. The microorganism according to claim 59, wherein the selected enzyme of glycogen biosynthesis is phosphoglucomutase (Pgm).

61. A microorganism comprising: a) a recombinant polynucleotide encoding an epimerase and a phosphatase; b) a mutation in a gene encoding an enzyme of the pentose phosphate pathway; c) a mutation in a gene encoding an enzyme of glycolysis; d) a mutation in a gene encoding an enzyme of the allose degradation pathway; and e) a mutation in a gene encoding an enzyme of the mannose biosynthesis pathway; and f) optionally, a recombinant polynucleotide encoding GalP, Glk, or both.

62. A microorganism comprising: a) a recombinant polynucleotide encoding allose-6-phosphate 3-epimerase (AlsE) and hexitol phosphate phosphatase B (HxpB); b) a mutation in glucose-6-phosphate 1-dehydrogenase (Zwf); c) a mutation in phosphofructokinase-1 (PfkA); d) a mutation in allose-6-phosphate isomerase (RpiB); e) a mutation in mannose-6-phosphate isomerase (ManA); and f) optionally, a recombinant polynucleotide encoding GalP, Glk, or both.

63. A microorganism comprising: a) a recombinant polynucleotide encoding allose-6-phosphate 3-epimerase (AlsE); b) a recombinant polynucleotide encoding hexitol phosphate phosphatase B (HxpB); c) a mutation in glucose-6-phosphate 1-dehydrogenase (Zwf); d) a mutation in phosphofructokinase-1 (PfkA); e) a mutation in allose-6-phosphate isomerase (RpiB); f) a mutation in mannose-6-phosphate isomerase (ManA); and g) optionally, a recombinant polynucleotide encoding GalP, Glk, or both.

64. The microorganism according to any one of claims 33-64, wherein the recombinant polynucleotide is stably integrated into the genome.

65. The recombinant microorganism according to any one of claims 1-64, which comprises an increased intracellular fructose-6-phosphate content as compared to a naturally-occurring microorganism.

66. The microorganism according to any one of claims 1-65, wherein the microorganism is Escherichia coli, Bacillus subtilis or Lactococcus lactis.

67. The microorganism according to any one of claims 16-24, 31-32 or 44-52, 59 or 60, wherein the mutation is a deletion.

68. The microorganism according to any one of claims 16-24, 31-32 or 44-52, 59 or 60, wherein the mutation reduces or eliminates the expression or activity of the enzyme.

69. A method for producing allulose, which comprises culturing the microorganism according to any one of claims 1-68 under conditions suitable for converting a substrate into allulose.

70. The method according to claim 69, wherein the substrate comprises glucose.

71. The method according to claim 69 or 70, wherein the allulose has a purity value of at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99%.

72. The method according to claim 71, wherein the purity value is 100%.

73. The method according to claim 71 or 72, wherein the purity value is determined by the following formula:

74. Allulose produced by a method comprising culturing the microorganism according to any one of claims 1-68 under conditions suitable for converting a substrate into allulose, wherein the allulose has a purity value of at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99%.

75. The allulose according to claim 74, wherein the purity value is 100%.

76. The allulose according to claim 74 or 75, wherein the purity value is determined by the following formula:

77. A method for preparing a food product comprising allulose, which comprises: a) culturing the microorganism according to any one of claims 1-68 under conditions suitable for converting a substrate into allulose; b) purifying the allulose; and c) blending the allulose with a food product to form a food product comprising allulose.

78. The method according to claim 77, wherein the food product is chewing gum, candy, chocolate or a savory food.

79. The method according to claim 77, wherein the food product is a beverage, yogurt, ice cream, a baked good or a nutrition bar.

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