Microorganisms for the production of low-calorie sugars

Abstract

The present disclosure relates to microorganisms useful for the biosynthesis of psicose. Methods for producing the disclosed microorganisms and methods for producing psicose are also provided. TIFF2025530206000101.tif93138

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 405,208, filed September 9, 2022, and U.S. Provisional Patent Application No. 63 / 450,582, filed March 7, 2023, the contents of each of which are incorporated by reference in their entirety and to which priority is claimed.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, and is incorporated by reference herein in its entirety. Said XML copy, created on September 5, 2023, is named 081906-1401075-250410PC_SL and is 284,563 bytes in size.

[0003] Technical Field The subject matter disclosed herein relates to compositions and methods for producing low calorie sugars in microorganisms. [Background technology]

[0004] background Current industrial D-psicose (allulose) production relies on a two-step in vitro enzymatic synthesis, beginning with the conversion of glucose to fructose via xylose isomerase (EC 5.3.1.5). This reaction is reversible with a ΔG° of -0.1 kJ / mol. This isomerization is followed by epimerization of fructose to psicose via either D-tagatose-3-epimerase (EC 5.1.3.31) or D-psicose-3-epimerase (EC 5.1.3.30). The major drawback of in vitro synthesis is that this reaction has a predicted ΔG° of +5 kJ / mol and is thermodynamically unfavorable. Because both reactions are reversible, this in vitro system ultimately results in a mixture of glucose, fructose, and psicose, which increases the cost of downstream separation and purification processes. Summary of the Invention

[0005] overview In one aspect, the present disclosure relates to a recombinant microorganism comprising an exogenous epimerase and an exogenous phosphatase, wherein the recombinant microorganism produces increased amounts of psicose compared to naturally occurring microorganisms.

[0006] In some embodiments, the epimerase is allulose-6-phosphate 3-epimerase (AlsE). In some embodiments, the epimerase is E. coli AlsE. In some embodiments, the epimerase comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the epimerase comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the epimerase consists of the amino acid sequence set forth in SEQ ID NO: 1.

[0007] In some embodiments, the phosphatase is hexitol phosphatase B (HxpB). In some embodiments, the phosphatase is E. 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.

[0008] In some embodiments, the recombinant microorganism contains exogenous galactose:H + In some embodiments, the GalP comprises an amino acid sequence 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 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.

[0009] In some embodiments, the recombinant microorganism further comprises a mutation in a gene encoding a pentose phosphate pathway enzyme compared to a naturally occurring microorganism. In some embodiments, the pentose phosphate pathway enzyme is glucose-6-phosphate 1-dehydrogenase (Zwf). In some embodiments, the recombinant microorganism further comprises a mutation in a gene encoding a glycolytic enzyme compared to a naturally occurring microorganism.

[0010] 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).

[0011] In some embodiments, the recombinant microorganism further comprises a mutation in a gene encoding an enzyme in an allolytic pathway. In some embodiments, the enzyme in the allolytic pathway is allose-6-phosphate isomerase (RpiB). In some embodiments, the recombinant microorganism further comprises a mutation in a gene encoding an enzyme in a mannose biosynthetic pathway. In some embodiments, the enzyme in the mannose biosynthetic pathway is mannose-6-phosphate isomerase (ManA).

[0012] 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 by a stationary phase promoter. In some embodiments, the exogenous nuclease is expressed by an inducible promoter.

[0013] 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).

[0014] In another aspect, the present disclosure relates to a microorganism comprising a recombinant polynucleotide 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 polynucleotide.

[0015] In some embodiments, the epimerase is allulose-6-phosphate 3-epimerase (AlsE). In some embodiments, the epimerase is E. coli AlsE. In some embodiments, the epimerase comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the epimerase comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the epimerase consists of the amino acid sequence set forth in SEQ ID NO: 1.

[0016] In some embodiments, the phosphatase is hexitol phosphatase B (HxpB). In some embodiments, the phosphatase is E. 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.

[0017] In some embodiments, the microorganism further comprises a mutation in a gene encoding a pentose phosphate pathway enzyme. In some embodiments, the pentose phosphate pathway enzyme is glucose-6-phosphate 1-dehydrogenase (Zwf). In some embodiments, the microorganism further comprises a mutation in a gene encoding a glycolytic enzyme. 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).

[0018] In some embodiments, the microorganism further comprises a mutation in a gene encoding an enzyme in an allolytic pathway. In some embodiments, the enzyme in the allolytic pathway is allose-6-phosphate isomerase (RpiB). In some embodiments, the microorganism further comprises a mutation in a gene encoding an enzyme in a mannose biosynthetic pathway. In some embodiments, the enzyme in the mannose biosynthetic pathway is mannose-6-phosphate isomerase (ManA).

[0019] 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 by a stationary phase promoter. In some embodiments, the exogenous nuclease is expressed by an inducible promoter.

[0020] 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).

[0021] In one aspect, the present disclosure relates to a microorganism comprising a recombinant polynucleotide encoding an epimerase and a phosphatase; a mutation in a gene encoding an enzyme of the pentose phosphate pathway; a mutation in a gene encoding an enzyme of glycolysis; a mutation in a gene encoding an enzyme of the allolytic pathway; and a mutation in a gene encoding an enzyme of the mannose biosynthetic pathway; and, optionally, a recombinant polynucleotide encoding GalP, Glk, or both.

[0022] In a further aspect, the present disclosure relates to a microorganism comprising a recombinant polynucleotide encoding allulose-6-phosphate 3-epimerase (AlsE) and hexitol phosphatase B (HxpB); a mutation in glucose-6-phosphate 1-dehydrogenase (Zwf); a mutation in phosphofructokinase-1 (PfkA); a mutation in allose-6-phosphate isomerase (RpiB); a mutation in mannose-6-phosphate isomerase (ManA); and, optionally, a recombinant polynucleotide encoding GalP, Glk, or both.

[0023] Furthermore, in one aspect, the present disclosure relates to a microorganism comprising a recombinant polynucleotide encoding allulose-6-phosphate 3-epimerase (AlsE); a recombinant polynucleotide encoding hexitol phosphatase B (HxpB); a mutation in glucose-6-phosphate 1-dehydrogenase (Zwf); a mutation in phosphofructokinase-1 (PfkA); a mutation in allose-6-phosphate isomerase (RpiB); a mutation in mannose-6-phosphate isomerase (ManA); and, optionally, a recombinant polynucleotide encoding GalP, Glk, or both.

[0024] In some embodiments, the recombinant polynucleotide is stably integrated into the genome. In some embodiments, the microorganism comprises an increased content of intracellular fructose-6-phosphate compared to a naturally occurring microorganism. In some embodiments, the microorganism is Escherichia coli, Bacillus subtilis, or Lactococcus lactis.

[0025] In some embodiments, the mutation is a deletion. In some embodiments, the mutation reduces or eliminates the expression or activity of the enzyme.

[0026] In one aspect, the present disclosure also relates to a method for producing psicose, comprising culturing a microorganism disclosed herein under conditions suitable for converting a substrate to psicose. In some embodiments, the substrate comprises glucose. In some embodiments, the psicose 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%. In some embodiments, the purity value is 100%. In some embodiments, the purity value is expressed by the formula: Determined by TIFF2025530206000002.tif9128.

[0027] Furthermore, the present disclosure relates to psicose produced by a method comprising culturing a microorganism disclosed herein. In some embodiments, the psicose 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%. In some embodiments, the purity value is 100%. In some embodiments, the purity value is determined by the formula disclosed herein.

[0028] Furthermore, the present disclosure relates to a method for producing a food product comprising psicose, the method comprising culturing a microorganism disclosed herein under conditions suitable for converting a substrate to psicose; purifying the psicose; and blending the psicose with a food product to form a food product comprising psicose. In some embodiments, the food product is a chewing gum, a confectionery, a chocolate, or a savory good. In some embodiments, the food product is a beverage, yogurt, ice cream, a baked good, or a nutritional bar. [Brief explanation of the drawings]

[0029] [Figure 1] The psicose production ability of E. coli is exemplified below. AL3601 is MG1655 carrying the Z1 fragment (lacIq tetR specR) and the T7 RNA polymerase gene (PlacUV5:T7RNAP) (see Table 1). When grown at 30°C in M9P medium (M9 minimal medium containing 5 g / L of yeast extract) containing 10 g / L of glucose, AL3601 was not observed to produce psicose. However, deletion of pfkA resulted in the production of 0.15 g / L of psicose. [Figure 2]The following illustrates a pathway for the biosynthetic production of psicose. Glucose is transported and phosphorylated to glucose-6-phosphate (G6P) by the phosphotransferase system (PTS) or GalP / Glk. G6P is then isomerized to fructose-6-phosphate (F6P) by glucose-6-phosphate isomerase. F6P is epimerized to psicose-6-phosphate by D-allulose-6-phosphate 3-epimerase (AlsE), which is then dephosphorylated to free psicose by hexitol phosphatase B (HxpB). Finally, 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). [Figure 3] Comparison of psicose production using the phosphatases HxpB and YbiV is shown. E. coli encodes the phosphatases HxpB and YbiV. Two plasmids, pAL1946 and pAL1947 (Table 2), were constructed to overexpress alsE and either hxpB or ybiV, respectively, under the inducible promoter PT7. AL3601 carrying pAL1946 or pAL1947 was grown at 30°C in M9P medium containing 10 g / L glucose and induced with 1 mM IPTG. The culture containing pAL1946 produced 1.0 g / L of psicose after 24 hours, while the culture containing pAL1947 produced 0.4 g / L. Cultures induced with IPTG reached a lower density than their uninduced counterparts. [Figure 4]Psicose production in AL3601 and the triple knockout (TKO) strain is shown. The pfkA, zwf, and rpiB genes were deleted in AL3601 to generate AL3729 (Table 1). Plasmid pAL1946, containing PT7:alsE-hxpB, was introduced into AL3601 and AL3729. Cultures were grown at 30°C in M9P medium supplemented with 10 g / L glucose and induced with 25 mM IPTG. The uninduced TKO strain produced 1.5 g / L of psicose after 24 hours, while the induced TKO strain produced 0.6 g / L. [Figure 5] A comparison of psicose production between the PLlacO1 and PT7 promoters is shown. The alsE and hxpB genes were expressed under either PT7 or PLlacO1. pAL1946 (Table 2) containing PT7:alsE-hxpB was introduced into strain AL3601 (Table 1), and pAL2001 containing PLlacO1:alsE-hxpB was introduced into strain AL1050 (Table 2). Cultures were grown at 30°C in M9P medium supplemented with 10 g / L glucose and induced with 1 mM IPTG. The culture using PLlacO1 produced 0.5 g / L of psicose after 24 hours, whereas the culture using PT7 produced 0.5 g / L. [Figure 6] A comparison of the promoters PLlacO1 and PT7 in TKO strains is shown. pAL1946 (Table 2) containing PT7:alsE-hxpB was introduced into AL3729 (AL3601 + TKO, Table 1), and pAL2001 (Table 2) containing PLlacO1:alsE-hxpB was introduced into AL3756 (AL1050 + TKO, Table 1). Cultures were grown at 30°C in M9P medium supplemented with 10 g / L glucose and induced with 1 mM IPTG for 24 hours. The uninduced TKO strain using PT7 produced 1.8 g / L of psicose, while the induced strain produced 0.6 g / L. The uninduced TKO strain using PLlacO1 produced 0.6 g / L of psicose, while the induced strain produced 1.4 g / L. [Figure 7]GC / MS analysis to identify by-products is shown. Gas chromatography-mass (GC-MS) analysis was used to identify by-products in psicose production. Analysis identified the by-product as mannose. The image on the top left shows the GC elution peak of the green by-product compared to a brown mannose standard. Both the by-product and the mannose standard eluted at approximately 667.5-668.0 seconds after injection. The image on the right shows the mass spectrum of the by-product peak taken at 667.567 seconds (top) compared to the mass spectrum of mannose (bottom). [Figure 8] Identification and elimination of mannose by-products 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. A production plasmid containing PLlacO1:alsE-hxpB was introduced into the QKO and TKO strains. Cultures were grown at 30°C in M9P medium supplemented with 10 g / L glucose and induced with 1 mM IPTG. After 24 h, 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. [Figure 9-1] Psicose production in the TKO and QKO strains is shown. Psicose production in the AL3756 (ΔpfkA Δzwf ΔrpiB) and AL3990 (ΔpfkA Δzwf ΔrpiB ΔmanA) strains was compared (Table 1). Each strain was transformed with pAL2001 containing PLlacO1:alsE-hxpB. 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 induced QKO strain produced 3.5 g / L, a 34% yield. The TKO strain produced 2.3 g / L of psicose, a 25% yield. [Figure 9-2] See the description of Figure 9-1. [Figure 10-1]Psicose production from 15 g / L of glucose in AL3990 (Tables 1 and 2) containing pAL2001 is shown. AL3990 (ΔpfkA Δzwf ΔrpiB ΔmanA) (Table 1) was transformed with pAL2001 containing PLlacO1:alsE-hxpB. Cultures were grown at 30°C in M9P medium supplemented with 15 g / L of glucose and induced with 1 mM IPTG. After 24 hours, the induced strain produced 2.3 g / L, a 40% yield. [Figure 10-2] See the description of Figure 10-1. [Figure 11-1] Dynamic regulation of carbon flux at high density using CRISPRi was demonstrated. CRISPRi was used to knock down pfkB, which is responsible for converting F6P to fructose-1,6-bisphosphate in glycolysis in the QKO strain. A gRNA was designed to target the promoter region of pfkB. Cells were grown at 37°C in M9P medium supplemented with 10 g / L glucose until an OD600 of 1.0 was reached. 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 derivative strain containing a gRNA sequence targeting the pfkB promoter produced 1.7 g / L of psicose, with a yield of 46%. Figure 11 (left) shows the amount of psicose produced. Figure 11 (center) shows the glucose consumed. Figure 11 (right) shows the change in optical density. [Figure 11-2] See the description of Figure 11-1. [Figure 12-1]Figure 12 shows psicose production without inducer using the stationary-phase-active promoter PgadB. Plasmid pAL2001 (Table 2) containing PLlacO1:alsE-hxpB and plasmid pAL2247 (Table 2) containing PgadB:alsE-hxpB were separately introduced into the QKO strain AL3990 (Table 1). Cells were grown at 30°C in M9P medium supplemented with 30 g / L glucose and induced with 1 mM IPTG (for the PLlacO1 strain) for 24 hours. The induced culture containing pAL2001 produced 6.8 g / L of psicose, with a yield of 57%, while the culture containing PgadB:alsE-hxpB produced 8.8 g / L, with a yield of 63%. Figure 12 (right) shows the psicose produced. Figure 12 (center) shows the glucose consumed. Figure 12 (left) shows the change in optical density. [Figure 12-2] See the description of Figure 12-1. [Figure 13-1] The effect of the sugar symporter GalP on psicose production is shown. The galactose-proton symporter GalP can be used to supplement glucose import. Once glucose is transported across the plasma membrane by GalP, it can be phosphorylated by the glucokinase Glk and absorbed into central carbon metabolism. Plasmid pAL2274 containing PLtetO1:galP-glk was co-introduced into QKO strain AL3990 with either pAL2001 (PLlacO1:alsE-hxpB) or pAL2247 (PgadB:alsE-hxpB) (Tables 1 and 2). Cultures were grown at 30°C in M9P medium supplemented with 30 g / L glucose and induced with 1 mM IPTG (for PLlacO1 strains) for 24 h. PLtetO1 was not induced because full induction of galP-glk impairs cell growth. The highest titer and yield were achieved with the culture containing pAL2247 and pAL2274, which produced 10.7 g / L of psicose with a yield of 61%. [Figure 13-2] See the description of Figure 13-1. [Figure 14-1]Figures 14A-14C show strategies for the biosynthesis of D-psicose. Figure 14A: Shows the current industrial method for D-psicose production, which leads to limited yields (approximately 50%) due to a positive ΔG'°. Figure 14B: Shows a proposed D-psicose biosynthetic pathway. The dephosphorylation step thermodynamically drives and promotes production due to a large negative ΔG'm at a cellular reactant concentration of 1 mM. Figure 14C: Shows a proposed pathway for the biosynthetic production of D-psicose in E. coli. Deleted steps are in blue. Overexpressed steps are in red. PTS, phosphotransferase system; AlsE, D-allulose 6-phosphate 3-epimerase; HxpB, hexitol phosphatase B. [Figure 14-2] See the description of Figure 14-1. [Figure 15-1] Figures 15A-15D show the ability of E. coli to produce D-psicose. Cells were grown in M9P medium containing 10 g L of glucose at 37°C to an OD of approximately 0.4, followed by growth at 30°C for 24 hours. At an OD of approximately 0.4, 1 mM IPTG was added (Figures 15B-15D). Figure 15A shows D-psicose production in MG1655 and AL3601 (Table 5) with and without deletion of pfkA and / or alsE. Figure 15B shows that various sugar phosphatases containing AlsE were tested for D-psicose in AL3601. Figure 15C shows that the alsE and hxpB operons were expressed under PT7 and PLlacO1 in AL3601 and AL1050 (Table 5), respectively. ΔOD indicates the difference in OD at 0 and 24 hours. Figure 15D: Comparison of the effects of gene deletion on D-psicose production. Error bars indicate sd (n = 3 biological replicates). [Figure 15-2] See the description of Figure 15-1. [Figure 16-1]Figures 16A-16D show the enhanced ability to produce D-psicose in E. coli. Figure 16A shows cells grown in M9P medium containing various concentrations of glucose at 37°C to an OD of approximately 0.4, followed by growth at 30°C for 24 hours. At an OD of approximately 0.4, 1 mM IPTG was added for the PLlacO1 construct. The alsE and hxpB operons were expressed under PLlacO1 (pAL2001) and PgadB (pAL2247) in AL3756 and AL3990 (strains 1 and 2, Table 4), respectively. Figure 16B shows growth of strains 1 and 2 in M9P medium containing 40 g L of glucose at 37°C to an OD of approximately 0 (no culture at 37°C), approximately 0.4, or approximately 1, followed by growth at 30°C for 24 hours. When the temperature was shifted to 30°C, 1 mM IPTG was added for the PLlacO1 construct. Figure 16C shows that the galP and glk operons were expressed under PLlacO1 (pAL2264, Table 6). Strain 4 (AL3990 harboring pAL2264 and pAL2247, Table 4) was grown in M9P medium containing 40 g L of glucose to an OD of approximately 1, followed by growth at 30°C for 24 hours. At an OD of approximately 1, 1 mM IPTG was added to induce PLlacO1:galP-glk. The specific titer (g L OD) represents the titer per final OD. Figure 16D shows a comparison of the effects of gene deletion on D-psicose production. ptsG, ptsH, and / or pgm were deleted in strain 4. D-psicose production was performed as described in Figure 16C. Error bars indicate s.d. (n = 3 biological replicates). [Figure 16-2] See the description of Figure 16-1. [Figure 17]Figures 17A and 17B show the dynamic regulation of glycolysis by CRISPRi. Figure 17A: pfkB was knocked down using CRISPRi. An sgRNA targeting the pfkB promoter region or an sgRNA without a targeting sequence was expressed from a constitutive promoter. dcas9 was expressed from an aTc-inducible Ptet. Strains 5, 6, and 7 (Table 4) were grown in M9P medium containing 40 g L of glucose at 37°C to an OD of approximately 1, after which 1 mM IPTG and 100 ng / mL aTc were added, and the cells were grown at 30°C for 24 hours. Figure 17B: D-psicose production at high cell density is shown. Strain 7 was grown in M9P medium containing 40 g L of glucose at 37°C to an OD of approximately 1, after which it was induced with 1 mM IPTG and 100 ng mL of aTc and grown for an additional 30 minutes. Cultures were then spun down and resuspended in M9P medium containing 40 g L-1, 1 mM IPTG and 100 ng mL-1 aTC to an OD600 of approximately 8 and grown for 24 h at 30 °C. Error bars indicate sd (n = 3 biological replicates). [Figure 18A] Figures 18A-18C show GC-MS identification of D-mannose by-products. Figure 18A: GC elution peaks: Showing the by-product (green) and mannose standard (brown). Figures 18B and 18C: Mass spectra: Showing the by-product peak (Figure 18B) and mannose standard (Figure 18C). [Figure 18B] See the description of Figure 18A. [Figure 18C] See the description of Figure 18A. [Figure 19]The effect of manA knockout on D-mannose production is shown. To reduce mannose production, manA was knocked out in AL3756 (Table 5) to generate strain AL3990 (Table 5). The production plasmid pAL2001 (Table 6), containing PLlacO1:alsE-hxpB, 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 h, strain 1 produced 2.5 g L-1 of D-mannose, and strain 2 produced 0.7 g L-1. Error bars indicate s.d. (n = 3 biological replicates). [Figure 20A] Figures 20A-20C show the characterization of stationary-phase promoters. Fluorescence and OD600 of strains harboring sfGFP expressed under the PgadB, PcbpA2, Pdps, and PihfA4 promoters were monitored for timing and activity relative to PLlacO1 induced with 1 mM IPTG. Error bars indicate s.d. (n = 3 biological replicates). [Figure 20B] See the legend to Figure 20A. [Figure 20C] See the legend to Figure 20A. [Figure 21] Figures 21A and 21B show the inhibition of fluorescence by CRISPRi. Figure 21A: Three sgRNAs were designed to target PLlacO1:sfgfp. The figure discloses SEQ ID NO: 58. Figure 21B: The dcas9 gene was cloned under the aTc-inducible promoter Ptet to generate plasmid pAL1952 (Table 6). Constitutively expressed sgRNAs A (pAL2066), B (pAL2173), and C (pAL2174) or an sgRNA without a targeting sequence (pAL2063) were individually cloned into a plasmid containing PLlacO1:sfgfp (Table 6). AL1050 harboring the CRISPRi system was grown at 30°C, and fluorescence was measured at 0 and 4 hours after induction with 1 mM IPTG and 100 ng mL-1 aTc, with or without aTc. Error bars indicate s.d. (n = 3 biological replicates). [Figure 22] Figures 22A and 22B show growth inhibition by CRISPRi. The effects of two different CRISPRi systems on growth were tested in AL4186 (Table 5). In the first CRISPRi system, dcas9 and sgRNA are on medium-copy (p15A ori) and high-copy (ColE ori) plasmids, respectively. In the second CRISPRi system, both dcas9 and sgRNA are on the same plasmid (p15A ori). Figure 22A: sgRNAs designed to target the central pfkB promoter region, PpfkB1 and PpfkB2, are shown. The figure discloses SEQ ID NO: 59. Figure 22B: Cells were grown at 30°C, and OD600 was measured at 0 and 24 hours. ΔOD600 indicates the difference in OD600 between 0 and 24 hours. Error bars indicate sd (n = 3 biological replicates). [Figure 23A] Figures 23A-23C show glucose consumption and psicose production over time. D-glucose consumption and D-psicose production by strain 7 (Table 4) were monitored for 10 hours at 30°C in M9P medium containing 3 g L (Figure 23A), 5 g L (Figure 23B), and 10 g L (Figure 23C) of glucose. Error bars indicate s.d. (n = 3 biological replicates). [Figure 23B] See the description of Figure 23A. [Figure 23C] See the description of Figure 23A. [Figure 24]D-psicose production at high cell density in strain AL4186 is shown. Strain AL4186 (MG1655 ΔpfkA Δzwf ΔrpiB ΔmanA Δpgm) was transformed with plasmids pAL2247 (PgadB:alsE-hxpB), pAL2264 (PLlacO1:galP-glk), and pAL2188 (Ptet:dcas9 pTargetF-pfkB). Cultures were grown in M9P medium containing 40 g L of glucose at 37°C to an OD of approximately 1, after which they were induced with 1 mM IPTG and 100 ng mL of aTc and grown for an additional 30 minutes. The culture was then spun down and resuspended in M9P medium containing 40 g L of glucose, 1 mM IPTG, and 100 ng mL of aTC to an OD of approximately 10 and grown at 30°C for 8 hours. Samples were taken at 0, 4, and 8 hours. Over the 8-hour period, the culture produced an average of 15.3 g L of D-psicose, with a specific titer of 1.4 g L of OD, a yield of 43%, and a productivity of 1.9 g L hr. The D-psicose titers produced at 0-4 and 4-8 hours were similar, at 7.3 and 8.0 g L, respectively, but the yield at 4-8 hours (53%) was higher than the yield at 0-4 hours (35%). Error bars indicate standard deviation (s.d.) (n = 3 biological replicates). [Figure 25] HPLC chromatograms of high cell density experiments with strain 7 (see Table 4) grown for 24 hours are shown, with D-glucose eluting at approximately 3.36 minutes and D-psicose eluting at approximately 5.45 minutes. [Figure 26]Figures 26A and 26B show the biochemical characteristics of the phosphate. Figure 26A: The AlphaFold predicted structure of HxpB is shown, with P6P (199 Å) positioned in the active site pocket (429 Å). The volumes were calculated using MoloVol and CAVER, respectively. Figure 26B: ASP173 interacts with the magnesium ion, which in turn positions the phosphate on 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 dotted lines) and position P6P for hydrolysis. DETAILED DESCRIPTION OF THE INVENTION

[0030] Detailed Description The market for rare sugars as foods, nutritional supplements, and health supplements is expanding. Among rare sugars, D-psicose has attracted particular attention. However, current D-psicose production methods are costly, inefficient, and thermodynamically unfavorable, limiting their potential for widespread use. Importantly, the present disclosure addresses various obstacles to D-psicose production, including thermodynamic barriers, limited yields, the need for purified enzymes, and the addition of cofactors. Another key discovery disclosed herein is that Escherichia coli naturally possesses a thermodynamically favorable pathway for D-psicose production, enabling improved D-psicose production by increasing expression of native genes and eliminating competing pathways without the introduction of heterologous genes. The subject matter disclosed herein facilitates industrial-scale production of D-psicose without the need for expensive enzyme purification or difficult separation of raw materials and products.

[0031] The present disclosure is based, in part, on the discovery that microorganisms can be produced that contain specific genetic modifications (e.g., gene deletions) to produce low-calorie sugars. In certain embodiments, the low-calorie sugar is psicose. For clarity, and not by way of limitation, the detailed description of the subject matter disclosed herein is divided into the following subsections: 1. Definition; 2. Psicose-producing microorganisms; 3. Methods for producing and manufacturing microorganisms; 4. A method for producing psicose; and 5. Food.

[0032] 1. Definition The terms used herein generally have their ordinary meaning in the art, within the context of this invention and in the specific context that each term is used in. Certain terms are discussed below or elsewhere herein to provide additional guidance to the practitioner in describing the methods and compositions of the invention, and how to make and use them.

[0033] As used herein, the use of the words "a" or "an" in conjunction with the word "comprising" in the claims and / or specification can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0034] The term "about" or "approximately" refers to within an acceptable error range for a particular value as determined by one skilled in the art, which will depend in part on the method by which the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 standard deviations or more than 3 standard deviations, according to the practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within one order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value.

[0035] As used herein, the terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The present disclosure similarly contemplates other embodiments that "comprising," "consisting of," and "consisting essentially of" the embodiments or elements presented herein, whether or not explicitly stated.

[0036] As used herein, the term "microorganism" refers to any organism that exists as a minute cell contained within the domains of archaea, bacteria, or eukaryotes, the latter including yeast and filamentous fungi, protozoa, algae, or higher protists. In certain embodiments, the term includes prokaryotic or eukaryotic cells or organisms having a microscopic size, including, but not limited to, all species of bacteria, archaea, and eubacteria, as well as eukaryotic microorganisms such as yeast and fungi. In certain embodiments, the term microorganism includes cells that can be cultured for the production of chemicals (e.g., sugars). In certain embodiments, the microorganism is a prokaryotic microorganism. In certain embodiments, the prokaryotic microorganism is a bacterium.

[0037] As used herein, the terms "bacterium," "bacteria," or "eubacteria" refer to the domain of prokaryotic organisms. In certain embodiments, bacteria include Gram-negative bacteria, Gram-positive bacteria, Proteobacteria, Cyanobacteria, Spirochetes and related species, Planctomycetes, Bacteroides, Chlamydia, Green sulfur bacteria, Green non-sulfur bacteria, Radioresistant Micrococcus, and Thermotoga and Thermosipho thermophiles.

[0038] As used herein, the term "gram-negative bacteria" includes cocci, non-enteric bacilli, and enteric bacilli. Genera of gram-negative bacteria include, but are not limited to, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, and S. cerevisiae. These include Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium.

[0039] As used herein, the term "Gram-positive bacteria" includes cocci, non-spore-forming bacilli, and spore-forming bacilli. Genera of Gram-positive bacteria include, but are not limited to, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

[0040] As used herein, the term "recombinant microorganism" refers to a microorganism that contains one or more recombinant polynucleotides.

[0041] As used herein, the term "exogenous" refers to a molecule that is not naturally found in and / or not naturally produced by a given yeast, bacterium, organism, microorganism, or cell. As used herein, the term "endogenous" refers to a molecule that is naturally found in and / or naturally produced by a given yeast, bacterium, organism, microorganism, or cell.

[0042] As used herein, the terms "nucleic acid molecule," "nucleotide sequence," or "polynucleotide" refer to a single- or double-stranded covalently linked sequence of nucleotides in which the 3' and 5' ends of each nucleotide are joined by a phosphodiester bond. Nucleic acid molecules can contain deoxyribonucleotide or ribonucleotide bases and can be produced synthetically in vitro or isolated from natural sources.

[0043] As used herein, a "recombinant polynucleotide" refers to a polynucleotide in which the exact nucleotide sequence of the polynucleotide is foreign to a given host (i.e., not naturally found in a given host). In certain embodiments, a recombinant polynucleotide sequence is found naturally in a given host, but in an unnatural (e.g., more or less than expected) amount, or even when the sequence of the polynucleotide comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, but not by way of limitation, a recombinant polynucleotide can have two or more sequences from unrelated polynucleotides or from endogenous nucleotides arranged to create a new polynucleotide. In certain embodiments, the present disclosure provides for the introduction of a recombinant polynucleotide into a microorganism, wherein the polynucleotide encodes a polypeptide not normally found in the microorganism. With reference to the genome of the microorganism, the polynucleotide sequence encoding the polypeptide is recombinant or heterologous.

[0044] As used herein, "gene" refers to the DNA region (including exons and introns) that encodes a gene product, as well as all DNA regions that regulate the production of the gene product, regardless of whether such regulatory sequences are adjacent to the coding sequence and / or transcription sequence.In certain non-limiting embodiments, genes include promoter sequences, terminators, translation regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions.

[0045] The terms "polypeptide," "peptide," "amino acid sequence," and "protein," used interchangeably herein, refer to a molecule formed from 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 products, expression in a recombinant expression system, chemical synthesis, or enzymatic synthesis. These terms can apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as to natural and unnatural amino acid polymers.

[0046] As used herein, the term "amino acid" refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as amino acids that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, 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., carbon atoms bonded to hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs can 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 amino acids, but function 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, hydroxyl proline, arginine, cystine, glutamine, lysine, valine, ornithine, taurine, and combinations thereof.

[0047] As used herein, the term "isolated" refers to material that has been removed from at least one component with which it is naturally associated (eg, removed from its original environment).

[0048] As used herein, the terms "reduce" and "reduction" refer to a measurable decrease in an endpoint (e.g., enzyme 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 reduction can be from about 10% to about 100%.

[0049] As used herein, the terms "increase," "elevate," and "elevation" refer to a measurable increase in an endpoint (e.g., enzyme 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 can be 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.

[0050] Techniques for determining the identity of nucleic acid and amino acid sequences are known in the art. Typically, such techniques involve determining the nucleotide sequence of mRNA for a gene and / or determining the amino acid sequence encoded thereby, and comparing these sequences with a second nucleotide or amino acid sequence. Genomic sequences can also be determined and compared in this manner. Generally, identity refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their percent identity. The percent identity of two sequences, whether nucleic acid or amino acid, is calculated by dividing the number of exact matches between the two aligned sequences by the length of the shorter sequence and multiplying by 100. Unless otherwise specified, percent identity is determined for two sequences when compared and aligned for maximum correspondence over a comparison window or designated region measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters. See, for example, the NCBI website at ncbi.nlm.nih.gov / BLAST. For example, BLASTN and BLASTP can be used with the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expectation=10; matrix=BLOSUM62; description=50 sequences; sort=HIGH SCORE; database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+Swiss protein+Spupdate+PIR. Details of these programs can be found on the GenBank website.

[0051] A "mutation" of a gene can include, for example, a nucleotide change, a deletion of one or more nucleotides (which can include the entire coding sequence and / or a promoter or other regulatory sequence), and an insertion of one or more nucleotides, which can occur in the coding sequence of a gene or its regulatory components (e.g., the gene's promoter). 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.

[0052] As used herein, the term "yield" refers to the amount of product recovered from a process or chemical reaction.For example, but not limited to, yield refers to the amount of allulose recovered from one of the cultures of the microorganisms disclosed herein.In certain embodiments, yield is expressed as a fraction or percentage based on the raw materials used, or as the ratio of the final product to the starting material, not taking into account side reactions.As used herein, the term "yield coefficient" is a measure of the amount of product produced relative to the raw materials consumed.In certain embodiments, yield coefficient refers to the production of allulose relative to a substrate (for example, glucose).

[0053] 2. Psicose-producing microorganisms The present disclosure provides genetically engineered microorganisms. In certain embodiments, the microorganisms disclosed herein can produce increased amounts of psicose, for example, compared to a naturally occurring control microorganism.

[0054] 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 forms an epimer with fructose at the 3-position. Its enantiomer, L-psicose, is not known in nature but has been synthesized. D-psicose has the formula: I have TIFF2025530206000003.tif24128.

[0055] D-psicose is 70% as sweet as sucrose (e.g., table sugar) but has only about 10% of the nutritional energy (e.g., calorie) value. As a result, D-psicose can be used as a substitute for sucrose and artificial sweeteners.

[0056] The present disclosure provides genetically engineered microorganisms that exhibit increased psicose production compared to naturally occurring microorganisms. Figure 2 illustrates the biochemical pathways regulated in an exemplary microorganism of the present disclosure. In this exemplary microorganism, glucose is transported and phosphorylated to glucose-6-phosphate (G6P) by a phosphotransferase system (PTS) or GalP / Glk. G6P is then isomerized to fructose-6-phosphate (F6P) by glucose-6-phosphate isomerase. F6P is then epimerized to psicose-6-phosphate by D-allulose-6-phosphate 3-epimerase (AlsE), which is then dephosphorylated to free psicose by hexitol phosphatase B (HxpB). Finally, free psicose can diffuse across the cell membrane into the supernatant. In some embodiments, the microorganisms disclosed herein also contain 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); allose-6-phosphate isomerase (RpiB) catalyzed allolytic pathway; and the mannose biosynthesis pathway catalyzed by mannose-6-phosphate isomerase (ManA).

[0057] 2.1. Psicose-producing enzymes In certain embodiments, the microorganisms disclosed herein comprise overexpression of at least one gene encoding an enzyme that catalyzes a reaction for the production of psicose. In certain embodiments, the microorganisms disclosed herein comprise a recombinant polynucleotide encoding at least one enzyme that catalyzes a reaction for the production of 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 several asymmetric centers. Non-limiting examples of epimerases include D-allulose-6-phosphate 3-epimerase, methylmalonyl-CoA epimerase, UDP-galactose 4-epimerase, UDP-glucose 4-epimerase, UDP-glucuronate 4-epimerase, UDP-glucuronate 5'-epimerase, ribose-5-phosphate epimerase, GDP-mannose 3,5-epimerase, L-ribulose phosphate 4-epimerase, UDP-N-acetylglucuronide 4-epimerase, and UDP-N-acetylglucuronide 4-epimerase. These include UDP-cosamine 2-epimerase, UDP-N-acetylglucosamine 4-epimerase, UDP-galactose 4-epimerase, UDP-glucose 4-epimerase, UDP-glucuronate 4-epimerase, UDP-glucuronate 5'-epimerase, GDP-mannose 3,5-epimerase, methylmalonyl-CoA epimerase, ribose-5-phosphate epimerase, and UDP-N-acetylglucosamine 2-epimerase.

[0058] In certain embodiments, the epimerase is D-allulose-6-phosphate 3-epimerase (AlsE) (UniProt #P32719). AlsE catalyzes the reversible epimerization of D-allulose 6-phosphate to D-fructose 6-phosphate. In certain embodiments, the AlsE is E. coli AlsE. In certain embodiments, the 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, the AlsE comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the AlsE consists of the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO: 1 is provided below. TIFF2025530206000004.tif17156

[0059] 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 provided below. TIFF2025530206000005.tif47156

[0060] In certain embodiments, the enzyme is a phosphatase, for example, a phosphatase that dephosphorylates psicose-6-phosphate to 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 hexose.

[0061] In certain embodiments, the phosphatase is hexitol phosphatase B (HxpB) (UniProt #P77247 or UniProt #Q7ADF8). HxpB catalyzes the dephosphorylation of D-psicose 6-phosphate. In certain embodiments, HxpB is E. 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 provided below. TIFF2025530206000006.tif16156

[0062] 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 provided below. TIFF2025530206000007.tif17156

[0063] 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 provided below. TIFF2025530206000008.tif47156

[0064] In certain embodiments, the phosphatase is a sugar phosphatase, e.g., YbiV, G6425 (EcoCyc) P75792 (UniProt), which catalyzes the dephosphorylation of D-psicose 6-phosphate. In certain embodiments, the YbiV is E. coli YbiV. In certain embodiments, the 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 set forth in SEQ ID NO: 36. In certain embodiments, the YbiV comprises the amino acid sequence set forth in SEQ ID NO: 36. In certain embodiments, the YbiV consists of the amino acid sequence set forth in SEQ ID NO: 36. SEQ ID NO: 36 is provided below. TIFF2025530206000009.tif21156

[0065] 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 provided below. TIFF2025530206000010.tif56156

[0066] In certain embodiments, one or more transporters that transport glucose into the cell can be expressed (i.e., overexpressed) in the cell, thereby increasing intracellular glucose. In some embodiments, galactose:H + One or both of a symporter (GalP) and a glucokinase (Glk) are expressed in the microorganism. For example, in some embodiments, GalP transports glucose into the cell, where it is phosphorylated by Glk to glucose-6-phosphate and incorporated into central carbon metabolism.

[0067] In certain embodiments, galactose:H +The symporter (GalP) is as described in EG12148 (EcoCyc) or POAEP1 (UniProt). In certain embodiments, the GalP is E. coli GalP. In certain embodiments, the 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, the GalP comprises the amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, the GalP consists of the amino acid sequence set forth in SEQ ID NO: 38. SEQ ID NO: 38 is provided below. TIFF2025530206000011.tif34156

[0068] 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 provided below. TIFF2025530206000012.tif95156

[0069] In certain embodiments, the glucokinase (Glk) is as described in EG12957 (EcoCyc) or P0A6V8 (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 provided below. TIFF2025530206000013.tif21156

[0070] 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 provided below. TIFF2025530206000014.tif65156

[0071] Without being bound by any 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 disclosed herein.For example, but not limited to, the microorganisms disclosed herein can include any enzyme that catalyzes the reversible epimerization of D-fructose 6-phosphate to D-psicose 6-phosphate.In another non-limiting example, the microorganisms disclosed herein can include any enzyme that dephosphorylates psicose-6-phosphate to free psicose.

[0072] 2.2. Competing Paths In certain embodiments, the microorganisms disclosed herein comprise mutations in one or more genes encoding one or more enzymes that regulate biochemical pathways that can reduce psicose production. In certain embodiments, the microorganisms disclosed herein comprise reduced expression of genes encoding enzymes that regulate biochemical pathways that can reduce psicose production. Physiologically, cells catalyze sugars through the pentose phosphate pathway and glycolysis to generate energy (e.g., ATP). The inventors of the present disclosure have discovered that deletion or reduced expression of genes encoding enzymes in specific metabolic pathways results in increased psicose production.

[0073] In certain embodiments, the microorganisms disclosed herein comprise a mutation in a gene encoding a pentose phosphate pathway enzyme. In certain embodiments, the microorganisms disclosed herein comprise reduced expression of a gene encoding a pentose phosphate pathway enzyme. In certain embodiments, the pentose phosphate pathway enzyme is selected from the group consisting of glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonase, phosphogluconate dehydrogenase, phosphopentose isomerase, phosphopentose epimerase, transketolase, and transaldolase. In certain embodiments, the pentose phosphate pathway enzyme is glucose-6-phosphate dehydrogenase (Zwf) (Entrez Gene ID: 946370). Zwf catalyzes the oxidation of glucose 6-phosphate to 6-phosphogluconolactone. In certain embodiments, the Zwf is Escherichia coli Zwf. A 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 provided below. TIFF2025530206000015.tif100156

[0074] 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 provided below. TIFF2025530206000016.tif34156

[0075] In certain embodiments, the Zwf is a Bacillus subtilis Zwf. A representative amino acid sequence of a Bacillus subtilis Zwf is found as 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 provided below. TIFF2025530206000017.tif34156

[0076] 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 provided below. TIFF2025530206000018.tif100156

[0077] In certain embodiments, the Zwf is a Lactococcus lactis Zwf. A representative amino acid sequence of a Lactococcus lactis Zwf is found as LLA12_RS12225: glucose-6-phosphate dehydrogenase, EC 1.1.1.49, or as set forth in SEQ ID NO: 11, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 11. SEQ ID NO: 11 is provided below. TIFF2025530206000019.tif34156

[0078] A representative nucleotide sequence of the Lactococcus lactis zwf gene 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 provided below. TIFF2025530206000020.tif100156

[0079] In certain embodiments, the microorganisms disclosed herein comprise a mutation in a gene encoding a glycogen biosynthetic enzyme. In certain embodiments, the microorganisms disclosed herein comprise reduced expression of a gene encoding a glycogen biosynthetic enzyme. In certain embodiments, the glycogen biosynthetic enzyme is selected from the group consisting of phosphoglucomutase (Pgm), UDP-glucose pyrophosphorylase, glycogen synthase, glycogen branching enzyme, and glycogenin. In certain embodiments, the glycogen biosynthetic enzyme is phosphoglucomutase (Pgm) (Entrez Gene ID: 946370). Pgm (EC 5.4.2.2) is an enzyme that transfers a phosphate group on an α-D-glucose monomer from position 1 to position 6 in the forward direction or from position 6 to position 1 in the reverse direction. In certain embodiments, the Pgm is E. coli Pgm. A representative nucleotide sequence of the 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 provided below. TIFF2025530206000021.tif109156

[0080] 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 provided below. TIFF2025530206000022.tif38156

[0081] In particular embodiments, the 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 provided below. TIFF2025530206000023.tif104156

[0082] In certain embodiments, B. 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, B. subtilis Pgm comprises the amino acid sequence set forth in SEQ ID NO: 50. SEQ ID NO: 50 is provided below. TIFF2025530206000024.tif34156

[0083] In certain embodiments, the 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 provided below. TIFF2025530206000025.tif47156

[0084] In certain embodiments, 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, Lactococcus lactis Pgm comprises the amino acid sequence set forth in SEQ ID NO: 52. SEQ ID NO: 52 is provided below. TIFF2025530206000026.tif16156

[0085] In certain embodiments, the microorganisms disclosed herein comprise a mutation in a gene encoding a glycolytic enzyme. In certain embodiments, the microorganisms disclosed herein comprise reduced expression of a gene encoding a glycolytic enzyme. In certain embodiments, the glycolytic enzyme is selected from the group consisting of phosphofructokinase A, phosphofructokinase B, fructose-bisphosphate aldolase, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, and pyruvate kinase. In certain embodiments, the glycolytic enzyme is phosphofructokinase B (PfkB). In certain embodiments, the glycolytic enzyme is pyruvate kinase. In certain embodiments, the glycolytic enzyme is phosphofructokinase A (PfkA) (Entrez Gene ID: 948412). PfkA catalyzes the phosphorylation of D-fructose 6-phosphate to fructose 1,6-bisphosphate by ATP, the first committed step in glycolysis. In a particular embodiment, the PfkA is E. coli PfkA. A representative nucleotide sequence of the gene pfkA is shown in SEQ ID NO: 7. SEQ ID NO: 7 is provided below. TIFF2025530206000027.tif65156

[0086] 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 provided below. TIFF2025530206000028.tif25156

[0087] In certain embodiments, the PfkA is Bacillus subtilis PfkA. A representative amino acid sequence of Bacillus subtilis PfkA is found as 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 provided below. TIFF2025530206000029.tif21156

[0088] 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 provided below. TIFF2025530206000030.tif65156

[0089] In certain embodiments, the PfkA is a Lactococcus lactis PfkA. A representative amino acid sequence of a Lactococcus lactis PfkA is found as LLA12_RS07020: ATP-dependent 6-phosphofructokinase, EC 2.7.1.11, or as set forth in SEQ ID NO: 13, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 13, which is provided below. TIFF2025530206000031.tif25156

[0090] A representative nucleotide sequence of the Lactococcus lactis gene pfkA 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 provided below. TIFF2025530206000032.tif69156

[0091] In a particular embodiment, the 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 provided below. TIFF2025530206000033.tif65156

[0092] 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 provided below. TIFF2025530206000034.tif21156

[0093] In certain embodiments, the microorganisms disclosed herein do not comprise a deletion or reduced expression of hexokinase. In certain embodiments, the microorganisms disclosed herein do not comprise a deletion, disruption, or reduced expression of glucokinase. In certain embodiments, the microorganisms disclosed herein do not comprise a deletion or reduced expression of glucose-6-phosphate isomerase.

[0094] In certain embodiments, the microorganisms disclosed herein comprise a mutation in a gene encoding an enzyme of the allolytic pathway. In certain embodiments, the microorganisms disclosed herein comprise reduced expression of a gene encoding an enzyme of the allolytic pathway. In certain embodiments, the enzyme of the allolytic pathway is allose-6-phosphate isomerase (RpiB) (Entrez Gene ID: 948602). RpiB catalyzes the interconversion of ribulose-5-P and ribose-5-P, and the interconversion of D-allose-6-phosphate (All6P) and D-allulose-6-phosphate. In certain embodiments, the RpiB is E. coli RpiB. A representative nucleotide sequence of the gene rpiB is set forth 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 provided below. TIFF2025530206000035.tif30156

[0095] 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 set forth in SEQ ID NO: 24. In certain embodiments, the E. coli RpiB comprises the amino acid sequence set forth in SEQ ID NO: 24. SEQ ID NO: 24 is provided below. TIFF2025530206000036.tif12156

[0096] In particular embodiments, RpiB is the Bacillus subtilis gene rpiB. A representative amino acid sequence of RpiB is found as A0A6M4JQ63 (Uniprot) / BSU36920 (KEGG), or is 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 provided below. TIFF2025530206000037.tif12156

[0097] 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 provided below. TIFF2025530206000038.tif30156

[0098] In certain embodiments, the RpiB is Lactococcus lactis RpiB. A representative amino acid sequence of the gene rpiB is found as LLA12_RS12460: ribose-5-phosphate isomerase, EC 5.3.1.6, or is set forth in SEQ ID NO: 15, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 15, and is provided below. TIFF2025530206000039.tif17156

[0099] A representative nucleotide sequence of the Lactococcus lactis gene rpiB 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 provided below. TIFF2025530206000040.tif47156

[0100] In certain embodiments, the microorganisms disclosed herein comprise a mutation in a gene encoding an enzyme in the mannose biosynthetic pathway. In certain embodiments, the microorganisms disclosed herein comprise reduced expression of a gene encoding an enzyme in the mannose biosynthetic pathway. In certain embodiments, the enzyme in 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 several important mannosyl transfer reactions. ManA also catalyzes the interconversion of fructose-6-phosphate and mannose-6-phosphate. In certain embodiments, the ManA is E. 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 and is provided below. TIFF2025530206000041.tif78156

[0101] In certain embodiments, 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, E. coli ManA comprises the amino acid sequence set forth in SEQ ID NO: 25. SEQ ID NO: 25 is provided below. TIFF2025530206000042.tif30156

[0102] In certain embodiments, the ManA is Bacillus subtilis ManA. A representative amino acid sequence of ManA is found in O31646 (Uniprot) / BSU12020 (KEGG), or is set forth in SEQ ID NO: 16, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 16. TIFF2025530206000043.tif21156

[0103] 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. TIFF2025530206000044.tif65156

[0104] In certain embodiments, one or both of the following are also deleted in B. subtilis, optionally in combination with the deletion of B. subtilis ManA:

[0105] (i) YvyI of Bacillus subtilis. A representative amino acid sequence of YvyI is found in P39841 (Uniprot) / BSU35790 (KEGG), or is 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 provided below. TIFF2025530206000045.tif21156

[0106] 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 provided below. TIFF2025530206000046.tif65156

[0107] (ii) GmuF of Bacillus subtilis. A representative amino acid sequence of GmuF is found in O05511 (Uniprot) / BSU05870 (KEGG), or is 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 provided below. TIFF2025530206000047.tif21156

[0108] 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 provided below. TIFF2025530206000048.tif65156

[0109] In certain embodiments, the ManA is Lactococcus lactis ManA. A representative amino acid sequence of Lactococcus lactis ManA is found as LLA12_RS03920: mannose-6-phosphate isomerase, EC 5.3.1.8, or is set forth in SEQ ID NO: 19, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 19. SEQ ID NO: 19 is provided below. TIFF2025530206000049.tif21156

[0110] 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 provided below. TIFF2025530206000050.tif65156

[0111] In certain embodiments, the gene deletion comprises a non-frameshift deletion, a frameshift deletion, or a combination thereof. In certain embodiments, the gene deletion can be achieved by an insertion (e.g., a non-frameshift insertion, a frameshift insertion, or a combination thereof). In certain embodiments, the gene deletion comprises a nonsense mutation.

[0112] 2.3. Cells The present disclosure provides recombinant microorganisms. Any culturable microorganism is suitable for use in the compositions and methods described herein. In certain embodiments, the microorganism is a bacterium. In particular embodiments, the microorganism is selected from the group consisting of Aceiobacter aceti, Achromobacter, Acidiphilium, Acinetobacter, Actinomadura, Actinoplanes, Aeropyrumpernix, Agrobacterium, Alcaligenes, Ananas comosus (M), Arthrobacter, Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, and the like. circulars, Bacillus clausii, Bacillus lentus, Bacillus lichenifirmis, Bacillus macerans, Bacillus stearothermophilus, Bacillus subtilis, Bifidobacterium, Brevibacillus brevis, Burkholderia cepacia, Candida cylindracea, Carica papaya (L) papaya (L), Cellulosimicrobium, Cephalosporium, Chaetomium erraticum, Chaetomium gracile.), Clostridium, Clostridium butyricum, Clostridium acetobutylicum, Clostridium thermocellum, Corynebacterium (glutamicum), Corynebacterium efficiens, Escherichia coli, Enterococcus, Erwina chrysanthemi, Gliconobacter, Gluconacetobacter, Haloarcula, Humicola insolens, Kitasatospora setae setae.), Klebsiella, Klebsiella oxytoca, Kocuria, Lactlactis, Lactobacillus, Lactobacillus fermentum, Lactobacillus sake, Lactococcus, Lactococcus lactis, Leuconostoc, Methylocystis, Methanolobus siciliae, Methanogenium organophilum.), Methanobacterium bryantii, Microbacterium imperiale, Micrococcus lysodeikticus, Microlunatus, Mucorjavanicus, Mycobacterium, Myrothecium, Nitrobacter, Nitrosomonas, Nocardia, Papaya carica, Pediococcus, Pediococcus halophilus, Paracoccus pantotrophus pantotrophus, Propionibacterium, Pseudomonas, Pseudomonas fluorescens, Pseudomonas denitrificans, Pyrococcus, Pyrococcus furiosus, Pyrococcus horikoshii, Rhizobium, Rhizomucor miehei, Rhizomucor pusillus Lindt, Rhizopus, Rhizopus delemar, Rhizopus japonicas, Rhizopus niveus niveus.), Rhizopus oryzae, Rhizopus oligosporus.), Rhodococcus, Sckroiina libertina, Sphingobacterium multivorum, Sphingobium, Sphingomonas, Streptococcus, Streptococcus thermophilus Y-1, Streptomyces, Streptomyces griseus, Streptomyces lividans, Streptomyces murinus, Streptomyces ruhiginosus, Streptomyces violaceolvera The microorganism is selected from the group consisting of Bacillus subtilis, Bacillus violaceoruber, Streptoverticillium mobaraense, Tetragenococcus, Thermus, Thiosphaera pantotropha, Trametes, Vibrio alginolyticus, Xanthomonas, Zymomonas, and Zymomonas mobilis. In certain embodiments, the microorganism is Escherichia coli. In certain embodiments, the microorganism is Bacillus subtilis. In certain embodiments, the microorganism is Lactococcus lactis.

[0113] 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), verotoxigenic 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.

[0114] In certain embodiments, the E. coli is NCTC 12757, NCTC 12779, NCTC 12790, NCTC 12796, NCTC 12811, ATCC 11229, ATCC 25922, ATCC 8739, DSM 30083, BC 5849, BC 8265, BC 8267, BC 8268, BC 8270, BC 8271, 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, BC 8327, BC 8331, BC 8335, BC 8338, BC 8341, BC 8344, BC 8345, BC 8346, BC 8347, BC 8348, BC 8863 and BC 8864.

[0115] In certain embodiments, Escherichia coli includes BC 4734 (O26:H11), BC 4735 (O157:H-), BC 4736, BC 4737 (n.d.), 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), BC 5580 (O157:H7), BC 5582 (O3:H), BC 5643 (O2:H5), BC 5644 (O128), BC 5645 (O55:H-), BC 5646 (O69:H-), 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 prototype:H-), BC 7834 (ONT:H-), BC 7835 (O103:H2), BC 7836 (O57:H-), BC 7837 (ONT:H-), BC 7838, BC 7839 (O128:H2), BC 7840 (O157:H-), BC 7841 (O23:H-), BC 7842 (O157:H-), BC 7843, BC 7844 (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 7855 (O157:H7), BC 7856 (O26:H-), BC 7857, BC 7858, BC 7859 (ONT:H-), BC 7860 (O129:H-), BC 7861, BC 7862 (O103:H2), BC 7863, BC 7864 (O prototype:H-), BC 7865, BC 7866 (O26:H-), BC 7867 (O prototype:H-), BC 7868, BC7869(ONT:H-), BC 7870(O113:H-), BC 7871(ONT:H-), BC 7872(ONT:H-), BC 7873, BC 7874(O prototype:H-), BC 7875(O157:H-), BC 7876(O111:H-), BC 7877(O146:H21), BC 7878(O145:H-), BC 7879(O22:H8), BC 7880(O prototype:H-), BC 7881(O145:H-), BC 8275(O157:H7), BC 8318(O55:K-:H-), BC 8325(O157:H7), BC 8332(ONT) and BC 8333.

[0116] In certain embodiments, the E. coli is selected from the group consisting of BC 8246 (O152:K-:H-), BC 8247 (O124:K(72):H3), BC 8248 (O124), BC 8249 (O112), 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), BC 8260 (O128a.c.:H35), BC 8261 (O164), BC 8262 (O164:K-:H-), BC 8263 (O164) and BC 8264 (O124).

[0117] In certain embodiments, the E. coli is selected from the group consisting of BC 5581 (O78:H11), BC 5583 (O2:K1), BC 8221 (O118), BC 8222 (O148:H-), BC 8223 (O111), BC 8224 (O110:H-), BC 8225 (O148), BC 8226 (O118), BC 8227 (O25:H42), BC 8229 (O6), BC 8231 (O153:H45), BC 8232 (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.

[0118] The specifications for this product are BC 7567(O86), BC 7568(O128), BC 7571(O114), BC 7572(O119), BC 7573(O125), BC 7574(O124), BC 7576(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) BC 8569(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) BC 8586(O158) BC 8588(O26) BC 8589(O86) BC 8590(O127) BC 8591(O128) 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), BC 8605(O158) 8606(O158), BC 8607(O158), BC 8608(O128), BC 8609(O55), BC 8610(O114), BC 8615(O158), BC 8616(O128), BC 8617(O26), BC8618 (O86), BC 8619, BC 8620, BC 8621, BC 8622, BC 8623, BC 8624 (O158) and BC 8625 (O158).

[0119] In a particular embodiment, the Bacillus subtilis is from strain 168.

[0120] In a particular embodiment, the Lactococcus lactis is derived from strain A12.

[0121] 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, Aspergillus oryzae, Aspergillus melleus, Aspergillus pulverulentus, Aspergillus saitoi, Aspergillus sojea, Aspergillus terreus, Aspergillus pseudoterreus, Aspergillus usamii, and Candida rugosa. rugosa, Issatchenkia orientalis, Kluyveromyces, Kluyveromyces fragilis, Kluyveromyces lactis.), Kluyveromyces marxianas, Penicillium, Penicillium camemberti, Penicillium citrinum, Penicillium emersonii, Penicillium roqueforti, Penicillium lilactinum, Penicillium multicolor, Rhodosporidium toruloides, Saccharomyces cerevisiae, Schizosaccharomyces pombe pombe, Trichoderma, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride, Trichosporon penicillaium, Yarrowia lipolytica, and Zygosaccharomyces rouxii.

[0122] In certain embodiments, the microorganism is a yeast cell. In certain embodiments, the yeast cell is Saccharomyces cerevisiae.

[0123] 2.4. Exemplary Microorganisms 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 and an exogenous phosphatase. In certain embodiments, the exogenous epimerase is allulose-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.

[0124] 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, and one, two, three, or four (4) deleted genes. In certain embodiments, the exogenous epimerase is allulose-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, allose-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.

[0125] In certain embodiments, the present disclosure provides a microorganism comprising a recombinant polynucleotide, the microorganism comprising 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 allulose-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.

[0126] In certain embodiments, the present disclosure provides a microorganism comprising a recombinant polynucleotide, the microorganism comprising 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 allulose-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 allose-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.

[0127] 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 a deletion of four (4) genes. In certain embodiments, the exogenous epimerase is allulose-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.

[0128] In certain embodiments, the present disclosure provides a microorganism comprising a recombinant polynucleotide, the microorganism comprising 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 allulose-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.

[0129] 3. Methods for Producing and Making Microbial Psicose The present disclosure also provides methods for preparing and / or producing any of the microorganisms disclosed herein.To introduce one or more recombinant polynucleotides of the present disclosure into microorganisms, many recombinant techniques generally known in the art can be used, including but not limited to protoplast fusion, transfection, transformation, conjugation and transduction.These techniques include conventional molecular biology techniques (e.g., recombinant techniques), microbiology, cell biology and biochemistry, which are within the skill of the art. Further information on these techniques can be found in Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (Gait, ed., 1984); Animal Cell Culture (Freshney, ed., 1987); Gene Transfer Vectors for Mammalian Cells (Miller & 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).

[0130] 3.1. Recombinant Polynucleotides In certain embodiments, the recombinant polynucleotides disclosed herein can be stably integrated into a microbial chromosome. In certain embodiments, the recombinant polynucleotides disclosed herein are stably integrated into a microbial chromosome using homologous recombination, translocation-based chromosomal integration, recombinase-mediated cassette exchange (RMCE; e.g., using the Cre-lox system), or an integration plasmid (e.g., a yeast integration plasmid). A variety of integration techniques suitable for a range of microorganisms are known in the art (e.g., Griffiths, AJF, Miller, JH, Suzuki, DT et al. An Introduction to Genetic Analysis. 7 th ed. New York: W. H. Freeman; 2000). In certain embodiments, the recombinant polynucleotides disclosed herein are maintained on an extrachromosomal plasmid (e.g., an expression plasmid or vector) in the recombinant microorganism of the present disclosure. A variety of extrachromosomal plasmids suitable for a range of microorganisms are known in the art, including, but not limited to, replicating plasmids (e.g., yeast replicating plasmids containing an autonomously replicating sequence ARS), centromeric plasmids (e.g., yeast centromere plasmids containing an autonomously replicating sequence, CEN), episomal plasmids (e.g., 2-pm plasmids), and / or artificial chromosomes (e.g., yeast artificial chromosomes YAC, or bacterial artificial chromosomes BAC).

[0131] Vectors 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 to introduce nucleic acids into one or more microorganisms. Vectors can include, but are not limited to, cloning vectors, expression vectors, shuttle vectors, plasmids, and cassettes. As used herein, the term "plasmid" refers to a circular double-stranded DNA construct used as a cloning vector and / or expression vector. In certain embodiments, a plasmid can be an extrachromosomal self-replicating genetic element (e.g., an episomal plasmid) when introduced into a microorganism. In certain embodiments, a plasmid can be integrated into a microorganism chromosome. In certain embodiments, a vector can direct the expression of an operably linked coding region, e.g., an "expression vector." These expression vectors enable the expression of exogenous polynucleotides and / or polypeptides in a microorganism. In certain embodiments, a vector enables the integration of one or more polynucleotides into the genome of a microorganism.

[0132] In certain embodiments, the vectors disclosed herein comprise a promoter. In certain embodiments, the vectors are bacterial or prokaryotic expression vectors. In certain embodiments, the vectors are yeast or fungal expression vectors.

[0133] In certain embodiments, the vectors disclosed herein comprise nucleotide sequences in a single operon.

[0134] Promoter In certain non-limiting embodiments, the recombinant polynucleotides disclosed herein include a regulatory sequence, an enhancer, or a 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, an enhancer, or a promoter.

[0135] 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. Biologically, a promoter is not transcribed, but rather regulates the assembly of components that initiate transcription of other nucleotide sequences. Furthermore, a promoter can restrict this assembly and subsequent transcription to specific conditions. For example, but not limited to, a promoter can enable transcription in response to one or more environmental, temporal, or developmental stimuli. Bacterial and fungal cells possess numerous proteins that sense external or internal conditions and initiate signal transduction cascades that culminate in the binding of the protein to a specific promoter and the subsequent initiation of transcription of a nucleic acid under the control of the promoter. In certain embodiments, the promoter is endogenous. In certain embodiments, the promoter is exogenous. In certain embodiments, the promoter is artificially designed for expression in a specific species.

[0136] 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 without interruption in response to internal or external stimuli. Constitutive promoters are commonly used in recombinant engineering to ensure the continuous expression of a desired nucleotide sequence. Constitutive promoters provide a robust amount of nucleic acid expression and are therefore used in many recombinant engineering 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 rrnB-derived P spc , P bla , P RNAI , P RNAII , P1 and P2, and the lambda phage promoter P L(Liang, ST et al. JMoi. Biol. 292(1):19-37 (1999)). In some embodiments, the promoter is active in the stationary phase of the microorganism. Exemplary stationary phase promoters can be found, for example, in Shimada, et al., JOURNAL OF BACTERIOLOGY, Nov. 2004, pp. 7112-7122; Pletnev at el., ACTA NATURAE | VOL. 7 No. 4 (27) 2015.

[0137] In certain 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 continuous expression upon exposure to a particular stimulus (e.g., IPTG). In certain embodiments, an inducible promoter drives graded expression levels that correlate with the amount of stimulus. Non-limiting examples of stimuli for inducible promoters include heat shock, exogenous compounds or lack thereof (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 Examples include:

[0138] In certain embodiments, the recombinant polynucleotide can comprise multiple promoters. In certain embodiments, the multiple promoters can be the same. For example, but not limited to, the recombinant polynucleotide can comprise 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 can be different. For example, but not limited to, the recombinant polynucleotide can comprise a nucleotide sequence encoding an alsE 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 can be P LlacO1 In certain 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 the promoter P of phage lambda, in which the CI binding site has been replaced by lacO1. L The hybrid design allows for strong promotion that can be repressed by the Lac inhibitor (i.e., repressor), LacI, or inducible by IPTG.

[0139] 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.

[0140] In certain embodiments, the promoter is PT7 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.

[0141] 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.

[0142] 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. TIFF2025530206000051.tif111156

[0143] 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 E. coli culture includes five distinct phases: lag phase, logarithmic growth phase, stationary phase, death phase, and long-term stationary phase. The lag phase occurs when cells are inoculated into the medium and adjust their metabolic processes to the new environment. Cells then rapidly grow and divide, entering the logarithmic growth phase. At this time, enzymes related to central carbon metabolism become most important, and the transcription of the corresponding genes is upregulated. When cells sense environmental stressors, such as a lack of medium nutrients, their growth and division slows, and the culture enters the stationary phase. The use of a stationary phase promoter prevents productive pathways from competing with central carbon metabolism for carbon flux during the logarithmic growth phase, a period when cells require carbon for rigorous growth and division.

[0144] In certain embodiments, the stationary phase promoter is P gadB In certain embodiments, P gadB The promoter comprises the nucleotide sequence shown in SEQ ID NO: 46 or SEQ ID NO: 53. In certain embodiments, P gadB The promoter consists of the nucleotide sequence shown in SEQ ID NO:53.

[0145] 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.

[0146] 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 ihfA4The promoter consists of the nucleotide sequence shown in SEQ ID NO:55.

[0147] 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. TIFF2025530206000052.tif111156

[0148] 3.1.3. Genetic markers In certain embodiments, the recombinant polynucleotides disclosed herein comprise genetic markers. These genetic markers allow for the selection of microorganisms carrying one or more desired polynucleotides (e.g., recombinant polynucleotides). In certain embodiments, the genetic marker is an antibiotic resistance marker selected from the group consisting of 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, zeocin resistance, and streptomycin resistance. In certain embodiments, the genetic marker comprises a coding sequence for an antibiotic resistance protein (e.g., β-lactamase for certain ampicillin resistance markers) and a promoter or enhancer element that drives expression of the coding sequence in the microorganisms of the present disclosure. In certain embodiments, the microorganisms of the present disclosure are grown under conditions in which the antibiotic resistance marker is expressed and confers resistance to the microorganism, thereby selecting microorganisms that have successfully integrated the marker. In certain embodiments, the genetic marker is an auxotrophic marker. In certain embodiments, the auxotrophic marker is a gene involved in vitamin, amino acid, fatty acid synthesis, or carbohydrate metabolism. In certain embodiments, the auxotrophic marker is a gene for synthesizing an amino acid. In certain embodiments, the auxotrophic marker is a gene 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 certain embodiments, the auxotrophic marker is a gene for synthesizing adenosine, biotin, thiamine, leucine, glucose, lactose, or maltose. In certain embodiments, the microorganisms of the present disclosure are grown under conditions in which the auxotrophic resistance marker is expressed in an environment or medium lacking the corresponding nutrient and allows the microorganism (lacking the endogenous ability to produce the nutrient) to grow, thereby selecting microorganisms that have successfully integrated the marker.

[0149] 3.2. Gene deletion and reduction of expression In certain embodiments, the present disclosure also provides a method for introducing the deletion of any gene or enzyme disclosed herein.These deletions can be made by any suitable gene editing method.In certain embodiments, deletions are made by the method comprising homologous recombination, zinc finger nuclease, meganuclease, transcription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeats (CRISPR) system, or a combination thereof.

[0150] In certain embodiments, the deletion is caused by the CRISPR system. The clustered regularly interspaced short palindromic repeats (CRISPR) system is a genome editing tool discovered in prokaryotic cells. When used for genome editing, this system includes Cas9 (a protein that can modify DNA using crRNA as a guide), CRISPR RNA (crRNA, which contains an RNA used to guide Cas9 to the correct part of host DNA and a region that binds to tracrRNA (generally in the form of a hairpin loop) and forms an active complex with Cas9), transactivating crRNA (tracrRNA, which binds to crRNA and forms an active complex with Cas9), and any part of a DNA repair template (DNA that guides the cellular repair process, allowing for the insertion of a specific DNA sequence). Multiple crRNAs and tracrRNAs can be packaged together to form a single guide RNA (sgRNA). This sgRNA can be combined with a Cas9 gene and made into a plasmid to be transfected into cells. In certain embodiments, the CRISPR system includes a base editor. In certain embodiments, the CRISPR system includes a transposase / recombinase. In certain embodiments, the CRISPR system comprises a prime editor. In certain embodiments, the CRISPR system comprises an epigenetic regulator. In certain embodiments, the CRISPR system comprises a CRISPRoff system. Further details regarding the CRISPR systems of the present disclosure can be found in Anzalone et al., Nature biotechnology 38.7 (2020): 824-844 and Nunez 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 by reference in their entirety.

[0151] In certain embodiments, the deletion is created using zinc finger nucleases. Zinc finger nucleases (ZFNs) are artificial restriction enzymes created by combining a zinc finger DNA binding domain and a DNA cleavage domain. The zinc finger domain can be engineered to target specific DNA sequences, allowing the zinc finger nuclease to target desired sequences within the genome. The DNA binding domain of an individual ZFN typically contains multiple individual zinc finger repeats, each capable of recognizing multiple base pairs. The most common method for creating new zinc finger domains is to combine small zinc finger "modules" with known specificities. The most common cleavage domain in ZFNs is the non-specific cleavage domain of the type II restriction endonuclease FokI.

[0152] In certain embodiments, deletions are created using the TALEN system. Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific sequences in DNA. The TALEN system operates on roughly the same principle as ZFNs. TALENs are created by combining a transcription activator-like effector DNA-binding domain with a DNA-cleavage domain. Transcription activator-like effectors (TALEs) consist of a repeating motif of 33-34 amino acids with two variable positions that have strong recognition for specific nucleotides. By assembling an array of these TALEs, the TALE DNA-binding domain can be designed to bind to a desired DNA sequence, thereby guiding the nuclease to cleave at a specific location in the genome.

[0153] In certain embodiments, the deletion is made by meganuclease. Meganuclease is an endodeoxyribonuclease that recognizes approximately 12 to approximately 40 base pairs of double-stranded DNA that occurs only once in the genome. Meganuclease is one of the most specific natural restriction enzymes. Meganuclease can replace, remove or modify sequences in a highly targeted manner, so it is also defined as molecular DNA scissors. Protein engineering allows the modification of its recognition sequence and target sequence.

[0154] In certain embodiments, the present disclosure also provides methods for reducing the expression of any of the genes or enzymes disclosed herein. In certain embodiments, reducing the expression of the genes and enzymes disclosed herein involves using oligonucleotides having sequences complementary to the mRNA of the genes disclosed herein (e.g., zwf, manA, rpiB, pfkA, pfkB, etc.). 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%, or at least about 99% identical to at least a portion of the zwf mRNA sequence. 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%, or at least about 99% identical to at least a portion of the zwf mRNA sequence. 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%, or at least about 99% identical to at least a portion of the pfkA mRNA sequence. 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%, or at least about 99% identical to at least a portion of the pfkB mRNA sequence. 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%, or at least about 99% identical to at least a portion of the pfkB mRNA sequence.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%, or at least about 99% identical to at least a portion of the rpiB mRNA sequence. 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%, or at least about 99% identical to at least a portion of the manA mRNA sequence. In certain embodiments, these oligonucleotides can be identical to at least a portion of the manA mRNA sequence. In certain embodiments, the antisense nucleic acid, shRNA, miRNA, or siRNA molecule can comprise DNA or atypical or non-natural residues, such as, but not limited to, phosphorothioate residues.

[0155] In some embodiments, reducing the expression of the genes and enzymes disclosed herein can involve the use of CRISPR, which can mutate coding sequences or promoters to reduce or eliminate expression of gene products, or CRISPRi can be targeted to the genes disclosed herein, thereby reducing expression of one or more of the genes. See, for example, Arroya-Olarte, et al., Microorganisms, 2021 Apr; 9(4): 844. Zhang et al., Front. Microbiol. (31 March 2021).

[0156] In certain embodiments, the reduction of gene and enzyme expression disclosed herein comprises using a CRISPRi system. The CRISPRi system can silence genes at the transcriptional level and exert fewer sequence-specific off-target effects than RNAi. In certain embodiments, the CRISPRi system comprises catalytically dead Cas9 (dCas9). dCas9 is a programmable transcription factor that can be targeted to a promoter through sgRNA and function as a repressor therein. 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 provided below. TIFF2025530206000053.tif91156

[0157] 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 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 a gene encoding an enzyme in a competitive pathway. For example, but not limited to, the sgRNA can target the zwf gene, pgm gene, pfkA gene, pfkB gene, ManA gene, or 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 operator, or a protein-coding sequence.

[0158] 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.

[0159] 3.3. Transformation and gene editing In certain embodiments, the present disclosure provides the use of the plasmids and vectors disclosed herein for transformation.The vectors and plasmids disclosed herein can be transformed into cells through any system known in the art.For example, but not limited to, the microorganisms disclosed herein can be transformed by particle bombardment, chemical transformation, Agrobacterium transformation, nanospike transformation, electroporation and viral transformation.

[0160] In certain embodiments, the vectors of the present disclosure can be introduced into microorganisms using various techniques, including transformation, transfection, transduction, viral infection, gene guns, 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, transformed microorganisms are referred to as recombinant microorganisms.

[0161] 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 protein and nucleotide sequences, or DNA transformation followed by intracellular expression of RNA and proteins (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 & 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 & Doudna, JA "Enhanced homology-directed human genome engineering by controlled timing of CRISPR / Cas9 delivery." Elife 3, e04766 (2014)).

[0162] 3.4. Recombinant Systems 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 may be native to the host cell or may be introduced into a cellular host. For example, but not limited to, the genes of the homologous recombination system may be introduced into a plasmid, introduced on a linear DNA fragment, introduced as an RNA or set of RNAs and translated, or introduced as a protein or set of proteins. In certain embodiments, the method includes a recombinant polynucleotide disclosed herein. In certain embodiments, the polynucleotide includes sequences (e.g., left and right homology arms) homologous to regions in a nucleic acid (e.g., a genome, a plasmid, etc.) such that the left and right homology arms are separated by a designed gene edit (e.g., a promoter, an insertion, a substitution, a SNP, a terminator, a degron, a sequence for a tag, a degradation signal, or a sequence for deletion). In certain embodiments, the recombinant polynucleotide includes a genetic marker, a counterselectable genetic marker (e.g., SacB or PheS), and an origin of replication (e.g., R6K).

[0163] In certain embodiments, a recombinant polynucleotide comprising homology arms and sequences for gene editing is introduced into a microorganism using any of the methods disclosed herein (e.g., transformation by electroporation, conjugation, etc.). In certain embodiments, after transformation, the resulting transformants can be plated on a medium to select transformants that express a selectable genetic marker. Recombination between the plasmid comprising the homology arms and the target locus in the nucleic acid (e.g., genome, plasmid, etc.) can occur at one of two homology sites that are targeted by the homology arms present on the plasmid and flank the designed gene edit. In certain embodiments, the resulting transformants can grow as colonies on a selective medium and be selected and plated on a second type of selective medium (e.g., a counterselective medium). In certain embodiments, the second type of selective medium allows for the selection of cells containing the desired gene edit.

[0164] In certain embodiments, the methods disclosed herein include using proteins from one or more recombination systems. The recombination system may be endogenous or exogenous to the microorganism. In certain embodiments, the proteins from one or more recombination systems can be introduced as nucleic acids (e.g., plasmids, linear DNA or RNA, or integrons) and integrated into the genome of the host cell, or stably expressed from extrachromosomal elements. In certain embodiments, the proteins from one or more recombination systems can be introduced as RNA and translated by the host cell. In certain embodiments, the proteins from one or more recombination systems can be introduced into the host cell as proteins. Non-limiting examples of recombination systems include the lambda Red recombination system, the RecET recombination system, the Red / ET recombination system, any homologs, orthologs, or paralogs of proteins from the lambda Red recombination system, the RecET recombination system, the Red / ET recombination system, the lambda Red-mediated recombination system, or any combination thereof.For details of the RecET recombination system, see 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, which are incorporated herein by reference in their entireties.

[0165] 4. Method for Producing Psicose The present disclosure also provides methods for producing psicose. Cell-free methods (e.g., in vitro synthesis) are thermodynamically unfavorable, with a predicted ΔG° of +5 kJ / mol. In certain embodiments, the methods for producing psicose disclosed herein include culturing a microorganism (e.g., one disclosed in Section 2) and purifying psicose.

[0166] 4.1. Cell culture The present disclosure provides a method for culturing the microorganisms disclosed herein. As used herein, "culturing" a cell refers to introducing an appropriate medium under appropriate conditions to promote cell growth. In certain embodiments, the culture is carried out using a liquid or solid growth medium. In certain embodiments, the culture is carried out under aerobic or anaerobic conditions based on the requirements of the microorganism and its desired metabolic state. In certain embodiments, the culture includes specific conditions such as temperature, pressure, light, pH, and cell density.

[0167] In certain embodiments, the method for producing psicose includes a medium for culturing recombinant bacteria. As used herein, "medium" refers to any composition or broth that supports the growth of the microorganisms disclosed herein. The medium can be liquid or solid. In certain embodiments, the medium includes nutrients, salts, buffers, elements, and other compounds that support cell growth and viability. Additionally, the medium can include sources of nitrogen, carbon, amino acids, carbohydrates, trace elements, vitamins, and minerals. In certain embodiments, the medium includes a complex extract (e.g., yeast extract). In certain embodiments, the medium is concentrated to support rapid growth. In certain embodiments, the medium is modified to support slower growth. In certain embodiments, the medium includes an agent (e.g., antibiotic) that can inhibit the growth of or kill contaminating organisms. In certain embodiments, the medium includes an inducible promoter or an agent (e.g., IPTG) that can activate an enzyme. Non-limiting examples of media encompassed by the present disclosure include M9 medium, lysogeny broth (LB), terrific broth (TB), and YT broth. In certain embodiments, the medium comprises a substrate that is converted to psicose by the recombinant microorganism.

[0168] In certain embodiments, the substrate is a sugar (e.g., glucose or fructose) that can be phosphorylated by the bacterium via a kinase (e.g., hexokinase) and converted to fructose-6-phosphate. In certain embodiments, the substrate is glucose. In certain embodiments, the glucose can be derived from cellulose, C5 sugars, hemicellulose, and / or xylose. In certain embodiments, the substrate is a component of the culture medium. In certain embodiments, the substrate is replenished to the culture medium. In certain embodiments, the substrate is continuously present in the culture medium. In certain embodiments, the substrate is replenished during the growth phase. In certain embodiments, the substrate is replenished during the stationary phase.

[0169] 4.2. Purification of psicose In certain embodiments, the methods of the present disclosure further include purifying the psicose produced by the microorganism of the present disclosure, for example, from the cell culture or cell 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 separately from fermentation, for example, from a batch culture or a fed-batch culture. Those skilled in the art will understand that the particular purification method used can depend, among other things, on the microorganism, culture conditions, and / or the particular product.

[0170] In certain embodiments, purifying the psicose includes separating or filtering the microorganism from the cell culture medium, separating the psicose from the culture medium (e.g., by chromatography), concentrating the water (e.g., by evaporation), and lyophilizing the psicose.

[0171] 4.3. Purity In certain embodiments, the method of the present disclosure allows obtaining psicose with high purity value.As used herein, the term "allulose purity" refers to the percentage value of the concentration of allulose compared to the total concentration of allulose, mannone and glucose.In other words, the term allulose purity refers to the relative value of allulose that does not contain mannose and / or glucose (for example, foreign sugars or contaminating sugars).In certain embodiments, allulose purity is calculated by the sugar concentration and the formula: Calculated using TIFF2025530206000054.tif9128.

[0172] To determine the purity (e.g., allulose purity) of a sample (e.g., post-production medium sample, post-purification sample), high-performance liquid chromatography (HPLC) can be used to analyze the concentrations of glucose, allulose, and mannose. Known concentrations of glucose, allulose, and mannose standards can be run on the HPLC, and the area under each corresponding peak can be integrated. For each sugar standard, the peak integrals can be plotted against concentration and fitted with a best-fit line. Production samples can be run on the HPLC simultaneously with the standards. Standards can be used to identify the corresponding sugar peaks in each production sample (e.g., post-production medium sample, post-purification sample). The peaks for each sample can be integrated, and their areas recorded. Using the line of best fit, the peak integrals can be used to determine the concentration of sugar in each sample.

[0173] In certain embodiments, the allulose purity is determined after culturing the microorganisms disclosed herein. In certain embodiments, the allulose purity has a percentage value (%) of about 50% to about 100%. In certain embodiments, the allulose 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 allulose purity has a percentage value of at least about 80%. In certain embodiments, the allulose purity has a percentage value of at least about 90%. In certain embodiments, the allulose purity has a percentage value of at least about 95%. In certain embodiments, the allulose purity has a percentage value of at least about 100%.

[0174] In certain embodiments, the allulose purity is determined after psicose purification. In certain embodiments, the allulose purity has a percentage value (%) of about 50% to about 100%. In certain embodiments, the allulose 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 allulose purity has a percentage value of at least about 80%. In certain embodiments, the allulose purity has a percentage value of at least about 90%. In certain embodiments, the allulose purity has a percentage value of at least about 95%. In certain embodiments, the allulose purity has a percentage value of at least about 100%.

[0175] In certain embodiments, the allulose purity meets or exceeds the standards set by the American Chemical Society (ACS) or the standards defined in the United States Pharmacopeia (USP).

[0176] 5. Food The present disclosure also provides delivery system methods for use in foods containing psicose prepared and / or produced by any of the microorganisms disclosed herein.

[0177] As used herein, the term "food" includes any food, such as those defined in 21 CFR 101.12. Non-limiting examples of such foods include frozen desserts, baked goods, fillings, nutritional drinks, beverages, salad dressings or similar dressings, sauces, icings, puddings and custards, batters, and the like. Various baked goods are disclosed in U.S. Patent No. 6,536,599, the disclosure of which is incorporated herein by reference in its entirety. Non-limiting examples of bakery goods include cookies, cakes, rolls, pastries, pie dough, brownies, breads, bagels, and the like. Psicose prepared and / or produced by any of the microorganisms disclosed herein is also suitable as an ingredient in frozen foods.

[0178] In certain embodiments, the food product is prepared by mixing psicose with any optional ingredients in an ingestible vehicle to form a homogeneous mixture.The final composition can be easily prepared using standard methods and equipment commonly known to those skilled in the art of the corresponding technology, such as confectionery technology.Equipment useful in accordance with the subject matter disclosed herein includes mixing devices well known in the art, and therefore, the selection of a specific equipment will be clear to those skilled in the art.

[0179] In certain aspects, the present application relates to modified edible food products produced by the methods disclosed herein. In certain aspects, the food products can be produced by processes for producing edible products known to those skilled in the art.

[0180] In certain embodiments, psicose prepared and / or produced by any of the microorganisms disclosed herein can be dissolved or dispersed in one of many known edible acceptable liquids, solids, or other carriers, such as water of neutral, acidic, or basic 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 shortening, fatty acids, certain low molecular weight oligomers of propylene glycol, glyceryl esters of fatty acids, and dispersions or emulsions of such hydrophobic substances in aqueous media, salts such as sodium chloride, vegetable flour, solvents such as ethanol, vegetable powder, or solid edible diluents such as vegetable flour, and then combined with a precursor to an edible product or pharmaceutical product or applied directly to the edible product or pharmaceutical product.

[0181] Those skilled in the art of preparing and selling food products are well aware of the wide variety of classes, subclasses, and species of edible compositions and utilize well-known and recognized terminology to refer to those edible compositions in their various preparation and marketing efforts. A list of such terminology is provided below, and it is specifically contemplated hereby that psicose prepared and / or produced by any of the microorganisms disclosed herein, alone or in any reasonable combination or mixture thereof, can be used to modify or enhance the taste of the edible compositions listed below.

[0182] In certain embodiments, foods that are mixed with psicose prepared and / or produced by any of the microorganisms disclosed herein include, by way of example, the wet soup category, the dried and prepared food category, the beverage category, the frozen food category, the snack food category, and the seasonings or seasoning blends described herein.

[0183] In certain embodiments, the psicose prepared and / or produced by any of the microorganisms disclosed herein is present in one or more of confectionery, chocolate confectionery, tablets, countlines, bagged selfies / softlines, boxed assortments, standard boxed assortments, twist wrap miniatures, seasonal chocolates, chocolates with toys, allsorts, other chocolate confectionery, mints, standard mints, strong mints, hard candies, lozenges, gum, and jelly. and chewing sweets, toffee, caramel and nougat, medicated sweets, lollipops, liquorice, other sugar confectionery, gum, chewing gum, sugar coated gum, sugar free gum, functional gum, bubble gum, bread, packaged / industrial bread, unpackaged / artisan bread, pastries, cakes, packaged / industrial cakes, unpackaged / homemade cakes, cookies, chocolate coated biscuits, sandwich biscuits, filled biscuits, savory biscuits and crackers, bread substitutes, breakfast cereals, rye cereals, home breakfast cereals, flakes, muesli, other rye cereals, children's breakfast cereals, hot cereals, ice cream, ice cream for store consumption, single portion ice cream with milk fat, single portion sorbet ice cream, multipack ice cream with milk fat, multipack sorbet ice cream, takeaway ice cream, takeaway ice cream with milk fat, ice cream desserts, bulk ice cream, takeaway sorbet ice cream, frozen yogurt, artisanal ice cream, dairy products, milk, fresh / pasteurised milk, full fat fresh / pasteurised milk, semi-skimmed fresh / Pasteurized milk, Long life / Ultra-high temperature milk, Whole fat Long life / Ultra-high temperature milk, Semi-skimmed Long life / Ultra-high temperature milk, Non-fat Long life / Ultra-high temperature milk, Goat's milk, Condensed / Evaporated milk, Plain condensed / Evaporated milk, Flavoured, Functional and other condensed milk, Flavoured milk drinks, Dairy only flavoured milk drinks, Flavoured milk drinks with fruit juice, Soy milk, Sour milk drinks, Fermented milk drinks, Milk for coffee, Milk powder, Flavoured milk powder drinks, Cream, Cheese, Processed cheese, Processed cheese for spreads, Processed cheese for non-spreads, Non-processed cheese,Non-processed cheese for spreads, Hard cheese, Packaged hard cheese, Unpackaged hard cheese, Yogurt, Plain / natural yogurt, Flavored yogurt, Yogurt with fruit, Probiotic yogurt, Drinking yogurt, Regular drinking yogurt, Probiotic drinking yogurt, Refrigerated and shelf-stable desserts, Dairy-based desserts, Soy-based desserts, Refrigerated snacks, Fromage frais and quark, Plain Fromage frais and quark, Flavored Fromage frais and quark, Savory Fromage frais and quark, sweet and savory snacks, fruit snacks, chips / crisps, extruded snacks, tortilla / corn chips, popcorn, pretzels, nuts, other sweet and savory snacks, snack bars, granola bars, breakfast bars, energy bars, fruit bars, other snack bars, meal replacements, diet 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, dried soup instant soup, refrigerated soup, ultra-pasteurized soup, frozen soup, pasta, canned pasta, dried pasta, refrigerated / fresh pasta, noodles, plain noodles, instant noodles, instant noodles in cups / bowls, instant noodles in pouches, 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, Oven Baked Potato Chips, Other Oven Baked Potato Products, Non-Oven Frozen Potatoes, Frozen Bakery Products, Frozen Desserts, Frozen Ready Meals, Frozen Pizza, Frozen Soups, Frozen Noodles, Other Frozen Foods, Dried Foods, Dessert Mixes, Dried Ready Meals, Dried Soups, Instant Soups, Dried Pasta, Plain Noodles, Instant Noodles, Cup / Bowl Instant Noodles, Pouch Instant Noodles, Refrigerated Foods, Refrigerated Processed Meat, Refrigerated Fish / Seafood Products, Refrigerated Processed Fish, Refrigerated Coated Fish,Refrigerated Smoked Fish, Refrigerated Lunch Kits, Refrigerated Ready Meals, Refrigerated Pizza, Refrigerated Soup, Refrigerated / Fresh Pasta, Refrigerated Noodles, Oils and Fats, Olive Oil, Vegetable and Seed Oils, Cooking Fats, Butter, Margarine, Spreadable Oils and Fats, Functional Spreadable Oils and Fats, Sauces, Dressings and Condiments, Tomato Paste and Puree, Bouillon / Stock Cubes, Stock Cubes, Gravy Granules, Liquid Stocks and Fonds, Herbs and Spices, Fermented Sauces, Soy Based Sauces, Pasta Sauces, Wet Sauces, Dry Sauces / Powder Mixes, Ketchup , mayonnaise, regular mayonnaise, mustard, salad dressing, regular salad dressing, low-fat salad dressing, vinaigrettes, dips, pickling products, other sauces, dressings and condiments, baby foods, formula, standard formula, follow-on formula, toddler formula, hypoallergenic formula, prepared baby food, dry baby food, other baby foods, spreads, jams and preserves, mixed with honey, chocolate spreads, nut-based spreads, and yeast-based spreads.

[0184] Chewing gum In certain embodiments, psicose prepared and / or produced by any of the microorganisms disclosed herein can be used in low-calorie gum formulations and can also be used in sugar chewing gum. Various details of chewing gum compositions are disclosed in U.S. Pat. No. 6,899,911, the disclosure of which is incorporated herein by reference in its entirety. The chewing gum compositions of the subject matter disclosed herein follow the general pattern outlined below. In general, chewing gum compositions typically contain a chewable gum base portion that is substantially water-free and water-insoluble, a water-soluble bulk portion, and generally water-insoluble flavors. The water-soluble portion dissipates during chewing, along with some of the flavor. The gum base portion remains in the mouth throughout chewing. The insoluble gum base generally contains elastomers, elastomer solvents, plasticizers, waxes, emulsifiers, and inorganic fillers. Plasticized polymers (such as polyvinyl acetate) that act as plasticizers to some extent are also often included. Other plasticizers that can be used include polyvinyl laurate, polyvinyl alcohol, and polyvinylpyrrolidone. Elastomers can include polyisobutylene, butyl rubber (isobutylene-isopropyl copolymer), and styrene-butadiene rubber, as well as natural latexes such as chicle. Elastomer solvents are often resins, such as terpene resins. Plasticizers, sometimes called softeners, are typically fats and oils, such as tallow, hydrogenated and partially hydrogenated vegetable oils, and cocoa butter. Commonly utilized waxes are paraffins, microcrystalline, and natural waxes, such as beeswax and carnauba. Microcrystalline waxes, especially those with a high degree of crystallinity, can be considered as thickening agents or texture modifiers.

[0185] In certain embodiments, the insoluble gum base comprises from about 5% to about 95% by weight of the gum. More preferably, the insoluble gum base comprises from 10% to 50% by weight of the gum, and most preferably from about 20% to about 35% by weight of the gum. The gum base typically also contains a filler component. Filler components may be calcium carbonate, magnesium carbonate, talc, dicalcium phosphate, etc. The filler may comprise from about 5% to about 60% by weight of the gum base. Preferably, the filler comprises from about 5% to about 50% by weight of the gum base.

[0186] Gum bases typically also contain softeners such as glycerol monostearate and glycerol triacetate. Gum bases may also contain optional ingredients such as antioxidants, colorants, and emulsifiers. The subject matter disclosed herein contemplates the use of any commercially acceptable gum base.

[0187] The water-soluble portion of chewing gum may further include softeners, sweeteners, flavors, physiological cooling agents, and combinations thereof. Sweeteners often serve as bulking agents in gum. Bulking agents typically comprise from about 5% to about 95% of the gum composition.

[0188] Softeners are added to chewing gum to optimize the chewiness and mouthfeel of the gum. Softeners, also known in the art as plasticizers or plasticizers, generally comprise about 0.5% to about 15% of the chewing gum. Softeners contemplated by the presently disclosed subject matter include glycerin, lecithin, and combinations thereof. Additionally, aqueous sweetener solutions, such as those containing sorbitol, hydrogenated starch hydrolysates, corn syrup, and combinations thereof, can be used as softeners and binders in the gum.

[0189] As mentioned above, psicose prepared and / or produced by any of the microorganisms disclosed herein can be used in low-calorie gum formulations. However, sugar-containing formulations are also within the scope of the present invention. Sugar sweeteners generally include saccharide-containing ingredients commonly known in the chewing gum art, including, but not limited to, sucrose, dextrose, maltose, dextrin, dry invert sugar, fructose, galactose, corn syrup solids, and the like, alone or in any combination. Psicose prepared and / or produced by any of the microorganisms disclosed herein can also be used in combination with sugarless sweeteners. Generally, sugarless sweeteners include commonly known sugar-free ingredients that have sweetening properties, including, but not limited to, sugar alcohols such as sorbitol, hydrogenated isomaltulose, mannitol, xylitol, lactitol, erythritol, hydrogenated starch hydrolysates, maltitol, and the like, alone or in any combination.

[0190] Depending on the release profile and shelf stability of the particular sweetener required, coated or uncoated high-intensity sweeteners can be used in the chewing gum compositions or in coatings applied to centers made from these gum compositions. High-intensity sweeteners, preferably aspartame, can be used at levels 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 may be added, and when aspartame is used, a combination of some free and encapsulated aspartame is preferred. Other high-intensity sweeteners that can be used in the gum centers are saccharin, thaumatin, alitame, saccharin salts, sucralose, stevia, and acesulfame K. Overall, the chewing gum composition preferably contains about 0.5% to about 90% sweetener. Most typically, the sweetener comprises at least one bulk sweetener and at least one intense sweetener. Optional ingredients such as colorants, emulsifiers, and pharmaceutical agents can also be added as separate components of the chewing gum composition or as part of the gum base.

[0191] Aqueous syrups, such as corn syrup and hydrogenated corn syrup, can be used, especially when the moisture content is reduced. This is preferably achieved by coevaporating the aqueous syrup with a plasticizer such as glycerin or propylene glycol to a moisture content of less than 10%. A preferred composition includes hydrogenated starch hydrolysate solids and glycerin. Such syrups and their preparation methods are discussed in detail in U.S. Pat. No. 4,671,967.

[0192] A method for making chewing gum according to the presently disclosed subject matter involves sequentially adding the various chewing gum ingredients to any commercially available mixer known in the art. Once the ingredients are thoroughly mixed, the gum is discharged from the mixer and shaped into the desired shape, for example, by rolling into a sheet and then cutting into sticks, extruding into chunks, or casting into pellets. Generally, the ingredients are mixed by first melting the gum base and adding it to a running mixer. It is also possible to melt the base in the mixer itself. At this point, colorants or emulsifiers may be added along with the syrup and a portion of the bulking agent. Another portion of the bulking agent can then be added to the mixer. The flavor system is typically added with the last portion of the bulking agent. If the flavor system is to be coated or otherwise modified, such as when incorporated into a delivery system to modify its release rate, it is preferable to add the flavor system after the last portion of the bulking agent. The entire mixing procedure typically requires 5 to 20 minutes, although longer mixing times may be required in some cases. Those skilled in the art will recognize that many variations of the above procedure can be performed.

[0193] When formed into pellets or spheres, the chewing gum composition can be coated. The coating initially exists as 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 carried out in a conventional panning apparatus. The gum center tablets to be coated are placed in the panning apparatus to form a moving mass.

[0194] The material or syrup that ultimately forms the coating is applied or distributed over the entire 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 multiple layers of coating. Psicose can be added to any or none of the coatings and / or layers.

[0195] In the panning procedure, 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 from about 140°F to about 200°F. Most preferably, the syrup temperature should be maintained constant throughout the process to prevent the polyol in the syrup from crystallizing. The syrup can be mixed with, sprayed onto, poured onto, or added to the gum center tablets by any method known to those skilled in the art.

[0196] In certain embodiments, the soft coating is formed by adding a powder coating after the liquid coating. The powder coating can include natural carbohydrate gum hydrolysates, maltodextrin, gelatin, cellulose derivatives, starch, modified starch, sugars, sugar alcohols, natural carbohydrate gums, and fillers such as talc and calcium carbonate.

[0197] Each coating component of the gum center can be applied in a single layer or multiple layers. Generally, multiple layers are achieved by applying a single coat, allowing the layer to dry, and then repeating the process. The amount of solids added with each coating step depends primarily on the consistency of the coating syrup. Any number of coatings may be applied to the gum center tablet. Preferably, no more than about 75 coatings are applied to the gum center. More preferably, fewer than about 60 coatings are applied, and most preferably, about 30 to about 60 coatings are applied. In any event, the presently disclosed subject matter contemplates applying an amount of syrup sufficient to obtain a coated chewing gum product containing about 10% to about 65% coating. Preferably, the final product contains about 20% to about 50% coating.

[0198] Those skilled in the art will recognize that multiple pre-measured aliquots of coating syrup can be applied to the gum centers to achieve multiple coating layers, however, it is contemplated that the amount of aliquots of syrup applied to the gum centers can vary throughout the coating procedure.

[0199] Once the syrup coating is applied to the gum centers, the syrup is dried in an inert medium. A preferred drying medium includes air. Preferably, forced drying air is contacted with the wet syrup coating at a temperature ranging from about 70°F to about 110°F. More preferably, the drying air has a temperature ranging from about 80°F to about 100°F. The present invention also contemplates that the drying air have a relative humidity of less than about 15%. Preferably, the relative humidity of the drying air is less than about 8%.

[0200] The drying air can be passed over and mixed with the syrup-coated gum centers by any method commonly known in the art. Preferably, the drying air is blown over and around the syrup-coated gum centers at a flow rate of about 2800 cubic feet per minute for large-scale operations. Lower flow rates would be used if smaller amounts of material are processed or if smaller equipment is used. If the flavor is applied after the syrup coating has dried, the subject matter disclosed herein contemplates drying the flavor with or without a drying medium.

[0201] The amount of psicose utilized herein is generally a matter of preference, depending on factors such as the type of final chewing gum composition, the particular flavor, the gum base utilized, and the desired flavor intensity. Therefore, the amount of psicose can be varied to achieve the desired result in the final product, and such variations are within the capabilities of those skilled in the art without undue experimentation. In gum compositions, psicose prepared and / or produced by any of the microorganisms disclosed herein is generally present in an amount of about 0.02% to about 5% by weight of the chewing gum composition, preferably about 0.1% to about 2% by weight, and more preferably about 0.8% to about 1.8% by weight.

[0202] 5.2. Sugar confectionery Another important aspect of the presently disclosed subject matter includes confectionery compositions incorporating psicose prepared and / or produced by any of the microorganisms disclosed herein, as well as methods for preparing the confectionery compositions. The preparation of confectionery formulations is well known in the art. Confectionery items have been classified as either "hard" or "soft" confectionery. Psicose prepared and / or produced by any of the microorganisms disclosed herein can be incorporated into confectionery by blending the compositions of the presently disclosed subject matter into conventional hard and soft confectionery.

[0203] Hard confections can be processed and formulated by conventional means. Generally, hard confections have a base composed of a mixture of sugar and other carbohydrate bulking agents maintained in an amorphous or glassy state. Hard confections can also be sugar-free. Hard confections can also be low-calorie. This form is generally considered a sugar syrup solid, having a moisture content of about 0.5% to about 1.5%. Such materials typically contain up to about 92% sugar, up to about 55% corn syrup, and about 0.1% to about 5% water by weight of the final composition. The syrup component is generally prepared from sucrose and corn syrup, but may contain other ingredients. In certain embodiments, the syrup component comprises psicose prepared and / or produced by any of the microorganisms disclosed herein. Additional ingredients, such as flavorings, sweeteners, acidulants, colorants, etc., can also be added.

[0204] Such confections can be routinely prepared by conventional methods, including, but not limited to, flame cookers, sous vide 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 confectionery arts; therefore, the selection of a particular equipment will be apparent to one skilled in the art.

[0205] The flame cooker involves the traditional method of producing a candy base, in which the desired amount of carbohydrate bulking agent is dissolved in water by heating in a kettle until the bulking agent melts. More bulking agent can then be added and cooked until a final temperature of 145°C to 156°C is achieved. The batch is then cooled and processed into a plastic-like mass, and additives such as flavors, colors, etc. are incorporated.

[0206] High-speed atmospheric cookers use a heat exchange surface, which spreads a candy film over the surface and heats the candy to 165–170°C within seconds. The candy is then rapidly cooled to 100–120°C and processed into a plastic-like mass, which allows for the incorporation of additives such as flavors, colors, etc. In sous vide cookers, carbohydrate bulking agents are boiled to 125–132°C, vacuumed, and further simmered to remove moisture without additional heating. Once cooked, the mass is semi-solid and has a plastic-like consistency. At this point, flavors, colors, and other additives are incorporated into the mass using routine mechanical mixing operations.

[0207] The optimum mixing required to achieve uniform incorporation of flavors, colors, and other additives during conventional hard confectionery manufacture is determined by the time required to obtain a uniform distribution of the ingredients. Generally, mixing times of 2 to 10 minutes have been found to be acceptable.

[0208] Once the candy mass is properly kneaded, it can be cut into workable portions or molded into the desired shape. Depending on the shape and size of the final product desired, various molding techniques can be utilized. A general discussion of the composition and preparation of hard confections can be found in H.A. Lieberman, Pharmaceutical Dosage Forms: Tablets, Volume 1 (1989), Marcel Dekker, Inc., New York, NY, pp. 419-582, the disclosure of which is incorporated herein by reference.

[0209] Compressed tablet confections contain certain ingredients and are formed into a structure under pressure. These confections generally contain sugar in an amount up to about 95% by weight of the composition, as well as typical tablet excipients such as binders and lubricants, as well as flavors, colors, etc. These confections can also be sugar-free.

[0210] Similar to hard confections, soft confections can be utilized in embodiments of the disclosed subject matter. The preparation of soft confections, such as nougat, involves traditional methods such as the combination of two major ingredients: (1) a high-boiling syrup, such as corn syrup, and (2) a relatively light-textured frappe, typically prepared from vegetable proteins, such as egg albumin, gum arabic, gelatin, soybean-derived compounds, unsweetened milk-derived compounds, such as milk proteins, and mixtures thereof. The frappe is generally relatively light, e.g., its density can range from about 0.5 to about 0.7 grams per cubic centimeter.

[0211] The high-boiling syrup of soft confections, or "bob syrup," is relatively viscous, has a higher density than the frappe ingredients, and often contains a significant amount of carbohydrate bulking agent. Traditionally, the final nougat composition is prepared by adding the "bob syrup" to the frappe under stirring to form the base nougat mixture. Additional ingredients, such as flavorings, additional carbohydrate bulking agents, coloring agents, preservatives, pharmaceuticals, mixtures thereof, and the like, can then be added, also under stirring. Soft confections can also be prepared sugar-free. A general discussion of the composition and preparation of nougat confections can be found in B.W. Minifie, Chocolate, Cocoa and Confectionery: Science and Technology, 2nd edition, AVI Publishing Co., Inc., Westport, Conn. (1983), pp. 576-580, the disclosure of which is incorporated herein by reference.

[0212] Generally, the frappe component is prepared first, after which the syrup component is slowly added with stirring at a temperature of at least about 65° C., preferably at least about 100° C. The mixture of ingredients is continued to be mixed to form a uniform mixture, after which the mixture is cooled to a temperature below 80° C., at which point flavors can be added. The mixture is further mixed for an additional period of time until it can be removed and formed into suitable confectionery shapes.

[0213] According to the present disclosure, an amount of psicose prepared and / or produced by any of the microorganisms disclosed herein can be incorporated into hard and soft confections. The exact amount of psicose used is typically a matter of preference, depending on factors such as the particular type of confection being prepared, the type of bulking agent or carrier used, the type of flavor used, and the strength of breath freshening desired. Therefore, 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. Generally, the amount of psicose typically present in hard or soft confections will be about 0.001% to about 20% by weight of the confection, preferably about 0.01% to about 15% by weight, more preferably about 0.01% to about 10% by weight, more preferably about 0.01% to about 5% by weight, and more preferably 0.01% to about 0.5% by weight.

[0214] The subject matter disclosed herein also extends to methods for producing improved confectionery. Psicose prepared and / or produced by any of the microorganisms disclosed herein can be incorporated into otherwise conventional hard or soft confectionery compositions using standard techniques and equipment known to those skilled in the art. Equipment useful with the subject matter disclosed herein includes mixing and heating equipment well known in the confectionery arts, and therefore, the selection of specific equipment will be apparent to those skilled in the art.

[0215] In such a method, the composition is prepared by mixing psicose into a confectionery composition together with other ingredients of the final desired composition. The other ingredients are typically incorporated into the composition as dictated by the properties of the desired composition, as is well known to those skilled in the art. The final confectionery composition is easily prepared using methods commonly known in the food and pharmaceutical arts. The confectionery mixture can then be formed into the desired confectionery shape.

[0216] Psicose prepared and / or produced by any of the microorganisms disclosed herein can be formulated with conventional ingredients to provide various textures suitable for specific applications. Such ingredients can be in the form of hard and soft confections, tablets, taffy, nougat, chewy candy, chewing gum, and center-filled candies, both sugar and sugar-free. Acceptable ingredients can be selected from a wide range of materials. Such materials include, but are not limited to, diluents, binders and adhesives, lubricants, disintegrants, bulking agents, humectants, buffers, and adsorbents. The preparation of such confectionery and chewing gum products is well known.

[0217] 5.3. Chocolate and fillings The subject matter disclosed herein can also be used with and / or in chocolate products, chocolate-flavored confections, and chocolate-flavored compositions. Chocolates also include those containing breadcrumb solids or solids produced completely or partially by breading. Various chocolates are disclosed, for example, in U.S. Patent Nos. 7,968,140 and 8,263,168, the disclosures of which are incorporated herein by reference in their entireties. A general discussion of the composition and preparation of chocolate confections can be found in B.W. Minifie, Chocolate, Cocoa, and Confectionery: Science and Technology, 2nd edition, AVI Publishing Co., Inc., Westport, Conn. (1982), the disclosure of which is incorporated herein by reference.

[0218] As used herein, the term "chocolate" refers to a solid or semi-plastic food product and is intended to refer to all chocolate or chocolate-like compositions containing a fat-based ingredient phase or fat-like composition. This term is intended to include standardized or non-standardized compositions that meet the US Standards of Identity (SOI), CODEX Alimentarius, and / or other international standards, as well as compositions that do not meet the US Standards of Identity or other international standards. Unless otherwise specified, this term includes dark chocolate, baking chocolate, sweet chocolate, bittersweet or semi-sweet chocolate, milk chocolate, buttermilk chocolate, skim milk chocolate, blended dairy chocolate, white chocolate, sweet cocoa and vegetable fat coating, sweet chocolate and vegetable fat coating, milk chocolate and vegetable fat coating, vegetable fat-based coating, white chocolate or pastels containing coatings made with cocoa butter or vegetable fat or combinations thereof, nutritionally modified chocolate-like compositions (chocolate or coatings made with reduced-calorie ingredients), and low-fat chocolate, aerated chocolate, compound coatings, non-standardized chocolate, and chocolate-like compositions.

[0219] Non-standardized chocolate occurs, for example, when nutritive carbohydrate sweeteners are partially or completely replaced, or when cocoa butter, cocoa butter substitutes, cocoa butter equivalents, cocoa butter extenders, cocoa butter substitutes, cocoa butter substitutes, or milk fats are partially or completely replaced, or when ingredients with flavors that simulate milk, butter, or chocolate are added, or when other additions or omissions in the formulation are made outside of FDA's standards for identity of chocolate, or a combination thereof. Chocolate-like compositions are fat-based compositions, such as carob, that can be used as a substitute for chocolate in applications such as panning, molding, or enrobing.

[0220] In the United States, chocolate is subject to the Standards of Identity established by the Food and Drug Administration (FDA) under the Federal Food, Drug, and Cosmetic Act. Definitions and standards for different types of chocolate are well established in the United States. Non-standardized chocolate is chocolate that contains ingredients outside the specified range of standardized chocolate.

[0221] In certain embodiments, chocolate can contain psicose prepared and / or produced by any of the microorganisms disclosed herein. Furthermore, chocolate can contain sugar syrup / solids, invert sugar, hydrolyzed lactose, maple sugar, brown sugar, molasses, honey, sugar substitutes, etc. Nutritive carbohydrate sweeteners with varying degrees of sweetness can be any commonly used in the art, including, but not limited to, sucrose derived from sugarcane or beet, dextrose, fructose, lactose, maltose, glucose syrup solids, corn syrup solids, invert sugar, hydrolyzed lactose, honey, maple sugar, brown sugar, molasses, etc. Sugar substitutes can be used in part to replace the nutritive carbohydrate sweetener. High-potency sweeteners include aspartame, cyclamate, saccharin, acesulfame-K, neohesperidin dihydrochalcone, sucralose, alitame, stevia sweetener, glycyrrhizin, thaumatin, etc., and mixtures thereof. Preferred high-potency sweeteners are aspartame, cyclamate, saccharin, and acesulfame-K. Examples of sugar alcohols may be any of those commonly used in the art, including sorbitol, mannitol, xylitol, maltitol, isomalt, lactitol, etc.

[0222] Chocolate may also contain a bulking agent. As used herein, the term "bulking agent" may be any of those commonly used in the art, including polydextrose, cellulose and its derivatives, maltodextrin, gum arabic, and the like.

[0223] The chocolate product may contain an emulsifier. Examples of safe and suitable emulsifiers include any of those commonly used in the art, such as lecithin derived from vegetable sources such as soybean, safflower, corn, etc., fractionated lecithin rich in phosphatidylcholine or phosphatidylethanolamine, or both, mono- and diglycerides, diacetyl tartaric 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 lecithin, lactylated fatty acid esters of glycerol and propylene glycol, polyglycerol esters of fatty acids, propylene glycol monoesters and diesters of fats and fatty acids, or emulsifiers that may become approved in the soft candy category as defined by the U.S. FDA. Additionally, other emulsifiers that can be used include polyglycerol polyricinoleate (PGPR), ammonium salts of phosphatidic acid, (e.g., YN) sucrose esters, oat extract, etc., any emulsifier or any blend found to be suitable for chocolate or similar fat / solid systems.

[0224] The term "chocolate-flavored confectionery" refers to foods that have a chocolate flavor / aroma, excluding "chocolate," and contain a cocoa fraction. These products are characterized as being stable at ambient temperature for extended periods (e.g., greater than one week) and microbiologically shelf-stable at 18-30°C under normal atmospheric conditions. Examples include chocolate-flavored hard candy, chewables, chewing gum, etc.

[0225] The term "chocolate-flavored composition" refers to a chocolate-flavored composition that contains a cocoa fraction and has a chocolate flavor / aroma, excluding "chocolate." Examples include chocolate-flavored cake mixes, ice cream, syrups, baking products, and the like. The term includes chocolate-flavored compositions (e.g., cakes, nougat, puddings, etc.) and compositions that do not have a chocolate flavor (e.g., caramel, etc.).

[0226] 5.4. Savory and other food products In certain embodiments, psicose prepared and / or produced by any of the microorganisms disclosed herein is incorporated into a savory product. In certain embodiments, the savory product is a food product having a savory flavor, including, but not limited to, spicy flavor, pepper flavor, dairy flavor, vegetable flavor, tomato flavor, dill flavor, meat flavor, poultry flavor, chicken flavor, and reaction flavors added or produced during heating of the food product.

[0227] In certain embodiments, psicose prepared and / or produced by any of the microorganisms disclosed herein is incorporated into a wet soup-category food product, including wet / liquid soup, regardless of concentration or container, including frozen soup. In certain embodiments, soup food refers to a food product prepared from meat, poultry, fish, vegetables, grains, fruit, and / or other ingredients, cooked in a liquid that may contain visible debris of some or all of these ingredients. Soup foods can be clear (as in a broth), thick (as in a chowder), smooth, pureed, chunky, ready-to-serve, semi-concentrated, or concentrated, hot or cold, and can be served as a first or main course of a meal or as a snack (sipped like a beverage). Soups can be used as ingredients for preparing other meal components and can range from broths (consomme) to sauces (cream- or cheese-based soups).

[0228] In certain embodiments, the psicose prepared and / or produced by any of the microorganisms disclosed herein is used in the preparation of: (i) pressed cubes, tablets, or concentrated liquid products in powder or granular form, including powders, granules, pastes, concentrated bouillons, bouillon and bouillon-like products, sold individually as a finished product or as an ingredient within a product, cooking aid products such as sauces and recipe mixes (regardless of technology); (ii) meal solution products such as dehydrated and freeze-dried soups, including dehydrated soup mixes, dehydrated instant soups, dehydrated cooked soups, dehydrated or room temperature preparations of ready-to-eat meals, meals, and single-serving dishes, including pasta, potato, and rice; and (iii) meal embellishment products, such as seasonings, marinades, salad dressings, salad toppings, dips, breadcrumbs, batter mixes, shelf-stable spreads, barbecue sauces, liquid recipe mixes, concentrates, sauces or sauce mixes, e.g., salad recipe mixes, whether dried, liquid, or frozen, sold as a finished product or as an ingredient within a product. It is included in the category of dried and prepared foods, including cereals containing gluten-free products.

[0229] In certain embodiments, the psicose prepared and / or produced by any of the microorganisms disclosed herein is incorporated into meat products. In certain embodiments, meat products include foods produced by processing the edible remains of any dead animal, including birds, fish, crustaceans, shellfish, and mammals. Meat products include, but are not limited to, cooked beef, lamb, pork, poultry, or seafood products. Examples of such meat products include, for example, bologna, frankfurters, sausages, luncheon meat, deli slices, loaf, bacon, meatballs, fish sticks, chicken fingers, and ground meat, such as meatloaf, meatballs, and hamburgers. Meat products may be combined with simulated meat products. Simulated meat products include, but are not limited to, meat substitutes, meat analogs, soy burgers, soy bologna, soy frankfurters, soy sausages, soy luncheon loaf, soy bacon, and soy meatballs. Simulated meat products may be combined with meat products.

[0230] In certain embodiments, the psicose prepared and / or produced by any of the microorganisms disclosed herein is incorporated into foods in the snack food category. In certain embodiments, snack foods include any foods that can be used as an informal snack, including but not limited to sweet snacks and snack bars and savory snacks and snack bars. Examples of snack foods include, but are not limited to, fruit snacks, chips / crisps, extruded snacks, tortilla / corn chips, popcorn, pretzels, nuts, and other sweet and savory snacks. Examples of snack bars include, but are not limited to, granola / muesli bars, breakfast bars, energy bars, fruit bars, and other snack bars.

[0231] In certain embodiments, psicose prepared and / or produced by any of the microorganisms disclosed herein is incorporated into refrigerated or frozen foods, such as, but not limited to, ice cream, impulse ice cream, single portion dairy ice cream, single portion water ice cream, multipack dairy ice cream, multipack water ice cream, take-out ice cream, take-out dairy ice cream, ice cream desserts, bulk ice cream, take-out water ice cream, frozen yogurt, artisanal ice cream, frozen ready meals, frozen pizza, chilled pizza, frozen soup, frozen pasta, frozen processed red meat, frozen processed poultry, frozen processed seafood / seafood, frozen processed vegetables, frozen meat substitutes, frozen potatoes, frozen bakery products, and frozen desserts.

[0232] 5.4. Medications The psicose prepared and / or produced by any of the microorganisms disclosed herein may be in the form of a pharmaceutical. A non-limiting example of a pharmaceutical form is a suspension. Pharmaceutical suspensions can be prepared by conventional formulation methods. Suspensions can contain auxiliary materials used in formulating suspensions in the art. The suspensions of the subject matter disclosed herein can include preservatives, buffers, suspending agents, antifoaming agents, sweeteners, flavoring agents, coloring or decoloring agents, solubilizers, and combinations thereof.

[0233] Flavoring agents, such as flavors well known to those skilled in the art, for example, natural and artificial flavors and mints, e.g., peppermint, menthol, citrus flavors such as orange and lemon, artificial vanilla, cinnamon, and various fruit flavors, both individual and mixed, can be utilized in amounts of from about 0.01% to about 5%, more preferably 0.01% to about 0.5%, by weight of the suspension.

[0234] 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 prepared and / or produced by any of the microorganisms disclosed herein with water to form a solution; (iii) optionally, mixing a flavoring with the thickener-water mixture to form a uniform thickener-flavoring; (iv) combining a sweetener solution with the thickener-flavoring and mixing until uniform; and (v) mixing optional auxiliary materials such as colorants, flavorings, decolorants, solubilizers, antifoaming agents, buffers, and additional water with the mixture of step (iv) to form a suspension.

[0235] The psicose prepared and / or produced by any of the microorganisms disclosed herein may be in chewable form. To achieve acceptable stability and quality as well as good taste and mouthfeel in chewable formulations, several considerations are important. These considerations include the amount of active substance per tablet, the flavoring agent used, the degree of tablet compressibility, and additional properties of the composition. Chewable pharmaceutical candies are prepared by procedures similar to those used to manufacture soft confectionery. A general discussion of confectionery lozenges and chewable tablet forms can be found in H.A. Lieberman and L. Lachman, Pharmaceutical Dosage Forms: Tablets Volume 1, Marcel Dekker, Inc., New York, NY (1989), pp. 367-418, the disclosure of which is incorporated herein by reference. In a typical procedure, a boiled sugar-corn syrup blend is formed, to which a frappe mixture is added. The boiled sugar-corn syrup blend can be prepared from sugar and corn syrup blended in a ratio of about 90:10 to about 10:90 parts by weight. The sugar-corn syrup blend is heated to a temperature above about 120°C to remove water and form a molten mass. Frappes are generally made from gelatin, milk proteins such as egg albumin and casein, and vegetable proteins such as soy protein, which are added to a gelatin solution and rapidly mixed at ambient temperature to form an aerated, spongy mass. The frappe is then added to the molten candy mass and mixed at a temperature of about 65°C to about 120°C until homogeneous. Psicose prepared and / or produced by any of the microorganisms disclosed herein can then be added to the homogeneous mixture while the temperature is reduced to about 65°C to 95°C, after which additional ingredients such as flavors and colorants can be added. The mixture is further cooled and formed into pieces of desired dimensions.

[0236] In other pharmaceutical embodiments, flavoring agents are incorporated into ingestible topical vehicles, which may be in the form of mouthwashes, rinses, ingestible sprays, suspensions, dental gels, etc. Typical non-toxic ingestible vehicles known in the pharmaceutical arts can be used in the presently disclosed subject matter. Preferred ingestible vehicles are water, ethanol, and water-ethanol mixtures. Water-ethanol mixtures are generally utilized 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 value of the ingestible vehicle is generally about 4 to about 7, preferably about 5 to about 6.5. Ingestible topical vehicles with pH values ​​less than about 4 generally irritate the ingestible cavity, while ingestible vehicles with pH values ​​greater than about 7 generally result in an unpleasant mouthfeel.

[0237] Ingestible topical flavoring agents can also contain conventional additives commonly used in such products. Conventional additives include fluoride-donating compounds, sweeteners, flavoring agents, coloring agents, humectants, buffers, and emulsifiers, provided that these additives do not interfere with the flavor characteristics of the composition. The coloring agents and humectants listed above, as well as the amounts of these additives used, can be used in ingestible topical compositions. Flavoring agents (flavors, flavoring materials) that can be used include flavors known to those skilled in the art, such as natural and artificial flavors. Suitable flavoring agents include mints such as peppermint, citrus flavors such as orange and lemon, artificial vanilla, cinnamon, various fruit flavors, both individual and mixed, and the like. The amount of flavoring agent used in ingestible topical compositions is usually a matter of preference, influenced by factors such as the type of final ingestible composition, the individual flavor used, and the desired flavor intensity. Therefore, the amount of flavoring agent can be varied to achieve the desired result in the final product, and such variations are within the ability of one skilled in the art without undue experimentation. Flavoring agents, when used, are generally utilized in amounts that may range, for example, from about 0.05% to about 6% by weight of the ingestible topical composition.

[0238] 5.5. Pet Food Products Psicose prepared and / or produced by any of the microorganisms disclosed herein can be used in a wide range of pet food products.

[0239] As used herein, the term "pet food" or "pet food product" refers to a product or composition intended for consumption by companion animals, such as cats, dogs, guinea pigs, rabbits, birds, and horses. For example, but not by way of limitation, the companion animal can be a "domestic" dog, such as Canis lupus familiaris. "Pet food" or "pet food product" includes any food, feed, snack, food supplement, liquid, beverage, treat, toy (chewable and / or consumable), meal substitute, or meal replacement.

[0240] In certain embodiments, the psicose prepared and / or produced by any of the microorganisms disclosed herein is added directly to a pet food product. In certain embodiments, the psicose prepared and / or produced by any of the microorganisms disclosed herein can be added before, during, or after the formulation or packaging of a pet food product.

[0241] Non-limiting examples of suitable pet food products include wet food products, dry food products, moist food products, pet food supplements (e.g., vitamins), pet drink products, snacks and treats, and the pet food categories described herein.

[0242] In certain embodiments, the pet food product is a dry food product. Dry or low-moisture nutritionally complete pet food products may contain less than about 15% moisture. In certain embodiments, the pet food product is a wet food product. Wet or high-moisture nutritionally complete pet food products may contain more than about 50% moisture. In certain embodiments, the pet food product is a nutritionally complete moist food product. Moist, e.g., semi-moist or semi-dry or soft-dry or soft-moist or medium or moderate moisture nutritionally complete pet food products contain about 15% to about 50% moisture.

[0243] In certain embodiments, the pet food product is a pet food snack product. Non-limiting examples of pet food snack products include snack bars, pet chews, crunchy treats, cereal bars, snacks, biscuits, and sweet products. [Example]

[0244] The subject matter disclosed herein may be better understood by reference to the following: The following examples are illustrative only and should not be construed as limiting in any way.

[0245] Example 1 – Biosynthesis of psicose from glucose E. coli can naturally produce trace amounts of D-psicose. In this example, D-psicose production is improved by overexpressing key genes, removing competing pathway genes, and optimizing production conditions.

[0246] Evaluation of psicose production potential in Escherichia coli First, we tested whether E. coli possesses an enzyme capable of producing psicose. Psicose production was tested in the production strain AL3601, depicted in Table 1 below. Cultures were grown at 30°C in M9P medium (M9 minimal medium containing 5 g / L yeast extract) containing 10 g / L glucose, and supernatant samples were collected 24 hours after inoculation and then analyzed by HPLC. Notably, no psicose was detected in AL3601 (FIG. 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 zwf gene 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 committed 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) as part of the first committed step of glycolysis. A single KO strain containing ΔpfkA produced 0.15 g / L of psicose, demonstrating that E. coli possesses the enzymes capable of producing psicose (Figure 1). No psicose was detected in the single KO strains containing Δzwf and ΔpfkB.

[0247] (Table 1) List of strains TIFF2025530206000055.tif115128

[0248] Determination of psicose-producing enzymes Next, because deletion of pfkA increased psicose production, it was hypothesized that fructose-6-phosphate (F6P) is an intermediate for psicose production in E. coli. The production pathway begins with the native absorption of glucose into E. coli via the phosphotransferase system (PTS) or GalP / Glk, which converts glucose to glucose-6-phosphate (G6P) (Figure 2). Next, G6P is isomerized to F6P via glucose-6-phosphate isomerase (Gpi) (EC 5.3.1.9). F6P can be converted to psicose-6-phosphate by epimerase (Figure 2). Phosphatase can dephosphorylate psicose-6-phosphate to free psicose, which is then exported from the cell (Figure 2). A 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 industrial psicose production.

[0249] Literature searches and genome mining identified one epimerase and two phosphatase candidates. D-allulose-6-phosphate 3-epimerase (AlsE) showed activity toward psicose. Two phosphatases (HxpB and YbiV) showed promiscuity toward various hexoses.

[0250] Two plasmids, pAL1946 and pAL1947 (Table 2), contain the inducible promoter P T7 The plasmids were constructed to overexpress the genes alsE and either hxpB or ybiV, respectively. Each plasmid was introduced into AL3601 and tested for psicose production. Cultures were grown at 30°C in M9P medium containing 10 g / L glucose and induced with 1 mM IPTG. After 24 hours, induced AL3601 / pAL1946 produced 1.0 g / L of psicose, while induced AL3601 / pAL1947 produced 0.4 g / L, indicating that HxpB is the superior phosphatase for psicose production (Figure 3).

[0251] (Table 2) Plasmid list TIFF2025530206000056.tif62128

[0252] Increasing psicose production by eliminating competing pathways To increase carbon flux through the psicose production pathway, a triple knockout (TKO) strain (see AL3729 in Table 1) containing Δzwf, ΔpfkA, and ΔrpiB was constructed to increase the pool of F6P for psicose production. The gene rpiB encodes the allose-6-phosphate isomerase (RpiB) enzyme, which reabsorbs 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 psicose production was tested. Cultures were grown at 30°C in M9P medium supplemented with 10 g / L glucose and induced with 25 μM IPTG. Uninduced AL3729 carrying pAL1946 produced the most psicose at 1.5 g / L after 24 hours, while induced AL3729 carrying pAL1946 produced 0.2 g / L (Figure 4). Induced AL3601 carrying pAL1946 produced 0.6 g / L of psicose after 24 hours, while uninduced AL3601 carrying pAL1946 produced 0.2 g / L.

[0253] Comparison of expression systems for psicose-producing enzymes AL3601 encodes the T7 RNA polymerase P lacUV5 :T7 RNAP (Table 1). T7 RNAP appears to be a growth hurdle for the test strain. To eliminate this growth burden, P T7 A weaker IPTG-inducible promoter than P LlacO1 A new psicose-producing plasmid (see pAL2001 in Table 2) was constructed that overexpresses alsE and hxpB under the conditions of psicose production. pAL2001 was introduced into strain AL1050 (see Table 1). Strain AL1050 has the same genotype as strain AL3601, but P lacUV5: lacking T7RNAP. Cultures were grown at 30°C in M9P medium containing 10 g / L glucose and induced with 1 mM IPTG. After 24 hours, the induced pAL2001-carrying AL1050 culture produced 0.5 g / L of psicose, while the induced pAL1946-carrying AL3601 culture produced 0.5 g / L. Growth of the induced and uninduced AL1050 cultures carrying pAL2001 appeared comparable. In comparison, AL3601 carrying pAL1946 grew less well, and the induced culture reached a significantly lower culture density (Figure 5). Based on the results that pAL2001 produced comparable titers of psicose without straining growth, we hypothesized that P LlacO1 The base expression system was selected as the psicose-producing plasmid in the next experiment.

[0254] Comparison of expression systems for psicose-producing enzymes in TKO strains P LlacO1 For TKO strains AL3756 and P T7 For the pAL2001-bearing AL3756 and pAL1946-bearing AL3729, psicose production was tested (see Table 1). pAL2001-bearing AL3756 and pAL1946-bearing AL3729 were grown at 30°C in M9P medium containing 10 g / L glucose and induced with 1 mM IPTG. After 24 hours, the induced pAL2001-bearing AL3756 produced 1.4 g / L of psicose, while the uninduced culture produced 0.6 g / L of psicose. The induced pAL1946-bearing AL3729 culture produced 0.6 g / L of psicose, while the uninduced culture produced 1.8 g / L of psicose (FIG. 6).

[0255] By-product identification and reduction When samples from the psicose-producing strain were analyzed, significant peaks were consistently observed on the HPLC chromatograms that did not match those of medium components or typical metabolite standards. The retention time and mass spectrum of the unknown peak were found to match those of a mannose standard (Figure 7). To further test the hypothesis that the by-product was mannose, we knocked out manA 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 psicose production was tested (Figures 8 and 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 of mannose but 3.5 g / L of psicose, a yield of 34%, while the TKO strain produced 2.5 g / L of mannose and 2.3 g / L of psicose.

[0256] AL3990 carrying pAL2001 was cultured in M9P medium supplemented with 15 g / L of 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 of psicose, a yield of 40.3% (Figure 10).

[0257] Dynamic regulation of production Balancing carbon flux between glycolysis and production is important for maximizing both culture health and psicose production. During logarithmic growth phase, cells require more energy to grow and divide rigorously. Genes related to glycolysis should be expressed, while genes related to psicose production should be repressed. When cells enter stationary phase and are not actively dividing, carbon flux can be diverted from glycolysis to psicose production.

[0258] We used CRISPR interference (CRISPRi) to downregulate pfkB in the QKO strain. CRISPRi involves the inactivating 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 achieved by downregulating the anhydrotetracycline (aTC)-inducible promoter P tet The dCas9 plasmid pAL1952 (see Table 2) containing the gene dCas9 under the control of P LlacO1 :alsE-hxpB and P J23119 : guide (pAL2179) or P J23119 Another production / guide plasmid was constructed containing either pAL1952 or an empty guide (pAL2160). The sgRNA guide encoded by pAL2179 directs dCas9 to the promoter region of pfkB. The AL3990 strain was transformed with pAL1952 and either pAL2160 or pAL2179, and psicose production was tested. OD was measured at 37°C in 10 mL of M9P medium supplemented with 10 g / L glucose. 600 The cultures were grown until the RI reached 1.0. 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 pAL1952- and pAL2179-bearing AL3990 culture produced 1.7 g / L of psicose (yield = 46%), whereas the aTC-uninduced, IPTG-induced culture produced 2.0 g / L of psicose (yield = 42%). The aTC-induced pAL1952- and pAL2160-bearing AL3990 culture produced 0.7 g / L of psicose (yield = 8%), whereas the uninduced culture produced 0.7 g / L of psicose (yield = 8%) (Figure 11).

[0259] Next, the stationary phase promoter P gadB An inducer-free production system using P gadBP regulates the expression of the glutamic acid decarboxylase B gene and is described in the literature as being active primarily during stationary phase. gadB By using P to control the expression of the genes alsE and hxpB, carbon flux can be directed towards production during the stationary phase. LlacO1 : Plasmid pAL2001 containing alsE-hxpB, and P gadB pAL2247 (Table 2) containing alsE-hxpB was individually introduced into QKO strain AL3990 (Table 1). Cultures were grown at 30°C in M9P medium supplemented with 30 g / L glucose and treated with 1 mM IPTG (P LlacO1 The induced culture containing pAL2001 produced 6.8 g / L of psicose, a yield of 56.5%, while the induced culture containing pAL2001 produced 6.8 g / L of psicose, a yield of 56.5%. gadB The culture containing alsE-hxpB produced 8.8 g / L, a yield of 63% (Figure 12). gadB allowed for higher expression of the production enzymes and higher flux through the allulose pathway (Figure 12). Collectively, these data suggest that inducer-independent promoters are beneficial for controlling the expression of the alsE and hpxB genes and shutting off carbon flux through glycolysis.

[0260] Increasing glucose uptake using the sugar symporator GalP In E. coli, the preferred method for glucose uptake is the phosphotransferase system (PTS). The PTS is dependent on activation by phosphoenolpyruvate (PEP), a downstream product of glycolysis. By inhibiting glycolysis either through gene knockout or CRISPRi, we theorized that intracellular stocks of PEP could be depleted, leading to reduced glucose consumption. To increase glucose consumption, we used galactose:H + Extra copies of the genes galP and glk, encoding the symporter (GalP) and glucokinase (Glk), were introduced. GalP transports glucose into the cell, where it is phosphorylated by Glk to glucose-6-phosphate, which is then incorporated into central carbon metabolism. LtetO1Plasmid pAL2274 (Table 2) was constructed to express galP and glk under the promoter. pAL2274 was transfected into the QKO strain AL3990 (Table 1) and pAL2001 (P LlacO1 :alsE-hxpB) or pAL2247(P gadB :alsE-hxpB) 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 of psicose, a yield of 46%. In comparison, IPTG-induced cultures containing pAL2001 and p2274 produced 6.6 g / L of psicose at a yield of 45%, while induced cultures containing only pAL2001 produced 5.8 g / L of psicose at a yield of 46% (Figure 13). Overexpression of GalP-Glk, when combined with the production plasmid pAL2247, appears to complement glucose import.

[0261] (Table 3) TIFF2025530206000057.tif38158TIFF2025530206000058.tif229158TIFF2025530206000059.tif229158TIFF2025530206000060.tif231158TIFF2025530206000061.tif229158TIFF2025530206000062.tif229158TIFF2025530206000063.tif231158TIFF2025530206000064.tif229158TIFF2025530206000065.tif229158TIFF2025530206000066.tif229158TIFF2025530206000067.tif229158TIFF2025530206000068.tif229158TIFF2025530206000069.tif229158TIFF2025530206000070.tif229158TIFF2025530206000071.tif229158TIFF2025530206000072.tif229158TIFF2025530206000073.tif229158TIFF2025530206000074.tif232158TIFF2025530206000075.tif229158TIFF2025530206000076.tif229158TIFF2025530206000077.tif229158TIFF2025530206000078.tif229158TIFF2025530206000079.tif225158TIFF2025530206000080.tif222158TIFF2025530206000081.tif231158TIFF2025530206000082.tif229158TIFF2025530206000083.tif229158TIFF2025530206000084.tif157158

[0262] Example 2 – Biosynthesis of psicose from glucose A devastating increase in sedentary lifestyles and access to high-calorie foods tripled global obesity rates between 1975 and 2016 (Bluher, Nat. Rev. Endocrinol. 2019 155 15, 288-298 (2019)). Nearly 40% of adults worldwide are considered overweight, a major risk factor associated with cardiovascular disease, diabetes, musculoskeletal disorders, and some cancers. In response to this public health crisis, an increasing number of people are seeking to treat or prevent disease by adopting healthier, lower-calorie diets. The food industry can play a role in helping people make better choices by replacing sucrose and high-fructose corn syrup with zero- or low-calorie sugar substitutes. As a result, the sugar substitute market is expected to reach US$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 to 2025. (2020)).

[0263] Rare sugars are monosaccharides rarely found in nature and have slightly different structures from common sugars such as glucose and fructose. Many rare sugars lack nutritional value and offer potential health benefits, making them attractive targets for sugar replacement. The GRAS, zero-calorie rare sugar D-psicose is the 3'-epimer of fructose and is 70% as sweet as sucrose. Marketed to consumers as "allulose," D-psicose possesses desirable browning, hygroscopicity, and solubility properties, as well as a desirable flavor profile. Furthermore, studies have linked D-psicose intake with antihyperglycemic, antihyperlipidemic, antiparasitic, and antioxidant health benefits. As with many rare sugars, a limitation in D-psicose research and industrial adoption is the lack of an economical, large-scale production system. D-psicose is found naturally in some fruits and grains, but only in such trace concentrations that it cannot be extracted (Oshima et al., Food Sci. Technol. Res. 12, 137-143 (2006)).

[0264] Although synthetic methods for producing D-psicose have been proposed, these methods suffer from poor stereoselectivity, yield, and purification (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 become 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 enhance production have focused on engineering these enzymes to improve catalytic efficiency, thermostability, 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 DPEase and DTEase, the method inherently suffers from yield limitations due to a lack of thermodynamic driving force and therefore has not achieved conversion yields greater than 50%. The epimerization of D-fructose to D-psicose is reversible, with a ΔG° of +5 kJ mol -1 This is predicted to be thermodynamically unfavorable, making it likely that D-fructose will be favored at equilibrium (Figure 14A). Low conversions result in a mixed solution of D-fructose and D-psicose, making isolation and purification a significant challenge. Methods that have achieved conversion rates greater than 50% have relied on the inclusion of toxic or expensive coenzymes.

[0265] To overcome this barrier, this example proposes that phosphorylation and dephosphorylation provide a thermodynamic incentive and driving force for D-psicose production (FIG. 14B). The predicted ΔG' at a typical physiological concentration of 1 mM m -31.1 kJ mol-1 Because of the ubiquitous ATPase activity, dephosphorylation of D-psicose-6-phosphate (P6P) to D-psicose is a highly favorable reaction. The enzymatic machinery and coenzymes required for sugar phosphorylation / dephosphorylation are readily available within living cells. Furthermore, most organisms, including the model organism Escherichia coli, utilize sugar phosphorylation / dephosphorylation as part of their sugar consumption and central carbon metabolism. In E. coli, carbon metabolism begins with the phosphotransferase system (PTS), where D-glucose is simultaneously phosphorylated and transported across the plasma membrane. Alternatively, glucose can be transported across the plasma membrane by the galactose proton symporter GalP and then phosphorylated by glucokinase Glk13,14. D-glucose-6-phosphate (G6P) can then be isomerized to D-fructose-6-phosphate (F6P), which can then be used in glycolysis. It is theorized herein that a portion of F6P is diverted from glycolysis and epimerized to P6P, which can then be dephosphorylated to D-psicose and exported from the cell.

[0266] In this example, it was discovered that E. coli naturally possesses enzymes capable of completing the D-psicose biosynthetic production pathway proposed above ( FIG. 14B ). Epimerization of F6P to P6P can be achieved using D-allulose-6-phosphate 3-epimerase (AlsE), and dephosphorylation of P6P can be achieved using the phosphatase hexitol-phosphatase B (HxpB). By utilizing native E. coli genes, this example successfully produced D-psicose from D-glucose in a yield of over 50% without the need for expression of heterologous enzymes.

[0267] The D-psicose production capacity of E. coli was improved by additionally expressing alsE and hxpB and eliminating or modulating competing metabolic pathways, including the pentose phosphate pathway (PPP), glycogen biosynthesis, glycolysis, the D-allosolysis pathway, and the D-mannose degradation pathway. D-glucose import was complemented by additionally expressing the native galactose proton symporter gene galP and the glucokinase gene glk. To further increase production while maintaining cell viability, multiple strategies for dynamic gene regulation and carbon flux allocation were explored. During the growth phase, cells require more energy for active growth, and glycolysis-related genes must be expressed. When cells enter stationary phase and are not actively growing, carbon flux can be diverted from glycolysis to D-psicose production. To balance carbon partitioning depending on the growth phase, two strategies for dynamically regulating the expression of key metabolic and D-psicose-producing genes were explored: inducer-free stationary-phase promoters and clustered regularly interspaced short palindromic repeats interference (CRISPRi).

[0268] Design of a thermodynamically favorable pathway for D-psicose production Currently, the primary method for producing D-psicose involves the 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 (FIG. 14A). To overcome the thermodynamic limitations of this method, this example proposes a pathway driven by sugar phosphorylation and dephosphorylation, a process that requires readily available coenzymes and enzymes in vivo (FIGS. 14B and 14C). Therefore, the model organism Escherichia coli was chosen as the host for the D-psicose biosynthetic pathway.

[0269] The proposed pathway begins with the absorption of D-glucose into E. coli via a PTS, which converts it to G6P (Figure 14C). Alternatively, D-glucose can be absorbed by the galactose proton symporter GalP, which is subsequently phosphorylated to G6P by glucokinase Glk (Figure 14C). G6P is then isomerized to F6P via glucose 6-phosphate isomerase (Gpi). Here, the proposed pathway diverges from native carbon metabolism. Many enzymes are promiscuous and utilize a variety of substrates. Therefore, it was theorized that, given the appropriate conditions and modifications to sugar metabolism, E. coli may naturally possess enzymes capable of producing D-psicose. F6P is epimerized to P6P, which could then be dephosphorylated to D-psicose in a final, thermodynamically favorable step (Figure 14C). It was theorized that placing this favored reaction at the end of the biosynthetic pathway would restore equilibrium, thereby promoting flux in the production pathway. Because D-psicose 6-phosphate is favorably dephosphorylated to D-psicose and exported from the cell, the equilibrium is restored by making more P6P from F6P.

[0270] Evaluation of Escherichia coli's intrinsic D-psicose production ability To evaluate the intrinsic ability of E. coli to produce D-psicose, production was tested in M9P medium using E. coli MG1655 and the MG1655-derived strain AL3601, which harbors a gene encoding T7 RNAP (Table 5, Methods). Without genetic manipulation, neither strain produced detectable levels of D-psicose (FIG. 15A).

[0271] It was hypothesized that carbon flux must be intentionally directed toward D-psicose production by the accumulation of the upstream metabolite F6P. Because F6P is selectively converted to D-fructose 1,6-bisphosphate by phosphofructokinases A and B (PfkA and PfkB, Figure 14C), the major metabolic pathway competing with F6P is glycolysis. To build up the intracellular F6P pool, the gene encoding PfkA, which accounts for approximately 90% of the phosphofructokinase activity, was deleted in MG1655 and AL3601 to generate strains AL4058 and AL3694, respectively (Table 5). The ΔpfkA strain produced 0.24 g L−1 in AL4058. -1 and 0.15 g L in AL3694 -1 D-psicose was produced, suggesting that E. coli contains the enzymes necessary to produce D-psicose, most likely from F6P (FIG. 15A). No D-psicose production was detected in cultures grown in glucose-free M9P medium.

[0272] Elucidation of the enzymes involved in D-psicose production D-allulose 6-phosphate 3-epimerase (AlsE) was identified as a potential candidate for the conversion of F6P to P6P. AlsE absorbs D-psicose into central carbon metabolism by converting P6P to F6P. Under high concentrations of F6P, AlsE exhibits the reverse activity, converting F6P to P6P. To confirm whether AlsE is involved in D-psicose production, we deleted alsE in AL3694 and AL4058, resulting in strains AL4063 and AL4082 (Table 5). Neither strain produced detectable levels of D-psicose, suggesting that AlsE is the epimerase responsible for D-psicose production (Figure 15A).

[0273] E. coli possesses several phosphatase enzymes that may be active against P6P. Candidate phosphatases were selected based on their broad activity toward various sugar substrates, and the following were tested: 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. To test the activity of each phosphatase for the conversion of P6P to D-psicose, the genes for each phosphatase were individually expressed from PT721 along with alsE on an expression plasmid (Table 6). This plasmid was then introduced into AL3601 (pfkA+). After 24 hours, the phosphatases were transformed with pAL1946 (P T7 The strain carrying alsE-hxpB was 0.55 g L -1 The highest D-psicose production was observed in pAL1947(P), suggesting that HxpB is a good candidate phosphatase for D-psicose production (Fig. 15B). T7 :alsE-ybiV) or pAL2351(P T7 :alsE-yidA) strains yielded 0.21 g L -1 and 0.20 g L -1 However, pAL2348(P T7 :alsE-hxpA), pAL2352(P T7 :alsE-yihX) and pAL2349(P T7 :alsE-yigL), no detectable D-psicose was produced (FIG. 15B).

[0274] Identification of a critical P6P-binding motif Using a combination of AlphaFold and Rosetta Molecular Suite, we evaluated the predicted binding modes between each of the six phosphatases and P6P. Phosphatases active against P6P (HxpB, YbiV, and YidA) were predicted to form at least two internal hydrogen bonds and one additional hydrogen bond with the terminal hydroxyl group of P6P (Figures 26A and 26B). Conversely, phosphatases inactive against P6P (HxpA, YihX, and YigL) were predicted not to form a hydrogen bond with the terminal hydroxyl group of P6P. The phosphatase HxpB, which yielded the highest D-psicose titer, was predicted to form hydrogen bonds between active site residues and four hydroxyl groups of P6P (Figure 26B). These predictions indicate that a minimum of three hydrogen bonds, including the hydrogen bond to the terminal hydroxyl group, are essential for binding P6P in a catalytically compatible orientation.

[0275] Comparison of expression systems for D-psicose production In AL3601, we utilized a T7 RNA polymerase expression system containing the T7 RNA polymerase (RNAP) gene under the IPTG-inducible PlacUV5 promoter. However, we observed that growth was reduced when IPTG was added to induce T7 RNAP expression (Figure 15C). Therefore, we tested an alternative expression system in which the alsE and hxpB operons are expressed from the IPTG-inducible promoter PLlacO1. Under inducible conditions (with IPTG), P LlacO1 The strain carrying alsE-hxpB is P T7 :alsE-hxpB strain produced D-psicose similarly to the strain with alsE-hxpB (FIG. 15C). LlacO1 In strains with alsE-hxpB, P T7 Compared to the strain with alsE-hxpB, better suppression was obtained under non-inducing (no IPTG) conditions and no growth burden was imposed under inducing conditions (Figure 15C). LlacO1 The expression system was used for further studies.

[0276] Increasing D-psicose production by eliminating competing pathways E. coli relies on two major glycolytic pathways to metabolize glucose: the PPP and glycolysis (also known as the Embdem Meyerhof-Parnas (EMP) pathway). The 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). In this example, we demonstrated that deletion of pfkA leads to improved D-psicose production compared to our unmodified base strain (Figure 15A).

[0277] In addition to the PPP and glycolysis, the allolytic pathway has the ability to divert carbon flux from D-psicose production by resorbing P6P into central carbon metabolism. The rpiB gene encodes allose-6-phosphate isomerase (RpiB), which can potentially convert P6P into aldehyde-D-allose 6-phosphate (Figure 14C).

[0278] To redirect carbon flux from central carbon metabolism to D-psicose production, gene knockouts ΔpfkA, Δzwf, and ΔrpiB were constructed in AL1050, resulting in strain 1 (Table 4). These three deletions resulted in D-psicose production of 2.31 g L−1. -1 This resulted in a four-fold increase in D-psicose production (FIG. 15D).

[0279] Table 4: List of major strains used in this example TIFF2025530206000085.tif77158 All strains and plasmids used in this study are listed in Tables 5 and 6, respectively.

[0280] Table 5: Strains used in this example TIFF2025530206000086.tif124158

[0281] Table 6: Plasmids used in this example TIFF2025530206000087.tif80156TIFF2025530206000088.tif218156 * See Figures 21A and 21B.

[0282] By knocking out pfkA, zwf, and rpiB, carbon flux was directed toward the D-psicose production pathway. In particular, knocking out pfkA and zwf should lead to increased intracellular F6P availability. These results reaffirmed our suspicion that F6P plays an important role as a precursor of P6P and that F6P accumulation is necessary to drive carbon flux through the production pathway.

[0283] Identification of by-products When a sample of strain 1 was analyzed, a significant peak was observed in the high-performance liquid chromatography (HPLC) chromatogram that did not match the D-glucose, D-fructose, or D-psicose standards or any of the medium components. Using gas chromatography-mass spectrometry (GC-MS), the retention time and mass spectrum of the unknown peak were found to match those of D-mannose (Figure 19).

[0284] Under standard conditions, the D-mannose pathway involves the incorporation of D-mannose 6-phosphate (M6P) into central carbon metabolism by the reversible isomerization of M6P to F6P via mannose-6-phosphate isomerase (ManA). Accumulation of F6P can reverse this reaction, resulting in the production of M6P and D-mannose. To test the hypothesis that this by-product is D-mannose, manA was deleted in strain 1, resulting in strain 2 (Table 4).

[0285] Deletion of manA resulted in a significant decrease in D-mannose production, with strain 2 in the presence of IPTG yielding 2.49 g L -1 compared to strain 1 with IPTG, which produced 0.69 g L -1 The strains produced only 1000 kJ of D-mannose (Figure 19). Complementing the reduced D-mannose production, D-psicose production in strain 2 was 1.5-fold higher than in strain 1 (Figure 15D). Although not completely eliminated, unwanted production of D-mannose was significantly reduced by the deletion of manA. Therefore, subsequent production was performed in strains containing ΔpfkA, Δzwf, ΔrpiB, and ΔmanA.

[0286] Although the D-mannose pathway was eliminated, there may be other D-fructose epimer pathways that continue to compete for carbon flux. Removing the PPP and limiting glycolysis significantly increases the intracellular F6P pool, and if it is not efficiently diverted to D-psicose biosynthesis, other epimerases or isomerases may act. In short, increased F6P availability may allow enzymes that are not normally observed to engage with the substrate and generate other sugar products.

[0287] Use of stationary phase promoters Building production pathways in microorganisms requires careful allocation of carbon between essential metabolic processes and production, especially when dealing with central carbon metabolism. The system disclosed herein can help maximize both cell viability and D-psicose production by dynamically balancing carbon flux between glycolysis and the D-psicose pathway.

[0288] The life cycle of an E. coli culture includes five distinct phases: lag phase, logarithmic growth phase, stationary phase, death phase, and long-term stationary phase. The lag phase occurs when cells are inoculated into the medium and adjust their metabolic processes in response to their new environment. Cells then rapidly grow and divide, entering the logarithmic growth phase. At this time, enzymes related to central carbon metabolism become most important, and transcription of the corresponding genes is upregulated. When cells sense environmental stressors, such as a lack of medium nutrients, their growth and division slows, and the culture enters the stationary phase. Here, culture density plateaus, and genes related to stress response are expressed. Transcription of these genes is regulated in part by the σ38 subunit of RNA polymerase, which recognizes the promoter regions of genes.

[0289] The carbon flux was balanced by placing the D-psicose production genes alsE and hxpB downstream of a stationary-phase active promoter, utilizing the native gene regulatory system of E. coli. This prevented the production pathway from competing with central carbon metabolism for carbon flux during logarithmic growth phase, the period when cells require carbon for rigorous growth and division.

[0290] Four promoters previously shown to be active during stationary phase, P gadB , P cbpA2 , P ihfA4 and P dps The promoters were selected for testing using green fluorescent protein (GFP) as a reporter. Each promoter was cloned upstream of sfgfp on an expression plasmid (Table 6). The strongest promoter, P gadB In addition to expression times that correlate with late logarithmic or early stationary phase, the IPTG-inducible promoter P LlacO1 The second most potent promoter, P cbpA2 is the induced P LlacO1 The timing of expression closely followed that of P, but the intensity of expression was approximately two-thirds that of P (Fig. 20B). ihfA4 and P dps MoP LlacO1The expression timing of P cbpA2 was lower (Fig. 20C).

[0291] Because it is strongly expressed during stationary phase, P gadB The alsE and hxpB operons were expressed using pAL2247 (Table 6). To evaluate the effect of initial glucose concentration, D-psicose production was measured at 10, 20, and 40 g L -1 Glucose concentrations (10, 20 and 40 g L -1 ) below, strain 3 (P gadB alsE-hxpB, Table 4) was isolated from strain 2 (P LlacO1 The D-psicose titer was consistently higher than that of the psicose-containing ... 600 ) represents the difference in ΔOD 600 The highest D-psicose titer was 40 g L -1 This resulted in strain 3 producing 6.92 g L -1 It is now possible to produce D-psicose of ΔOD 600 5.2, whereas strain 2 grew at 4.55 g L -1 and ΔOD 600 The strain was grown at an initial glucose concentration of 40 g L for further studies. -1 was used.

[0292] Next, the effect of the timing of the shift from 37°C to 30°C on D-psicose was examined. Preliminary tests showed that 37°C was suitable for cell growth, while 30°C was suitable for production. To determine the optimal timing for the shift from 37°C to 30°C, the culture was incubated at 37°C for 1 hour at OD . 600 OD was grown to approximately 0 (no incubation at 37°C), approximately 0.4, or approximately 1, and then grown at 30°C and induced as needed. All strains showed a slower OD 600 Cultures shifted to 30°C at 10°C produced higher titers of D-psicose and had larger ΔOD 600 The OD 600When transferred at approximately 1, strain 3 produced 9.13 g L -1 The highest titer of D-psicose was produced, and ΔOD 600 Strain 2 grew at 5.42 g L -1 of D-psicose was produced, and ΔOD 600 It multiplied at 4.4.

[0293] P LlacO1 Although it is a stronger promoter than P gadB It was theorized that the timing of expression of P prevents the D-psicose-producing enzyme from siphoning carbon from central metabolism during the critical growth period. This allows the culture to grow more robustly and produce higher titers of D-psicose. The use of an endogenous growth-phase-associated promoter eliminates the need for expensive chemical inducers and allows the culture to self-regulate pathway expression in response to growth and cell viability. gadB :alsE-hxpB (pAL2247, Table 6) was then used in further production experiments.

[0294] Complementation of glucose import using GalP and Glk Sustained glucose import, especially during the stationary phase of growth, is beneficial for D-psicose production. One consequence of limiting carbon flux through glycolysis by knocking out pfkA is a reduction in downstream metabolites, such as phosphoenolpyruvate (PEP). PEP is of particular concern because it is utilized by the PTS to uptake and phosphorylate glucose. Reduced PEP availability due to reduced glycolytic flux could affect the ability to absorb glucose and produce D-psicose. Furthermore, increased G6P or F6P pools in the ΔpfkA mutant have been shown to lead to degradation of the ptsG mRNA, which encodes the membrane receptor IICBGlc for the PTS complex.

[0295] To enhance glucose uptake without using the PTS in the ΔpfkA background, galP and glk were additionally expressed from a plasmid. The galP gene encodes the galactose proton symporter GalP, which can import glucose. The glk gene encodes the glucokinase Glk, which phosphorylates glucose to G6P (Figure 14C). These genes were cloned downstream of PLlacO1 to generate the plasmid pAL2264 (Table 6). Strain 4 (P LlacO1 Induction of galP-glk expression in strain 3 (Table 4) containing galP-glk significantly increased D-psicose production, with the strain achieving a specific titer of 3.2 g L -1 OD 600 -1 and 13.81 g L at a yield of 55%. -1 (Figure 16C). In comparison, strain 4 without IPTG produced 8.67 g L -1 of D-psicose with a specific titer of 1.3 g L -1 OD 600 -1 The yield was 49%. The yield was calculated based on the possibility of producing 1 mol of D-psicose per 1 mol of D-glucose consumed, with a theoretical maximum yield of 100%. Interestingly, the expression of galP and glk significantly increased the ΔOD 600 The overexpression of membrane proteins such as GalP has been shown to impose a burden on cell growth due to competition for membrane transport machinery.

[0296] Manipulation of glucose utilization and metabolism It has been shown that the PTS utilizes approximately 50% of PEP for transporting and phosphorylating glucose. To further balance the intracellular PEP supply and promote glucose import using GalP-Glk, we attempted to disable the PTS in AL3990 by knocking out the gene ptsG, which encodes the membrane receptor IICBGlc, and the gene ptsH, which encodes the phosphorylation carrier protein HPr (Table 5).

[0297] Another source of glucose from the D-psicose pathway is glycogen biosynthesis. Glycogen is stored for use during starvation, but is not required for the subject matter disclosed herein. Deletion of pgm, which encodes phosphoglucomutase Pgm, renders E. coli unable to produce glycogen. Therefore, pgm was deleted in the production strain (Table 5).

[0298] Strain 4 with ΔptsG appeared to be detrimental to psicose production (ΔptsG, ΔptsG ΔptsH, and ΔptsG Δpgm), whereas strain 4 with ΔptsH did not affect psicose production ( FIG. 16D ). Δpgm was beneficial for psicose production, especially at specific titers ( FIG. 16D ). Strain 5 (strain 4 with Δpgm, Table 4) produced 14.66 g L -1 of D-psicose with a specific titer of 5.5 g L-1 OD 600 -1 and the yield was 58% (Figure 16D).

[0299] Inhibiting glycogen biosynthesis by knocking out pgm deprives central metabolism of carbon sources that would normally be used to support cell growth during periods of starvation. The combined effects of growth inhibition and enhanced production result in an increase in specific titer from strain 4 to strain 5 (Figure 16D).

[0300] While some studies have shown that removing the PTS by gene knockout can help restore the intracellular PEP balance and restore growth, we found that knocking out either ptsG or ptsH was either detrimental or neutral to D-psicose production. The phosphorylated and dephosphorylated forms of IICBGlc and HPr are involved in signaling cascades linked to global gene expression, not only through carbon metabolism but also through the expression of RNA polymerase sigma subunits, including the aforementioned σ38 subunit and the log-phase-associated σ70 subunit. Given that PgadB is used to express alsE and hxpB, removing part of the PTS may reduce the expression of D-psicose production pathway genes.

[0301] Dynamic regulation of glycolysis using CRISPRi Deletion of pfkA successfully redirected carbon flux toward D-psicose production, while glycolysis remained active through PfkB. Because completely shutting down glycolysis by deleting pfkB did not allow cells to grow under culture conditions, we attempted to dynamically limit pfkB expression only when necessary. Cells require glycolytic carbon flux to build biomass during logarithmic growth phase. During stationary phase, glycolytic carbon flux can be reduced and redirected toward D-psicose production. To dynamically regulate pfkB expression, we implemented a CRISPRi system targeting pfkB on the genome.

[0302] The CRISPRi system utilizes dCas9, an inactivating Cas9 gene, which, when recruited by a single guide RNA scaffold (sgRNA), can precisely target and block transcription initiation by RNA polymerase. The dcas9 gene is then transduced under the aTc-inducible promoter P. tet A constitutively expressed sgRNA sequence targeting the gene of interest was cloned under the P. To confirm the functionality of the CRISPRi system and determine where the sgRNA should be targeted to achieve maximal inhibition of expression, LlacO1Three different sgRNA sequences were designed to suppress the expression of sfGFP under the P (Figure 17A). LlacO1 The upstream, middle, and downstream sequences of P were targeted (Figure 17A). LlacO1 We found that sgRNAs targeting the midstream of pfkB produced the greatest difference in fluorescence. Sequential sgRNAs targeting the pfkB promoter region were designed with homology to the midstream of the promoter sequence.

[0303] Further exploration of the CRISPRi system involved expressing dcas9 and sgRNA from the same or separate plasmids (Figures 22A and 22B). tet Constitutively expressed sgRNA sequences targeting the promoter regions of dcas9 and pfkB, or those without targeting sequences, were cloned onto the same plasmid (Table 6). tet dcas9 was cloned on one plasmid, and a constitutively expressed sgRNA sequence targeting the promoter region of pfkB or one without a targeting sequence was cloned on a separate plasmid (Table 6). Each CRISPRi system was introduced into AL4186 (Table 5) at 100 ng mL -1 Growth was measured 24 hours after induction with aTc. The separate plasmid system most likely caused greater growth inhibition because the sgRNA was expressed from a high-copy-number plasmid rather than a low-copy-number plasmid as in the single-plasmid system. When production was tested using the separate plasmid CRISPRi system in strain AL3990 harboring pAL2247 (Tables 5 and 6), very little D-psicose was produced, regardless of aTc induction. Therefore, the single-plasmid CRISPRi system was used for production.

[0304] The single-plasmid CRISPRi system was introduced into strain 5 to generate strain 6 (empty guide) and strain 7 (sgRNA targeting pfkB, Table 4). CRISPRi (strain 7) reduced cell growth and significantly improved specific titers with or without aTC, although the titers of strain 7 were lower than those of strains 5 and 6 (Figure 17A). 100 ng mL -1 Strain 7 with aTc was 11.40 g L -1 of D-psicose with a specific titer of 3.6 g L -1 OD 600 -1 and a yield of 62%. Strain 7 without aTc produced 13.65 g L -1 of allulose with a specific titer of 3.8 g L -1 OD 600 -1 and the yield was 60%.

[0305] D-Psicose production under high culture density conditions To investigate the D-glucose consumption rate and D-psicose production rate of strain 7 (Table 4), 3, 5, and 10 g L -1 The substrate concentration was monitored for 10 hours at a medium glucose concentration of 3 g L (Figures 23A-23C). It was found that the D-psicose production rate was similar throughout the culture, regardless of the medium glucose concentration. -1 The culture fed 1000 ml of glucose consumed all of the glucose during the experiment, suggesting that D-psicose has the advantage of being easier to extract and purify from the medium in an industrial environment.

[0306] To decouple growth and production and minimize production limitations due to glucose availability, strain 7 was grown for shorter periods under high cell density conditions where available D-glucose was in excess (Figure 17B). Strain 7 (Table 4) was grown at OD 600 After growth to approximately 100 ng mL -1 The cells were induced with aTc and 1 mM IPTG and grown for an additional 30 min, after which they were pelleted and cultured at 40 g L -1 Glucose, 100 ng mL -1OD at 100 s in M9P containing aTc and 1 mM IPTG 600 The culture was resuspended to approximately 10°C. Samples were taken and analyzed at 0, 4, and 8 hours. Over 8 hours, strain 7 produced 15.3 g L−1 of spores. -1 of D-psicose was produced with a specific titer of 1.4 g L -1 OD 600 -1 , yield 43% and productivity 1.9 g L -1 hr -1 The titers of D-psicose produced at 0 to 4 hours and 4 to 8 hours were similar, but were 7.3 and 8.0 g L−1 (FIG. 17B). -1 In the production system disclosed herein, the yield at 4 to 8 hours (53%) was higher than that at 0 to 4 hours (35%). gadB Because the system relies on the activation of ATP, the high yield may be a result of the cells responding to high-density culture conditions. Overall, this production system produced comparable high yields of bioethanol (>0.5 g ethanol) despite being cultured under non-optimized test tube conditions. 産物 g 基質 -1 ) and high productivity (>1 g L -1 hr -1 ) and achieved industrially suitable production.

[0307] conclusion In this study, we applied whole-cell catalysis as a strategy for producing the rare sugar D-psicose in an industrially relevant manner. Living cells possess the ability to assemble stereo- and regioselective enzymes, provide the necessary cofactors, and secrete an easily purified product, all under environmentally friendly production conditions. Whole-cell catalysis technology and infrastructure are already established industrially, and the model organism Escherichia coli can provide a feed ingredient that does not compete with commercial food production.

[0308] The use of static and dynamic gene regulation strategies, along with the elimination of competing pathways and the additional expression of native E. coli genes (alsE, hxpB, galP, and glk), resulted in a strain capable of producing D-psicose from readily available raw materials using a thermodynamically favorable biosynthetic pathway. Under test tube conditions, the highest titer of D-psicose produced was 16.59 g L−1. -1 and the specific titer is 5.0 g L -1 OD 600 -1 The highest yield achieved was 62%, exceeding current industry standards. Furthermore, the strain's ability to consume all D-glucose present in the medium greatly simplifies downstream purification requirements. Overall, this engineered strain represents a valuable step toward cost-effective production of D-psicose, providing the food industry with a viable source for creating low-glycemic index products desired by consumers.

[0309] Overall, this genetically engineered strain represents an important step in the efficient, cost-effective production of D-psicose and other rare sugars. The ability to produce rare sugars in large quantities will help address rising global obesity rates by providing low-calorie sugar alternatives to ultra-processed foods. Increased production of rare sugars will also provide the agricultural industry with access to sustainable pesticides and the pharmaceutical industry with access to pharmaceutically appropriate monosaccharides. The strategies developed in this study have the potential to drive a radical shift in our ability to generate, measure, and control the human-food relationship, fostering a world in which easily attainable metabolic health leads to happier, healthier, and longer lives for all.

[0310] method reagent All enzymes involved in molecular cloning experiments were purchased from New England Biolabs (NEB). All synthetic oligonucleotides were synthesized by Integrated DNA Technologies. Sanger sequencing was provided by Genewiz, a company from Azenta Life Sciences. D-psicose and D-mannose were purchased from Sigma-Aldrich. D-glucose was purchased from Fisher Scientific.

[0311] 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 for D-psicose production were constructed using sequence- and ligation-independent cloning (SLIC). The constructed plasmids were verified by Sanger sequencing. The construction guide for the plasmids used in this study is detailed in Table 8.

[0312] Table 7: Oligonucleotides used in this example TIFF2025530206000089.tif90156TIFF2025530206000090.tif223156TIFF2025530206000091.tif223156TIFF20255302060 00092.tif223156TIFF2025530206000093.tif229156TIFF2025530206000094.tif228156TIFF2025530206000095.tif249156

[0313] Table 8. Plasmid construction guide TIFF2025530206000096.tif252156 * Q5-Site-directed mutagenesis (NEB)

[0314] Genome modifications, such as gene deletions and insertions, were constructed using CRISPR-Cas9-mediated homologous recombination. Linear DNA repair fragments for gene deletions and insertions were constructed by amplifying genomic or plasmid DNA via PCR assembly. Plasmids encoding sgRNAs for CRISPR-Cas9-mediated homologous recombination were constructed using Q5 site-directed mutagenesis (New England Biolabs) with the pTargetF plasmid (Addgene #62226) as a template. All genome modifications were verified by Sanger sequencing. The guide for CRISPR-Cas9-mediated gene modifications used in this study is detailed in Table 9.

[0315] Table 9. Guide to CRISPR-Cas9-mediated gene deletion and insertion TIFF2025530206000097.tif184159

[0316] Culture medium Overnight cultures were grown at 37°C in 3 mL of Luria-Bertani (LB) medium. 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 mix (2.86 mg H3BO3, 1.81 mg MnCl2 4H2O, 0.079 mg CuSO4 5H2O, 49.4 μg Co(NO3)2 6H2O), various concentrations of glucose, and appropriate antibiotics. M9P medium for psicose production is prepared using 5 g L -1The medium consisted of M9 minimal medium supplemented with yeast extract and appropriate antibiotics. When cultures were grown in M9P medium without glucose, D-psicose production was not 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 was measured.

[0317] Fluorescence assay The overnight culture was inoculated into 300 μL of LB medium in a 96-well black-walled fluorescent assay plate at 1% OD. 600 Cells were grown at 37°C and 250 rpm to an OD of approximately 0.4. Cultures were then induced with IPTG as needed and grown at 37°C and 250 rpm for 24 hours. 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.

[0318] D-psicose production For normal cell density production experiments, overnight cultures were inoculated into 3 mL of M9P medium at 1% OD. 600 Cells were grown at 37°C until an OD of approximately 1 was reached, then induced with IPTG and aTc as needed and grown at 30°C for 24 hours. For high cell density production experiments in M9P medium, overnight cultures were inoculated into 50 mL of M9P medium at 2%. Cells were grown at 37°C until an OD of approximately 1 was reached. Cultures were then induced with IPTG and aTc as needed and grown for an additional 30 minutes. Cultures were centrifuged at 5,000 g for 15 minutes, resuspended in M9P medium with IPTG and aTc as needed, and grown to the target OD of approximately 1. 600 The cultures were grown at 30°C for 24 hours.

[0319] HPLC analysis Analysis of D-psicose, glucose, and mannose concentrations was performed using a Shimadzu high-performance liquid chromatography (HPLC) system equipped with a refractive index detector (RID) 10 A and a Rezex™ RCU-USP sugar alcohol column (Phenomenex). The mobile phase consisted of 100% MilliQ water. The column oven temperature was 83°C and the RID cell temperature was 40°C for 7 minutes at a flow rate of 0.5 mL min. -1 Samples were run at 1 μL injection volume. To prepare samples for HPLC analysis, 300 μL of culture was centrifuged at 17,000 g for 5 minutes. The supernatant was applied 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.

[0320] 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 GC-MS analysis, 4 μL of the spun-down culture supernatant was dried and dissolved in 40 mg mL of pyridine (Sigma-Aldrich). -1Derivatization was performed by adding 10 μL of methoxyamine hydrochloride (Sigma-Aldrich) and shaking at 30 °C for 1.5 hours. Subsequently, 90 μL of N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) (Sigma-Aldrich) was added along with 13 fatty acid methyl esters (FAMEs) as retention indicator markers, and the mixture was shaken at 80 °C for 30 minutes. The samples were immediately transferred to crimp-top vials and injected into each GC-MS instrument. A LECO Pegasus IV TOF MS was connected to an Agilent 7890 GC system equipped with a Restek RTX-5Sil MS column (29.70 m long, 0.25 mm id, 0.25 μM df, 95% dimethyl / 5% diphenylpolysiloxane film) with an additional 10 m guard column. 1 μL of the derivatized sample was injected at an injection temperature of 275 °C and a constant flow rate of 1 mL min. -1 The sample was injected into the GC in splitless mode. The initial oven temperature was held at 50°C for 1 minute, then at 20°C for 1 minute. -1 The temperature was ramped to 330 °C at a rate of 100 Hz and held for 5 min, resulting in a total run time of 20 min. The mass spectrometer was operated in electron ionization mode at +70 eV. Mass spectra were acquired from 85–500 m / z at a scan rate of 17 Hz and a source temperature of 250 °C. Binbase was used for metabolite annotation and reporting.

[0321] Example 3 - Biosynthesis of psicose from glucose under high-density conditions An additional source of glucose from the D-psicose pathway comes from glycogen biosynthesis, which produces glycogen for use during starvation. Deletion of pgm, encoding the phosphoglucomutase Pgm, renders E. coli unable to produce glycogen (Eydallin, G. et al. Genome-wide screening of genes affecting glycogen metabolism in Escherichia coli K-12. FEBS Lett. 581, 2947-2953 (2007)). Therefore, we deleted pgm in the glycogen-producing strain AL3990 to generate AL4186 (MG1655 ΔpfkA Δzwf ΔrpiB ΔmanA Δpgm).

[0322] The D-glucose consumption rate and D-psicose production rate of strain 7 (see Table 4) were measured at medium glucose concentrations of 3, 5, and 10 g L−1. -1 The D-glucose consumption rate and D-psicose production rate were found to be similar in all cultures, regardless of the medium glucose concentration. -1 Cultures fed 1000 ml of glucose consumed all of the glucose for the duration of the experiment.

[0323] To decouple growth and production and minimize production limitations due to glucose availability, we transformed the α-glucose-containing β-glucose (α-glucose)-containing β ... gadB :alsE-hxpB), pAL2264(P LlacO1 :galP-glk) and pAL2188(P tet Strain AL4186 (also identified as strain 7 in Table 4) transformed with dcas9 (pTargetF-pfkB) was grown at OD 600 Approximately 1 to 40 g L -1 After growth at 37°C in M9P medium containing glucose, 100 ng mL -1 The cells were induced with aTc and 1 mM IPTG and grown for an additional 30 min, after which they were pelleted and cultured at 40 g L -1Glucose, 100 ng mL -1 The cells were resuspended in M9P containing aTc and 1 mM IPTG to an OD600 of approximately 10. Samples were taken and analyzed at 0, 4, and 8 hours.

[0324] Over 8 hours, the culture yielded 15.3 g L -1 of D-psicose was produced with a specific titer of 1.4 g L -1 OD 600 -1 , yield 43% and productivity 1.9 g L -1 hr -1 See Figure 24. The titers of D-psicose produced at 0 to 4 hours and 4 to 8 hours were similar, but were 7.3 and 8.0 g L−1. -1 The yield at 4 to 8 hours (53%) was higher than that at 0 to 4 hours (35%). The productivity at 4 to 8 hours was 2.0 g L−1. -1 h -1 Because the production system disclosed herein relies on activation of the stationary phase promoter PgadB, the high yield may be a result of the cells responding to high-density culture conditions. Overall, this system produced comparable high yields of bioethanol (>0.5 g ethanol) despite being cultivated under non-optimized test tube conditions. 産物 g 基質 -1 ) and high productivity (>1 g L -1 hr -1 ) and achieved industrially suitable production.

[0325] The samples were then analyzed for glucose, allulose, and mannose concentrations using high performance liquid chromatography (HPLC) to determine purity. Known concentrations of glucose, allulose, and mannose standards were run on the HPLC, and the area under each corresponding peak was integrated. For each sugar standard, the peak integrals were plotted against concentration and fitted with a best fit line. Simultaneously with the standards, production samples were run on the HPLC. The standards were used to identify the corresponding sugar peaks in each production sample. The peaks for each sample were integrated, and their areas recorded. Using the best fit line, the peak integrals were used to find the sugar concentration in each sample. The sugar concentration and the following equation: The purity of allulose was calculated using TIFF2025530206000098.tif9128.

[0326] Strain 7 was able to consume all of the glucose in the medium at 24 hours ( FIG. 25 ), indicating approximately 100% allulose purity. Because separating D-psicose from a mixture of glucose and / or fructose is costly, complete consumption of the substrate is a desirable characteristic for microbial production. Depleting the medium of glucose facilitates the extraction and purification of D-psicose in an industrial environment.

[0327] While the subject matter disclosed herein and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations can be made therein without departing from the spirit and scope of the invention, as defined by the appended claims. Moreover, the scope of this application is not intended to be limited to the particular embodiments of the processes, machines, manufacture, compositions of matter, means, methods, and steps described herein. As those skilled in the art will readily understand from this disclosure of the subject matter disclosed herein, any now-existing or later-developed process, machine, manufacture, composition of matter, means, method, or step that performs substantially the same function or achieves substantially the same result as the corresponding embodiment described herein can be utilized by the subject matter disclosed herein. Accordingly, it is intended that the appended claims include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

[0328] Patents, patent applications, publications, product descriptions, and protocols 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, which produces increased amounts of psicose compared to naturally occurring microorganisms.

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

3. 3. The recombinant microorganism of claim 1 or 2, wherein the epimerase is E. coli AlsE.

4. 4. The recombinant microorganism of any one of claims 1 to 3, wherein the epimerase comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO:

1.

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

1.

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

1.

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

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

9. 9. The recombinant microorganism of any one of claims 1 to 8, wherein 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.

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

4.

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

4.

12. Exogenous galactose:H + 12. The recombinant microorganism of any one of claims 1 to 11, further comprising a symporter (GalP) and a glucokinase (Glk).

13. 13. The recombinant microorganism of claim 12, wherein the GalP is E. coli GalP and the Glk is E. coli Glk.

14. 14. The recombinant microorganism of claim 13, wherein 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 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.

15. 14. The recombinant microorganism of claim 13, wherein GalP comprises the amino acid sequence set forth in SEQ ID NO: 38 and Glk comprises the amino acid sequence set forth in SEQ ID NO:

40.

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

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

18. 18. The recombinant microorganism of any one of claims 1 to 17, further comprising mutations in genes encoding enzymes of glycolysis compared to a naturally occurring microorganism.

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

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

21. 21. The recombinant microorganism of any one of claims 1 to 20, further comprising a mutation in a gene encoding an enzyme of the allolytic pathway.

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

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

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

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

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

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

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

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

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

31. 31. The recombinant microorganism of any one of claims 25 to 30, further comprising 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.

32. 32. The recombinant microorganism of 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 expression of the epimerase and the phosphatase results in increased production of psicose compared to a microorganism lacking the recombinant polynucleotide.

34. 34. The microorganism of claim 33, wherein the epimerase is allulose-6-phosphate 3-epimerase (AlsE).

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

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

1.

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

1.

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

1.

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

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

41. 41. The microorganism of any one of claims 33 to 40, wherein 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.

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

4.

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

4.

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

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

46. 46. ​​The microorganism of any one of claims 33 to 45, further comprising a mutation in a gene encoding an enzyme of glycolysis.

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

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

49. 49. The microorganism of any one of claims 33 to 48, further comprising a mutation in a gene encoding an enzyme of the allolytic pathway.

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

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

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

53. 53. The microorganism of any one of claims 33 to 52, further comprising an exogenous nuclease and an sgRNA.

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

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

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

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

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

59. 59. The microorganism of any one of claims 33 to 58, further comprising 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. 60. The microorganism of claim 59, wherein the selected enzyme of glycogen biosynthesis is phosphoglucomutase (Pgm).

61. (a) a recombinant polynucleotide encoding an epimerase and a phosphatase; (b) mutations in genes encoding enzymes of the pentose phosphate pathway; (c) mutations in genes encoding enzymes of glycolysis; (d) mutations in genes encoding enzymes of the allosolytic pathway; and (e) mutations in genes encoding enzymes in the mannose biosynthetic pathway; and (f) optionally, a recombinant polynucleotide encoding GalP, Glk, or both; Microorganisms including.

62. (a) a recombinant polynucleotide encoding allulose-6-phosphate 3-epimerase (AlsE) and hexitol phosphatase B (HxpB); (b) mutations in glucose-6-phosphate 1-dehydrogenase (Zwf); (c) phosphofructokinase-1 (PfkA) mutation; (d) mutations 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; Microorganisms including.

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

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

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

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

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

68. 61. The microorganism of 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. 69. A method for producing psicose, comprising culturing the microorganism of any one of claims 1 to 68 under conditions suitable for converting a substrate to psicose.

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

71. 71. The method of claim 69 or 70, wherein the psicose 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. 72. The method of claim 71, wherein the purity value is 100%.

73. The purity value has the formula:

73. The method of claim 71 or 72, wherein the method is determined by

74. 69. Psicose produced by a method comprising culturing the microorganism of any one of claims 1 to 68 under conditions suitable for converting a substrate into psicose, wherein the psicose 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. 75. The psicose of claim 74, wherein the purity value is 100%.

76. The purity value has the formula:

76. The psicose according to claim 74 or 75, wherein the psicose is determined by

77. (a) culturing the microorganism of any one of claims 1 to 68 under conditions suitable for converting a substrate into psicose; (b) purifying the psicose; and (c) mixing the psicose with the food product to form a psicose-containing food product. A method for producing a food product containing psicose, comprising:

78. 78. The method of claim 77, wherein the food product is chewing gum, a confectionery, chocolate, or a savory good.

79. 78. The method of claim 77, wherein the food product is a beverage, yogurt, ice cream, baked goods, or a nutritional bar.

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