Microorganisms for the production of allose

Abstract

The present disclosure relates to microorganisms useful in the biosynthesis of D-allose. Also provided are methods of producing the disclosed microorganism and methods of producing D-allose.

Description

MICROORGANISMS FOR THE PRODUCTION OF ALLOSECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 537, 136, filed September 7, 2023, the content of which is incorporated by reference in their entirety, and to which priority is claimed.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on September 4, 2024, is named is 081906-1460211 253410PC SL, and is 248,312 bytes in size.TECHNICAL FIELD

[0003] The presently disclosed subject matter relates to compositions and methods for producing a low-calorie sugar in microorganisms. The presently disclosed subject matter further relates to compositions and methods for producing allose in microorganisms.BACKGROUND

[0004] Current industrial D-allose production is through challenging multi-step, in vitro enzymatic synthesis. These methods comprises isomerization of psicose to allose via L-rhanmose isomerase (RI) (EC 5.3.1.14), ribose-5-phosphale isomerase (Rpl) (EC 5.3.1.6), or D-glucose isomerase (EC 5.3.1.5 or isomerization of psicose-6-phosphate to allose-6-phosphate via Rpl. Major drawbacks to these methods include that multiple separation and purification processes are required, the use of thermodynamically unfavorable reactions, and the addition of expensive cofactors and other enzymes. Thus, there is a need for novel methods and compositions for the production of D-allose.SUMMARY

[0005] In one aspect, the present disclosure relates to a recombinant microorganism comprising an exogenous epimerase, an exogenous isomerase, and an exogenous phosphatase, wherein the recombinant microorganism produces an increased amount of allose as compared to a naturally occurring microorganism.

[0006] In some embodiments, the epimerase is an allulose-6-phosphate 3-epimerase (AlsE). In some embodiments, the epimerase is an E. coli AlsE. In some embodiments, the epimerase comprises an amino acid sequence 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 a hexilol phosphatase A (HxpA). In some embodiments, the phosphatase is an E. coli HxpA. In some embodiments, the phosphatase comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 62. In some embodiments, the phosphatase comprises the amino acid sequence set forth in SEQ ID NO: 62. In some embodiments, the phosphatase consists of the amino acid sequence set forth in SEQ ID NO: 62. In some embodiments, the phosphatase comprises an adenine-thymine-guanine (ATG) start codon.

[0008] In some embodiments, the exogenous phosphatase is fructose-1-phosphate phosphatase (YqaB). In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is E. coli. In some embodiments, the phosphatase comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 60. In some embodiments, the phosphatase comprises the amino acid sequence set forth in SEQ ID NO: 60. In some embodiments, the phosphatase consists of the amino acid sequence set forth in SEQ ID NO: 60.

[0009] In some embodiments, the recombinant microorganism further comprises an exogenous isomerase. In some embodiments, the isomerase is an E. coli RpiB. In some embodiments, the isomerase comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, the isomerase comprises the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, the isomerase consists of the amino acid sequence set forth in SEQ ID NO: 24.

[0010] In some embodiments, the recombinant microorganism further comprises exogenous galactose:H’ symporter (GalP) and glucokinase (Glk). In some embodiments, the GalP is an E. coli GalP and the Glk is E. coli Glk. 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. In some embodiments, the recombinant microorganism further comprises exogenous glucose-6-phosphate isomerase (Gpi).

[0011] In some embodiments, the recombinant microorganism further comprises a mutation of a gene encoding for an enzyme of the pentose phosphate pathway compared to a naturally occurring microorganism. In some embodiments, the enzyme of the pentose phosphate pathway is glucose- 6-phosphate 1-dehydrogenase (Zwf).

[0012] In some embodiments, the recombinant microorganism further comprises a mutation of a gene encoding for an enzyme of the glycolysis compared to a naturally occurring microorganism.In some embodiments, the enzyme of the glycolysis is phosphofructokinase-1 (PIkA), phospho fructokinase-2 (PfkB), or pyruvate kinase (PykF). in some embodiments, the enzyme of the glycolysis is ph ospho fructo kinase- 1 (PfkA).

[0013] In some embodiments, the recombinant microorganism further comprises a mutation of a gene encoding for an aldolase. In some embodiments, the aldolase is deoxyribose-phosphate aldolase (DeoC).

[0014] In some embodiments, the recombinant microorganism further comprises a mutation of at least one gene encoding for a phosphatase. In some embodiments, the at least one gene encoding for a phosphatase is selected from fructose- 1 -phosphate phosphatase (YqaB), sugar phosphatase YbiV, hexitol phosphatase B (HxpB), sugar phosphatase YidA, or combinations thereof.

[0015] In some embodiments, the recombinant microorganism further comprises a mutation of at least one gene encoding for an enzyme of allose metabolism. In some embodiments, the at least one gene encoding for an enzyme of allose metabolism is selected from D-allose import ATP- binding protein (AlsA), D-allose-binding periplasmic protein (AlsB), D-allose transport system permease protein (AlsC), D-allose kinase (AlsK), HTH-type transcriptional regulator (AlsR), or combinations thereof.

[0016] In some embodiments, the recombinant microorganism further comprises a mutation of a gene encoding for an enzyme of mannose biosynthesis pathway. In some embodiments, the enzyme of mannose biosynthesis pathway is mannose-6-phosphate isomerase (ManA).

[0017] In some embodiments, the recombinant microorganism further comprises a mutation of a gene encoding for an enzyme of the phosphotransferase system (PTS).

[0018] In some embodiments, the microorganism further comprising a mutation of a gene encoding for 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).

[0019] In some embodiments, the exogenous epimerase, the exogenous isomerase, and the exogenous phosphatase are expressed by a stationary phase promoter. In some embodiments, the exogenous epimerase, the exogenous isomerase, and the exogenous phosphatase are expressed by an inducible promoter.

[0020] In another aspect, the present disclosure relates to a microorganism comprising a recombinant polynucleotide encoding an epimerase, an isomerase, and a phosphatase, wherein expression of the epimerase, the isomerase, and the phosphatase results in an increased production of allose as compared to a microorganism lacking the recombinant polynucleotide.

[0021] In some embodiments, the epimerase is an allulose-6-phosphale 3-epimerase (AlsE). In some embodiments, the epimerase is an E. coli AlsE. In some embodiments, the epimerase comprises an amino acid sequence 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.

[0022] In some embodiments, the phosphatase is a hexitol phosphatase A (HxpA). In some embodiments, the phosphatase is an E. coli EIxpA. In some embodiments, the phosphatase comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 62. In some embodiments, the phosphatase comprises the amino acid sequence set forth in SEQ ID NO: 62. In some embodiments, the phosphatase consists of the amino acid sequence set forth in SEQ ID NO: 62. In some embodiments, the phosphatase comprises an adenine-lhymine-guanine (ATG) start codon.

[0023] In some embodiments, the phosphatase is a fructose-1-phosphate phosphatase (YqaB). In some embodiments, the phosphatase is an E. coli YqaB. In some embodiments, the phosphatase comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 60. In some embodiments, the phosphatase comprises the amino acid sequence set forth in SEQ ID NO: 60. In some embodiments, the phosphatase consists of the amino acid sequence set forth in SEQ ID NO: 60.

[0024] In some embodiments, the recombinant microorganism further comprises an exogenous isomerase. In some embodiments, the isomerase is an E. coli RpiB. In some embodiments, the isomerase comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, the isomerase comprises the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, the isomerase consists of the amino acid sequence set forth in SEQ ID NO: 24.

[0025] In some embodiments, the recombinant microorganism further comprises exogenous galaclose: H' symporter (GalP) and glucokinase (Glk). In some embodiments, the GalP is an E. coli GalP and the Glk is E. coli Glk. 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. In some embodiments, the recombinant microorganism further comprises exogenous glucose-6-phosphate isomerase (Gpi).

[0026] In some embodiments, the microorganism further comprises a mutation of a gene encoding for an enzyme of the pentose phosphate pathway. In some embodiments, the enzyme of the pentose phosphate pathway is glucose-6-phosphate 1 -dehydrogenase (Zwf). In some embodiments, the microorganism further comprises a mutation of a gene encoding for an enzyme of the glycolysis. In some embodiments, the enzyme of the glycolysis is phosphofructokinase- 1 (PfkA), phosphofructokinase-2 (PfkB), or pyruvate kinase (PykF). In some embodiments, the enzyme of the glycolysis is phosphofructokinase- 1 (PfkA),

[0027] In some embodiments, the recombinant microorganism further comprises a mutation of a gene encoding for an aldolase. In some embodiments, the aldolase is deoxyribose-phosphate aldolase (DeoC).

[0028] In some embodiments, the recombinant microorganism further comprises a mutation of at least one gene encoding for a phosphatase. In some embodiments, the at least one gene encoding for a phosphatase is selected from fructose- 1 -phosphate phosphatase (YqaB), sugar phosphatase YbiV, hexitol phosphatase B (HxpB), sugar phosphatase (YidA), or combinations thereof.

[0029] In some embodiments, the recombinant microorganism further comprises a mutation of at least one gene encoding for an enzyme of allose metabolism. In some embodiments, the at least one gene encoding for an enzyme of allose metabolism is selected from D-allose import ATP- binding protein (AlsA), D-allose-binding periplasmic protein (AlsB), D-allose transport system permease protein (AlsC), D-allose kinase (AlsK), HTH-type transcriptional regulator (AlsR), or combinations thereof.

[0030] In some embodiments, the microorganism further comprises a mutation ofa gene encoding for an enzyme of mannose biosynthesis pathway. In some embodiments, the enzyme of mannose biosynthesis pathway is mannose-6-phosphate isomerase (ManA).

[0003] ] In some embodiments, the recombinant microorganism further comprises a mutation of a gene encoding for an enzyme of the phosphotransferase system (PTS).

[0032] In some embodiments, the microorganism further comprising a mutation of a gene encoding for 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).

[0033] In some embodiments, the exogenous epimerase, the exogenous isomerase, and the exogenous phosphatase are expressed by a stationary phase promoter. In some embodiments, the exogenous epimerase, the exogenous isomerase, and the exogenous phosphatase are expressed by an inducible promoter.

[0034] In one aspect, the present disclosure relates to a microorganism comprising a recombinant polynucleotide encoding an epimerase, an isomerase, and a phosphatase; a mutation of a gene encoding for an enzyme of the pentose phosphate pathway; a mutation of a gene encoding for an enzyme of the glycolysis; a mutation of a gene encoding for an aldolase; a mutation of a gene encoding for an enzyme of mannose biosynthesis pathway; optionally a recombinant polynucleotide encoding GalP, Glk, or both; optionally a recombinant polynucleotide encoding Gpi; and optionally a mutation of a gene encoding for an enzyme of the phosphotransferase system (PTS). In some embodiments, the microorganism further comprises a mutation of at least one enzyme encoding for a phosphatase. In some embodiments, the microorganism further comprises a mutation of at least one enzyme of allose metabolism.

[0035] In a further aspect, the present disclosure relates to a microorganism comprising a recombinant polynucleotide encoding an allose-6-phosphate isomerase (RpiB) and a hexitol phosphatase A (HxpA); a mutation of glucose-6-phosphate I -dehydrogenase (Zwf); a mutation of phosphofructokinase- 1 (PfkA); a mutation of deoxyribose-phosphate aldolase (DeoC); a mutation of mannose-6-phosphate isomerase (ManA); and optionally a recombinant polynucleotide encoding GalP, Glk, or both. In some embodiments, the microorganism further comprises a mutation of at least one gene selected from fructose-1-phosphate phosphatase (YqaB), sugar phosphatase YbiV, hexitol phosphatase B (HxpB), sugar phosphatase (YidA), ATP-binding protein (AlsA), D-allose-b inding peri pl asm ic protein (AlsB), D-allose transport system permease protein (AlsC), D-allose kinase (AlsK), HTH-type transcriptional regulator (AlsR), or combinations thereof.

[0036] In a further aspect, the present disclosure relates to a microorganism comprising a recombinant polynucleotide encoding an allulose-6-phosphale 3-epimerase (AlsE), an allose-6- phosphale isomerase (RpiB), and a fructose-1-phosphate phosphatase (YqaB); a mutation of glucose-6-phosphate 1 -dehydrogenase (Zwf); a mutation of phosphofructokinase- 1 (PfkA); a mutation of deoxyribose-phosphate aldolase (DeoC); a mutation of mannose-6-phosphate isomerase (ManA); optionally a recombinant polynucleotide encoding GalP, Glk, or both; optionally a recombinant polynucleotide encoding Gpi; and optionally a mutation of a gene encoding for an enzyme of the phosphotransferase system (PTS). In some embodiments, the microorganism further comprises a mutation of at least one gene selected from sugar phosphatase YbiV, hexitol phosphatase B (HxpB), sugar phosphatase (YidA), ATP-binding protein (AlsA), D- allose-binding periplasmic protein (AlsB), D-allose transport system permease protein (AlsC), D- allose kinase (AlsK), HTH-type transcriptional regulator (AlsR), or combinations thereof.

[0037] Further, in one aspect, the present disclosure relates to a microorganism comprising a recombinant polynucleotide encoding an allulosc-6-phosphate 3-epimerase (AlsE); a recombinantpolynucleotide encoding an allose-6-phosphate isomerase (RpiB); a recombinant polynucleotide encoding a hexitol phosphatase A (HxpA); a mutation of giucose-6-phosphate 1-dehydrogenase (Zwl); a mutation of phosphofructokinase- 1 (PfkA); a mutation of deoxyribose-phosphate aldolase (DeoC); a mutation of mannose-6-phosphate isomerase (ManA); optionally a recombinant polynucleotide encoding GalP, Glk, or both; optionally a recombinant polynucleotide encoding Gpi; and optionally a mutation of a gene encoding for an enzyme of the phosphotransferase system (PTS). In some embodiments, the microorganism further comprises a mutation of at least one gene selected from fructose- 1 -phosphate phosphatase (YqaB), sugar phosphatase YbiV, hexitol phosphatase B (HxpB), sugar phosphatase (YidA), ATP-binding protein (AlsA), D-allose-binding periplasmic protein (AlsB), D-allose transport system permease protein (AlsC), D-allose kinase (AlsK), HTH-type transcriptional regulator (AlsR), or combinations thereof.

[0038] Further, in one aspect, the present disclosure relates to a microorganism comprising a recombinant polynucleotide encoding an allulose-6-phosphate 3-epimerase (AlsE); a recombinant polynucleotide encoding an allose-6-phosphate isomerase (RpiB); a recombinant polynucleotide encoding a fructose- 1 -phosphate phosphatase (YqaB); a mutation of glucose-6-phosphate 1- dehydrogenase (Zwl); a mutation of phosphofructokinase- 1 (PfkA); a mutation of deoxyribose- phosphate aldolase (DeoC); a mutation of mannose-6-phosphale isomerase (ManA); optionally a recombinant polynucleotide encoding GalP, Glk, or both; optionally a recombinant polynucleotide encoding Gpi; and optionally a mutation of a gene encoding for an enzyme of the phosphotransferase system (PTS). In some embodiments, the microorganism further comprises a mutation of at least one gene selected from sugar phosphatase YbiV, hexitol phosphatase B (HxpB), sugar phosphatase (YidA), ATP-binding protein (AlsA), D-allose-binding periplasmic protein (AlsB), D-allose transport system permease protein (AlsC), D-allose kinase (AlsK), HTH- type transcriptional regulator (AlsR), or combinations thereof.

[0039] In some embodiments, the mutation is a deletion. In some embodiments, the mutation reduces or eliminates expression or activity of the enzyme. In some embodiments, the microorganism is E. coli, bacillus subtilis or Lactocoecus lactis.

[0040] In one aspect, the present disclosure also relates to a method for producing allose comprising culturing the microorganism disclosed herein under conditions suitable for converting a substrate to allose. In some embodiments, the substrate comprises glucose.

[0041] Additionally, the present disclosure relates to methods of making a food product comprising allose, the method comprising culturing the microorganism disclosed herein under conditions suitable for converting a substrate to allose; and admixing the allose with one or morefoods to form a food comprising al lose. In some embodiments, the food product is a beverage, yogurt, ice cream, a baked good, or a nutritional bar.BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figures 1A and 1B illustrate a pathway for the biosynthetic production of allose. Glucose is imported and phosphorylated to glucose-6-phosphate (G6P) by the phosphotransferase system (PTS) or GalP / Glk. G6P is then isomerized to fructose-6-phosphate (FOP) by Glucose-6- phosphate isomerase. F6P is then epimerized to psicose-6-phosphate (P6P) by D-allulose-6- phosphate 3-epimerase (AlsE). P6P is then isomerized to allose-6-phosphate (A6P) by allose-6- phosphate isomerase (RpiB), which is then dephosphorylated to free allose by native phosphatases, e.g., Hexitol phosphatase A (HxpA). Finally, free allose can diffuse across the cell membrane into the supernatant. Competing pathways include the pentose phosphate pathway, catalyzed by glucose-6-phosphate dehydrogenase (Zwf), glycolysis, which is catalyzed by phospho fruclokinases A and B (PfkA and B), and the mannose biosynthesis pathway, catalyzed by mannose-6-phosphate isomerase (ManA). Other competing pathways include the glycogen biosynthesis pathway, catalyzed by phosphoglucomutase (Pgm), the allose degradation pathway, catalyzed DeoC, and allose import by allose transporter / phosphorylation machinery, AlsA, AlsB, AlsC, and AlsK.

[0043] Figures 2A-2C illustrate strategies for the biosynthesis of D-allose. Figure 2A shows the current method for D-allose production which leads to limited yield (—50%) due to an overall positive ΔG'°. Figure 2B shows the proposed D-allose biosynthetic pathway, in which the dephosphorylation step thermodynamically drives production forwards due to a large negative ΔG'munder cellular reactant concentrations of 1 mM. Figure 2C shows the proposed pathway for the biosynthetic production of D-allose in E. coli. Deleted steps are in blue. Overexpressed steps are in red. PTS, the phosphotransferase system: AlsE, D-allulose 6-phosphale 3-epimerase; RpiB, D-ribose-6-phosphate-isomerase B, HxpA, hexitol phosphatase A.

[0044] Figure 3 shows screening for a D-allose-6-phosphale phosphatase in E. coli. Cells were grown in MOP media with 10 g L-1glucose at 37°C to OD600~0.4, then grown at 30 C for 24 h. Al OD600~0.4, 1 mM IPTG was added. Various sugar phosphatase genes with alsE and rpiB under were tested for D-allose production (Table 5). (-) indicates that no phosphatase gene wasadditionally expressed. Error bars indicate s.d. (n = 3 biological replicates).

[0045] Figure 4 shows The effect of gene deletions on the D-allose production. Cells were grown in M9P media with 10 g L-1glucose to OD600-0.4 at 37 °C, then grown at 30 °C for 24 h. At OD600-0.4, 1 mM IPTG was added. The operon of alsE, rpiB, and hxpA were expressed under (pAL2310, Tabled). Yield (%) was calculated based on the possibility that for every 1 molof D-glucose consumed, 1 mol of D-allose could be produced, leading to a theoretical maximum yield of 100%. Error bars indicate s.d. (n = 3 biological replicates).

[0046] Figure 5 shows D-allose production via fusion of AlsE and RpiB. Cells were grown in M9P media with 10 g L-1glucose to OD600-0.4 at 37 °C, then grown at 30 °C for 24 h. At OD600-0.4, 1 mM 1PTG was added. The operons of peptide linked alsE-rpiB and hxpA were expressed under(pAL2562-pAL2565, Table 5) in AL4387 (Table 1). Peptide linkers consisted of (Gly-Ser-Gly)n (“(Gly-Ser-Gly)3” disclosed as SEQ ID NO: 79) or (Gly-Gly-Gly-Gly-Ser)n(n= 1,3) (“(Gly-Gly-Gly-Gly-Ser)l” and “(Gly-Gly-Gly-Gly-Ser)3” disclosed as SEQ ID NOS 80 and 81 , respectively). Error bars indicate s.d. (n = 3 biological replicates).[00471 Figure 6 shows modulation of the expression of the D-allose pathway genes. Cells were grown in M9P media with 10 g L-1glucose to OD600-0.4 at 37 °C, then grown at 30 °C for 24 h. At OD600~0.4, ] mM IPTG was added for constructs. The genes alsE, rpiB, and hxpA wereexpressed under either the or and the order of the genes was varied. hxpA* indicatesthat the hxpA gene start codon has been changed from GTG to ATG. These plasmids (pAL2580, pAL2581, pAL2609, pAL2623, and pAL2624 (Table 5)) were transformed into AL4387 (Table4). Error bars indicate s.d. (n = 3 biological replicates).

[0048] Figures 7A-7B show supplementation of glucose import with GalP and Glk and modulation of the psicose : allose production ratio. Figure 7 A shows galP and glk genes expressed under(pAL2264, Table 5). pAL2310, pAL2609, pAL2623, and pAL2624 (Table 2) were transformed into AL4387 with pAL2264. Cells were grown in M9P media with 40 g L-1glucose to OD600-0.4, then grown at 30 °C for 24 h. Al OD600-0.4, 1 mM IPTG was added to induce plasmids. Yield (%) was calculated based on the possibility that for every 1 mol of D-glucose consumed, 1 mol of D-psicose could be produced, leading to a theoretical maximum yield of 100%. Specific liter (g-1L-1OD600-1) indicates titer per final OD600. Error bars indicate s.d. (n = 3 biological replicates). Figure 7B shows the genes alsE, rpiB, and hxpA or yqaB expressed under (pAL2310 and pAL2313, respectively, Table 5) in AL4387. Cells were grown inM9P media with 10 g L-1glucose at 37°C to OD600-0.4, then grown at 30 °C for 24h. Al OD600—0.4, I mM IPTG was added. Error bars indicate s.d. (n = 3 biological replicates).

[0049] Figures 8A-8C show high cell density D-allose production. Cultures of this strain were grown in M9P media with 40 g L-1glucose at 37 °C until an OD600of -1, after which they were induced with 1 mM IPTG and grown for another 30 min. Cultures were then spun down and resuspended in M9P media with 40 g L-1glucose and 1 mM IPTG to an OD600of -10 and grown al 30 °C. Every 24 h, ] 0% of the culture volume was removed and replaced with M9P media containing 400 g L-1glucose and 1 mM IPTG. Figure 8A shows production of D-allose and D- psicosc in Strain 9 (AL4387, Tabic 4) harboring * and(pAL2609 and pAL2264, respectively, Table 5) over 120 h. Figure SB shows consumption of D- glucose over 120 h. Figure SC shows measurement of OD600over 120 h. Error bars indicate s.d. (n = 3 biological replicates),DETAILED DESCRIPTION

[0050] The market for rare sugars as a foodstuff, dietary supplement, and health aid is expanding. Among rare sugars, D-allose has attracted particular attention. However, the current method of producing D-allose is costly, inefficient, and thermodynamically unfavorable, limiting its potential for wide-spread use. Significantly, the present disclosure addressed various obstacles in D-allose production including thermodynamic barriers, limited yield, the requirement for purified enzymes and the addition of cofactors. Another significant finding disclosed herein was that E. coli naturally possesses a thermodynamically favorable pathway for D-allose production allowing the improvement of D-allose production by increasing the expression of native genes and eliminating competing pathways without the introduction of heterologous genes. The present disclosed subject matter facilitates the industrial-scale production of D-allose without the need for costly enzyme purification or difficult feedstock and product separation.

[0005] ] The present disclosure is based, in part, on the discovery that microorganisms including specific genetic modifications (e.g., gene deletion) can be made for producing low-calorie sugars. In certain embodiments, the low-calorie sugar is allose. For clarity and not by way of limitation, the detailed description of the presently disclosed subject matter is divided into the following subsections:1. Definitions;2. Microorganisms Producing Allose;3. Methods for Producing and Generating Microorganisms; and4. Methods for Producing Allose.1. Definitions

[0052] The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the methods and compositions of the invention and how to make and use them.

[0053] As used herein, the use of the word “a" or “an” when used in conjunction with the term “comprising” in the claims and / or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0054] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per 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 more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.

[0055] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The present disclosure also contemplates other embodiments “comprising,” “consisting of’, and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0056] As used herein, the term “microorganism” refers to any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria, or eukarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista. In certain embodiments, the term includes prokaryotic or eukaryotic cells or organisms having a microscopic size including, but without any limitation, bacteria, archaea, and eubacleria of all species as well eukaryotic microorganisms such as yeast and fungi. In certain embodiments, the term microorganism includes cells that can be cultured for the production of a chemical (e.g., sugar). In certain embodiments, the microorganism is a prokaryotic microorganism. In certain embodiments, the prokaryotic microorganism is a bacterium.

[0057] As used herein, the terms “bacterium,” “bacteria,” or “eubacleria,” refers to a domain of prokaryotic organisms. In certain embodiments, bacteria include gram-negative bacteria, gram- positive bacteria, proteobacteria, cyanobacteria, spirochetes, and related species, planclomyces, bacleroides, chlamydia, green sulfur bacteria, green non-sulfur bacteria, radioresistant micrococci, and lhermologa and Thermosipho thermophiles.

[0058] As used herein, the term “gram- negative bacteria” includes cocci, nonenteric rods, and enteric rods. The genera of gram-negative bacteria include, for example, and without any limitation, Neisseria, Spirillum, Pasteurella, Brucella, Yersinia, Erancisella, Haemophilus, Bordetella, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, Bacleroides, Acetobacter, Aerobacler, Agrobacterium, Azolobacier, Spirilla, Serratia. Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusohacterium .

[0059] As used herein, the term “grant-positive bacteria” includes cocci, nonsporulating rods, and sporulaling rods. The genera of gram-positive bacteria include, for example, and without anylimitation, Actinomyces, Bacillus, Clostridium, Corynebacterium, Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

[0060] As used herein, the term “recombinant microorganism” refers to a microorganism that contains one or more recombinant polynucleotides.

[0006] ] The term “exogenous,” as used herein, refers to molecules that are not naturally found in and / or produced by a given yeast, bacterium, organism, microorganism, or cell in nature. The term “endogenous,” as used herein, refers to molecules that are naturally found in and / or produced by a given yeast, bacterium, organism, microorganism, or cell in nature.

[0062] The term “nucleic acid molecule,” “nucleotide sequence,” or “polynucleotide,” as used herein, refers to a single or double-stranded covalently-linked sequence of nucleotides in which the 3’ and 5’ ends on each nucleotide are joined by phosphodiester bonds. The nucleic acid molecule can include deoxyribonucleolide bases or ribonucleotide bases and can be manufactured synthetically in vitro or isolated from natural sources.

[0063] As used herein, “recombinant polynucleotide” refers to a polynucleotide wherein the exact nucleotide sequence of the polynucleotide is foreign to (i.e., not naturally found in) a given host. In certain embodiments, a recombinant polynucleotide sequence is naturally found in a given host, but in an unnatural (e.g., greater than or less than expected) amount, or additionally if the sequence of a polynucleotide comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, but without any limitation, a recombinant polynucleotide could have two or more sequences from unrelated polynucleotides or from endogenous nucleotides arranged to make a new polynucleotide. In certain embodiments, the present disclosure provides the introduction of a recombinant polynucleotide into a microorganism, wherein the polynucleotide encodes for a polypeptide that is not normally found in the microorganism. With reference to the microorganism’s genome, then, the polynucleotide sequence that encodes the polypeptide is recombinant or heterologous.

[0064] A “gene,” as used herein, refers to a DNA region (including exons and introns) encoding a gene product, as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and / or transcribed sequences. In certain non-limiting embodiments, a gene includes promoter sequences, terminators, translational 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.

[0065] The terms “polypeptide,” “peptide,” “amino acid sequence” and “protein,” used interchangeably herein, refer to a molecule formed from the linking of at least two amino acids.The link between one amino acid residue and the next is an amide bond and is sometimes referred to as a peptide bond. A polypeptide can be obtained by a suitable method known in the art, including isolation from natural sources, expression in a recombinant expression system, chemical synthesis, or enzymatic synthesis. The terms can apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0066] The term “amino acid,” as used herein, can be naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids ate those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gammacarboxyglutamate, and O-phosphoserine. Amino acid analogs and derivatives can refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. Such analogs can have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics mean chemical compounds that have a structure that is different from the general chemical structure of amino acid, but that function in a manner similar to a naturally occurring amino acid. 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.

[0067] The term “isolated,” as used herein, refers to a material that is removed from at least one component with which it is naturally associated (e.g., removed from its original environment).

[0068] As used herein, the terms “reduce” and “reduction” refer to a measurable lessening of an end-point (e.g., enzymatic activity, production of compound, expression of a protein) 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 %.

[0069] As used herein, the term “increase,” “elevate” and “elevation” refers to a measurable augmentation of an end-point (e.g., enzymatic activity, production of compound, expression of a protein) 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 certainembodiments, the increase can be about i 00-fold or more, about 1000-fold or more, or about 10, 000-fold or more.

[0070] As used herein, the term “isomerase” refers to any enzyme of EC class 5 that catalyzes geometric or structural changes within one molecule. According to the type of isomeric change they catalyze, they can be called racemases or epimerases (EC subclass 5.1); cis - trans - isomerases (EC subclass 5.2); intramolecular oxidoreductases (EC subclass 5.3); intramolecular transferases (mutases) (EC subclass 5.4); or intramolecular lyases (EC subclass 5.5); other isomerases are placed in EC subclass 5.99.|()O71] As used herein, the term “epimerase” refers to a class of enzymes that catalyze the inversion of asymmetric groups in a substrate with several centers of asymmetry.

[0072] The term “dehydrogenase,” as used herein, refers to any enzyme that catalyzes the removal of hydrogen atoms (e.g., dehydrogenation) in biological reactions. Dehydrogenases occur in many biochemical pathways and are involved in driving the electron transport chain reactions of cell respiration. In certain embodiments, dehydrogenases work in conjunction with the hydrogen- accepting coenzymes NAD and FAD.

[0073] As used herein, the term “phosphatase” refers to a class of enzymes that catalyze the removal of a phosphate group from an organic compound. In certain embodiments, the phosphatase catalyzes the removal of a phosphate group from a sugar. In certain embodiments, the sugar is a hexose.

[0074] The term “aldolase,” as used herein, refers to enzymes of the sub-subclass EC 4.1.2 (i.e., the aldehyde-lyases) catalyzing aldol condensations and their reversal.

[0075] Techniques for determining nucleic acid and amino acid sequence identity are known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA for a gene and / or determining the amino acid sequence encoded thereby and comparing these sequences to a second nucleotide or amino acid sequence. Genomic sequences can also be determined and compared in this fashion. In general, identity refers to an exact nucleolide-lo- nucleotide or amino acid-to-amino acid correspondence of two polynucleotides 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 sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. Unless indicated otherwise, percent identity is determined for two sequences when compared and aligned for maximum correspondence over a comparison window or designated region as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters. See, e.g., the NCBI web site at ncbi.nlm.nih.gov / BLAST. For example, BLASTN and BLASTP can be used using thefollowing default parameters: genetic code=slandard; filler=none: slrand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS trans lalions+Swiss protein+Spupdate+PIR. Details of these programs can be found on the GenBank website.

[0076] A “mutation” in a gene can include, for example, nucleotide changes, deletions of one or more nucleotides (which can include the entire coding sequence and / or promoter or other regulatory sequences) and insertions of one or more nucleotides, which can occur in the coding sequence of the 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) as well as alterations of the genome that reduce or knockout expression of a gene product.

[0077] The terms “linker”, “peptide linker”, or “flexible peptide linker”, as used herein, refer to short polypeptide sequences, typically between 1 amino acid residue and 20 amino acid residues, that occur between polypeptides. Linkers are often composed of flexible residues like glycine and serine so that the adjacent polypeptides are free to move relative to one another. Conventionally, longer linkers are used when it is necessary to ensure that two adjacent domains do not sterically interfere with one another. Non-limiting examples of linkers include (Gly-Ser-Gly)n (“(Gly-Ser- Gly)3” disclosed as SEQ ID NO: 79) and (Gly-Gly-Gly-Gly-Ser)n where n = 1 or 3 (“(Gly-Gly- Gly-Gly-Ser)]” and “(Gly-Gly-Gly-Gly-Ser)3” disclosed as SEQ ID NOS 80 and 81, respectively).2. _ Microorganisms Producing Allose

[0078] The present disclosure provides genetically engineered microorganisms. In certain embodiments, the presently disclosed microorganisms can produce an increased amount of allose, e.g., an increased amount compared to a control microorganism that is naturally-occurring.

[0079] D-Allose is a natural but rare monosaccharide. It is an aldohexose that has the same empirical formula as common monosaccharides such as glucose and fructose. Il is epimeric with glucose at the 3-position. Ils enantiomer, L-a!lose, is unknown in nature but has been synthesized. D-allose has the following formula:

[0080] D-Allose is 80% as sweet as sucrose (e.g., table sugar), but it has a reduced nutritional energy (e.g., calorie) value (Mooradian et al., Clinical nutrition eSPen 18 (2017): 1-8.). As a result, D-allose can be used as a replacement for sucrose and artificial sweeteners.

[0081] The present disclosure provides genetically engineered microorganisms that have increased production of allose as compared to naturally-occurring microorganisms. Figure 1 illustrates the biochemical pathways modulated in an exemplary microorganism of the present disclosure. In this exemplary microorganism, glucose is imported and phosphorylated to glucose- 6-phosphate (G6P) by the phosphotransferase system (PTS) or GalP / Glk. G6P is then isomerized to fmctose-6-phosphate (F6P) by Glucose-6-phosphate isomerase. F6P is then epimerized to psicose-6-phosphale(P6P)by D-allulose-6-phosphate 3-epimerase (AlsE). P6P is then isomerized to allose-6-phosphate (A6P) by allose-6-phosphate isomerase (RpiB), which is then dephosphorylaled to free allose by native phosphatases, e.g., Hexitol phosphatase A (HxpA). Finally, free allose can diffuse across the cell membrane into the supernatant. Competing pathways include the pentose phosphate pathway, catalyzed by glucose-6-phosphate dehydrogenase (Zwl), glycolysis, which is catalyzed by phosphofructokinases A and B (PfkA and B), and the mannose biosynthesis pathway, catalyzed by mannose-6-phosphate isomerase (ManA).

[0082] Glucose can be imported into the cell and phosphorylated using the phosphotransferase system (PTS). In certain embodiments, the microorganism comprises a mutation which reduces the activity of the PTS. In certain embodiments, the microorganism comprises a mutation of a gene encoding for an enzyme of the phosphotransferase system (PTS).2.1. Allose Production Enzymes

[0083] In certain embodiments, the presently disclosed microorganisms include overexpression of at least one gene encoding an enzyme catalyzing reactions for the production of allose. In certain embodiments, the presently disclosed microorganisms include a recombinant polynucleotide encoding at least one enzyme catalyzing reactions for the production of allose. In certain embodiments, the enzyme is an epimerase, for example an epimerase that converts fructose-6-phosphate (F6P) to psicose-6-phosphate. Non-limiting examples of epimerase include D-allulose-6-phosphate 3-epimerase, methylmalonyl-CoA epimerase, UDPgalaclose 4- epimerase, UDPglucose 4-epimerase, UDPglucuronate 4-epimerase, UDPglucuronate 5’- epim erase, ribose- 5 -phosphate epimerase, GDPmannose 3, 5 -epimerase, L-ribulosephosphale 4- epimerase, UDP-N-acelylghicosamine 2-epimerase, (J DP-N-acetyl glucosamine 4-epimerase, UDPgalaclose 4-epimerase, UDPglucose 4-epimerase, UDPglucuronate 4-epimerase, UDPglucuronate 5 ’-epimerase, GDPmannose 3,5-epimerase, methylmalonyl-CoA epimerase, ribosc-5 -phosphate epimerase, and UDP-N-acctylglucosaminc 2-cpimcrasc.

[0084] In certain embodiments, the epimerase is a D-allulose-6-phosphate 3-epimerase (AlsE) (UniProt No. P32719). AlsE catalyzes the reversible epimerization of D-allulose-6-phosphale to D-fruclose-6-phosphate. In certain embodiments, AlsE is an E. coli AlsE. In certain embodiments, AlsE comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%. at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, AlsE comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, AlsE consists of the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO: 1 is provided below:

[0085] 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:

[0086] In certain embodiments, the enzyme is an isomerase, for example an isomerase that catalyzes the conversion of psicose-6-phosphate to allose-6-phosphate. In certain embodiments, the isomerase is allose-6-phosphate isomerase (RpiB) (Entrez Gene ID: 948602). RpiB catalyzes the interconversion of ribulose-5-P and ribose-5-P, as well the interconversion of D-allose-6- phosphate (A116P) and D-allulose-6-phosphate. In certain embodiments, RpiB is an E. coli RpiB. A representative nucleotide sequence of gene rpiB is set forth in SEQ ID NO: 8 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 8. SEQ ID NO: 8 is provided below:

[0087] In certain embodiments, 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 S EQ ID NO: 24, In certain embodiments, E. coli RpiB comprises the amino acid sequence set forth in SEQ ID NO: 24. SEQ ID NO: 24 is provided below:

[0088] In certain embodiments, RpiB is a 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 at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 14. SEQ ID NO: 14 is provided below:

[0089] A representative nucleotide sequence of Bacillus subtilis gene rpiB is set forth in SEQ ID NO: 28 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 28. SEQ ID NO: 28 is provided below:

[0090] In certain embodiments, RpiB is a Lactococcus lacfi.s RpiB. A representative amino acid sequence of gene rpiB is found as LLAI2 RSI2460: ribose-5-phosphate isomerase, EC5.3.1.6 or is set forth in SEQ ID NO: 15 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 15, which is provided below.

[0091] A representative nucleotide sequence of a Lactococcus lactis gene tpiB is set forth in SEQ ID NO: 34 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 34. SEQ ID NO: 34 is provided below:

[0092] In certain embodiments, the enzyme is a phosphatase, for example a phosphatase that dephosphorylates allose-6- phosphate to free allose. In certain embodiments, the phosphatase catalyzes the removal of a phosphate group from a sugar. In certain embodiments, the sugar is a hexose.

[0093] In certain embodiments, the phosphatase is a Hexitol phosphatase A (HxpA) (UniProt No. P77625). HxpA catalyzes the dephosphorylation of D-allose-6-phosphate. In certain embodiments, HxpA is an E. coli HxpA. In certain embodiments, HxpA 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: 62. In certain embodiments, HxpA comprises the amino acid sequence set forth in SEQ ID NO: 62. In certain embodiments, HxpA consists of the amino acid sequence set forth in SEQ ID NO: 62. SEQ ID NO: 62 is provided below:

[0094] In certain embodiments, the gene hxpA 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: 63. In certain embodiments, hxpA comprises the nucleotide sequence set forth in SEQ ID NO: 63. In certain embodiments, hxpA consists of the nucleotide sequence set forth in SEQ ID NO: 63. SEQ ID NO: 63 is provided below:

[0095] In certain embodiments, the gene hxpA comprises a guanine-thymine-gtianine (GTG) start codon. In certain embodiments, the gene hxpA comprises an adenine-thymine-guanine (ATG) start codon. In certain embodiments, the gene hxpA 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: 76. In certain embodiments, hxpA comprises lhe nucleotide sequence set forth in SEQ ID NO: 76. In certain embodiments, hxpA consists of the nucleotide sequence set forth in SEQ ID NO: 76. SEQ ID NO: 76 is provided below:

[0096] In certain embodiments, the phosphatase is a sugar phosphatase, e.g., phosphosugar phosphatase (YigL), (UniProt P27848). In certain embodiments, YigL is an E. coli YigL. In certain embodiments, YigL 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 lhe amino acid sequence set forth in SEQ ID NO: 74. In certain embodiments, YigL comprises lhe amino acid sequence set forth in SEQ ID NO: 74. In certain embodiments, YigL consists of lhe amino acid sequence set forth in SEQ ID NO: 74. SEQ ID NO: 74 is provided below:

[0097] In certain embodiments, the gene yigL 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: 75. In certain embodiments, yigL comprises the nucleotide sequence set forth in SEQ ID NO: 75. In certain embodiments, yigL consists of the nucleotide sequence set forth in SEQ ID NO: 75. SEQ ID NO: 75 is provided below:

[0098] In certain embodiments, the phosphatase is fructose- 1 -phosphate phosphatase (YqaB) (Entrez Gene ID: 945776). YqaB catalyzes the dephosphorylation of allulose-6-phosphale. In certain embodiments, YqaB is an E. coli YqaB. A representative nucleotide sequence of gene yqaB is set forth in SEQ ID NO: 61 or SEQ ID NO: 80, or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 6] or SEQ ID NO: 80, which are provided below.

[0099] In certain embodiments, E. coli YqaB 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: 60. In certain embodiments, E. coli YqaB comprises the amino acid sequence set forth in SEQ ID NO: 60.

[0100] In certain embodiments, one or more transporter that transports glucose into the cell can be expressed (i.e., overexpressed) in the cell, thereby increasing glucose in the cell. IN some embodiments, one or both of galactose:H1symporter (GalP) and glucokinase (Glk) are expressed in the microorganism. For example, in some embodiments, GalP transports glucose into the cell where it is phosphorylated to glucose-6-phosphate by Glk and assimilated into central carbon metabolism.

[0101] In certain embodiments, the galactose:H' symporter (GalP) is as described in EG12148 (EcoCyc) or P0AEP] (UniProt). In certain embodiments, GalP is an E. coli GalP. In certain embodiments, GalP comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, ai least about 97%, at least about98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, GalP comprises the amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, GalP consists of the amino acid sequence set forth in SEQ ID NO: 38. SEQ ID NO: 38 is provided below:

[0102] 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:

[0103] In certain embodiments, the glucokinase (Glk) is as described in EG12957 (EcoCyc) or P0A6V8 (UniProt). In certain embodiments, Glk is an E. coli Glk. In certain embodiments, Glk comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about90%, 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, Glk comprises the amino acid sequence set forth in SEQ ID NO: 40. In certain embodiments, Glk consists of the amino acid sequence set forth in SEQ ID NO: 40. SEQ ID NO: 40 is provided below:

[0104] 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:

[0105] Without being bound by any theory, the inventors of the present disclosure believe that any enzyme performing similar function to the enzyme described above can be used in the presently disclosed microorganisms. For example, but w'ilhoul any limitation, the presently disclosed microorganism can include any enzyme that catalyzes the reversible epimerization of D-fructose-6-phosphate to D-psicose-6-phosphale. In another non-limiting example, the presently disclosed microorganism can include any enzyme that catalyzes the reversible isomerization of D-psicose-6-phosphate to D-allose-6-phsophate. In another non-limiting example, the presentlydisclosed microorganism can include any enzyme that dephosphorylates allose-6-phosphale to Tree allose.2.2. _ Competing Pathways

[0106] In certain embodiments, the presently disclosed microorganisms include a mutation of one or more gene encoding one or more enzymes regulating biochemical pathways that can reduce the production of allose. In certain embodiments, the presently disclosed microorganisms include a reduced expression of a gene encoding enzymes regulating biochemical pathways that can reduce the production of allose. Physiologically, cells catalyze sugars through the pentose phosphate pathway and glycolysis to produce energy (e g., ATP). The inventors of the present disclosure discovered that deletion or reduced expression of gene encoding enzymes of certain metabolic pathways results in increased allose production.

[0107] In certain embodiments, the presently disclosed microorganisms include a mutation of a gene encoding an enzyme of the pentose phosphate pathway. In certain embodiments, the presently disclosed microorganisms include a reduced expression of a gene encoding an enzyme of the pentose phosphate pathway. In certain embodiments, the enzyme of the pentose phosphate pathway is selected from the group consisting of glucose-6-phosphale dehydrogenase, 6- phosphogluconolactonase, phosphogluconate dehydrogenase, phosphopentose isomerase, phosphopenlose epimerase, transketolase, and transaldolase. In certain embodiments, the enzyme of the pentose phosphate pathway is glucose-6- phosphate dehydrogenase (Zwf) (Entrez Gene ID: 946370). Zwf catalyzes the oxidation of glucose-6-phosphate to 6-phosphogluconolactone. In certain embodiments, Zwf is an E. coli Zwf. A representative nucleotide sequence of the gene zwf is set forth in SEQ ID NO: 6 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 6. SEQ ID NO: 6 is provided below:

[0108] In certain embodiments, 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, E. coli Zwf comprises the amino acid sequence set forth in SEQ ID NO: 20. SEQ ID NO: 20 is provided below:

[0109] In certain embodiments, Zwf is a Bacillus subtilis Zwf. A representative amino acid sequence of Bacillus subfilis Zwf is found as P54547 (Uniprot) / BSU23850 (KEGG) or as set forth in SEQ ID NO: 10 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 10. SEQ ID NO: 10 is provided below:

[0110] A representative nucleotide sequence of Bacillus subtilis zwf gene is set forth in SEQ ID NO: 26 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 26. SEQ ID NO: 26 is provided below:

[0111] In certain embodiments, Zwf is a Ixtctococcus lactis Zwf. A representative amino acid sequence of Lactococcus laelix Zwf is found as LLA12 RS 12225: glucose-6-phosphate dehydrogenase, EC1.1.1.49, or as set forth in SEQ ID NO: 1 1 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 11. SEQ ID NO: 11 is provided below:

[0112] A representative nucleotide sequence of a Lactococcus lactis zwf gene is set forth in SEQ ID NO: 33 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 33. SEQ ID NO: 33 is provided below:

[0113] In certain embodiments, the presently disclosed microorganisms include a mutation of a gene encoding an enzyme of the glycogen biosynthesis. In certain embodiments, the presently disclosed microorganisms include a reduced expression of a gene encoding an enzyme of the glycogen biosynthesis. In certain embodiments, the enzyme of the glycogen biosynthesis is selected from the group consisting of phosphoglucomutase (Pgm), UDP-glucose pyrophosphorylase, glycogen synthase, glycogen branching enzyme, and glycogenin. In certain embodiments, the enzyme of the glycogen biosynthesis is phosphoglucomutase (Pgm) (Entrez Gene ID: 946370). Pgm (EC 5.4.2.2) is an enzyme that transfers a phosphate group on an a-D- glucose monomer from the 1 to the 6 position in the forward direction or the 6 to the 1 position in the reverse direction. In certain embodiments, Pgm is an E. coli Pgm. A representative nucleotide sequence of gene pgm is set forth in SEQ ID NO: 42 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 47. SEQ ID NO: 47 is provided below:

[0114] In certain embodiments, 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, E. coli Pgm comprises the amino acid sequence set forth in SEQ ID NO: 48. SEQ ID NO: 48 is provided below:

[0115] In certain embodiments, Pgm is a Bacillus subiilis Pgm. A representative nucleotide sequence of gene pgm is set forth in SEQ ID NO: 49 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 49. SEQ ID NO: 49 is provided below:

[0116] In certain embodiments, Bacillus subiilis 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, Bacillus subtilis Pgm comprises the amino acid sequence set forth in SEQ ID NO: 50. SEQ ID NO: 50 is provided below:[0117| In certain embodiments, Pgm is a Bacillus subtilis Pgm. A representative nucleotide sequence of gene pgm is set forth in SEQ ID NO: 81 or at least 90%, 95%, 95%, or 99% identical to SEQ 1 D NO: 81. SEQ ID NO: 81 is provided below:

[0118] In certain embodiments, Bacillus subtilis Pgm comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 82, In certain embodiments, Bacillus subtilis Pgm comprises the amino acid sequence set forth in SEQ ID NO: 82. SEQ ID NO: 82 is provided below:

[0119] In ceriain embodiments, Pgm is a Lactococcus laclis Pgm. A representative nucleotide sequence of gene pgm is set forlh in SEQ ID NO: 51 or al leasi 90%, 95%, 95%, or 99%i identical to SEQ ID NO: 51. SEQ ID NO: 51 is provided below:

[0120] In certain embodiments, Lactococcus laclis 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, Laclococcus laclis Pgm comprises the amino acid sequence set forth in SEQ ID NO: 52. SEQ ID NO: 52 is provided below:

[0012] ] In certain embodiments, the presently disclosed microorganisms include a mutation of a gene encoding an enzyme of glycolysis. In certain embodiments, the presently disclosed microorganisms include a reduced expression of a gene encoding an enzyme of the glycolysis. In certain embodiments, the enzyme of the glycolysis is selected from the group consisting ofphosphofruclokinase A, phosphofructokinase B, fructose-biphosphale aldolase, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, and pyruvate kinase. In certain embodiments, the enzyme of glycolysis is phosphofructokinase B (PfkB). In certain embodiments, the enzyme of glycolysis is pyruvate kinase. In certain embodiments, the enzyme of glycolysis is phosphofructokinase A (PfkA) (Entrez Gene ID: 948412). PfkA catalyzes the phosphorylation ofD-fructose-6-phosphate to fructose 1 ,6-bisphosphate by ATP, the first committing step of glycolysis. In certain embodiments, PfkA is an E. coli PfkA, A representative nucleotide sequence of gene pfkA is set forth in SEQ ID NO: 7. SEQ ID NO: 7 is provided below:

[0122] In certain embodiments, E. coli PIkA 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 sei forth in SEQ ID NO: 2i. In certain embodiments, E. coli PfkA comprises the amino acid sequence set forth in SEQ ID NO: 21. SEQ ID NO: 21 is provided below:

[0123] In certain embodiments, PfkA is a Bacillus subfilis PfkA, A representative amino acid sequence of Bacillus subfilis PfkA is found as 034529 (Uniprot) / BSU29190 (K.EGG) or as set forth in SEQ ID NO: 12 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 12. SEQ ID NO: 12 is provided below:

[0124] A representative nucleotide sequence of a Bacillus subtilis gene pfkA is set forth in SEQID NO: 27 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 27. SEQ ID NO: 27 is provided below:

[0125] In certain embodiments, PfkA is a Lactococcus laciis PfkA. A representative amino acid sequence of Lactococcus lactis PfkA is found as LLA12_RS07020: ATP-dependent 6- phospho fructokinase, EC2.7.1.11 , or as set forth in SEQ ID NO: 13 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 13, which is provided below:

[0126] A representative nucleotide sequence of a Lactococcus laciis gene pfkA is set forth in SEQ ID NO: 32 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 32. SEQ ID NO: 32 is provided below:

[0127] In certain embodiments, PfkB is an coli PfkB. A representative nucleotide sequence of genepJkB is set forth in SEQ ID NO: 22. SEQ ID NO: 22 is provided below:

[0128] In certain embodiments, E. co / i 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, E. coli PfkB comprises the amino acid sequence set forth in SEQ ID NO: 23. SEQ ID NO: 23 is provided below:

[0129] In certain embodiments, the presently disclosed microorganisms do not include a deletion or a reduced expression of hexokinase. In certain embodiments, the presently disclosed microorganisms do not include a deletion, a disruption, or a reduced expression of glucokinase. In certain embodiments, the presently disclosed microorganisms do not include a deletion or a reduced expression of glucose-6-phosphate isomerase.

[0130] In certain embodiments, the presently disclosed microorganisms include mutation of a gene encoding an enzyme of the mannose biosynthesis pathway. In certain embodiments, the presently disclosed microorganisms include a reduced expression of a gene encoding an enzyme of the mannose biosynthesis pathway. In certain embodiments, the enzyme of the mannose biosynthesis pathway is mannose-6-phosphate isomerase (ManA) (Enlrez Gene ID: 944840). ManA is involved in the synthesis of the GDP-mannose and dolichol-phosphate-mannose required for a number of critical mannosyl transfer reactions. ManA also catalyzes the inlerconversion of fructose-6-phosphaie and mannose-6-phosphate. In certain embodiments, ManA is an E. coll ManA. A representative nucleotide sequence of gene manA is set forth in SEQ ID NO: 9 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 9, which is provided below'.

[0013] ] 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:

[0132] In certain embodiments, ManA is a Bacillus subtil is Man A. A representative amino acid sequence of ManA is found at 031646 (Uniprol) / BSU12020 (KEGG) or is set forth in SEQ ID NO: 16 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 16.

[0133] A representative nucleotide sequence of a Bacillus subiilis gene mauA is set forth in SEQ ID NO: 29 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 29.

[0134] In certain embodiments, in Bacillus subtilis, optionally in combination with deletion of Bacillus subtilis ManA, one or both of the following are also deleted.[01351 (i) Yvyl of Bacillus subtilis. A representative amino acid sequence of Yvyl is found at P39841 (Uniprol) / BSU35790 (KEGG) or is set forth in SEQ ID NO: 17 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 17. SEQ ID NO: 17 is provided below:

[0136] A representative nucleotide sequence of Bacillus subtilis gene yvyl is set forth in SEQ ID NO: 30 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 30. SEQ ID NO: 30 is provided below:

[0137] (ii) GmuF of Bacillus subtilis. A representative amino acid sequence of GmuF is found al 0055 H (Uniprol) / BSU05870 (KEGG) or is set forth in SEQ ID NO: 18 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: IS. SEQ ID NO: 18 is provided below:

[0138] A representative nucleotide sequence of a Bacillus subtilis gene gmuB is set forth in SEQ ID NO: 31 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 31 . SEQ ID NO: 31 is provided below:

[0139] In certain embodiments, ManA is a 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 at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 19. SEQ ID NO: 19 is provided below:

[0140] A representative nucleotide sequence of a Lactococcus lactis gene manA is set forth in SEQ ID NO: 35 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 35. SEQ ID NO: 35 is provided below:

[0014] ] In certain embodiments, the phosphatase is a Hexitol phosphatase B (HxpB) (UniProl No. P77247 or UniProt No. Q7ADF8). HxpB catalyzes the dephosphorylalion of D-psicose-6- phosphate. In certain embodiments, HxpB is an 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:

[0142] 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:

[0143] 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:

[0144] In certain embodiments, the phosphatase is a sugar phosphatase, e.g., YbiV, G6425 (EcoCyc) P75792 (UniProt) and catalyzes the dephosphorylation of D-psicose-6-phosphate. In certain embodiments, YbiV is an E. coli YbiV. In certain embodiments, YbiV comprises an amino acid sequence that is at least about 80%. at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 36. In certain embodiments, YbiV comprises the amino acid sequence set forth in SEQ ID NO: 36. In certain embodiments, YbiV consists of the amino acid sequence set forth in SEQ ID NO: 36. SEQ ID NO: 36 is provided below:

[0145] In certain embodiments, the geneybiV 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, ybi I' comprises thenucleotide sequence set forth in SEQ ID NO: 37. In certain embodiments, ybiE consists of the nucleotide sequence set forth in SEQ ID NO: 37. SEQ ID NO: 37 is provided below:[0146| In certain embodiments, the presently disclosed microorganisms include mutation of a gene encoding an aldolase. In certain embodiments, the presently disclosed microorganisms include a reduced expression of a gene encoding an aldolase. In certain embodiments, the aldolase is deoxyribose-phosphate aldolase (DeoC) (Entrez Gene ID: 948902). DeoC catalyzes the reversible conversion of allose-6-phosphate to erythrose-4-phosphate and glycolaldehyde. In certain embodiments, DeoC is an E. coli DeoC. A representative nucleotide sequence of gene deoC is set forth in SEQ ID NO: 59 or at least 90%, 95%. 95%, or 99% identical to SEQ ID NO: 59, which is provided below.

[0147] In certain embodiments, E. coli DeoC 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: 58. In certain embodiments, E. coli DeoC comprises the amino acid sequence set forth in SEQ ID NO: 58. SEQ ID NO: 58 is provided below:

[0148] In certain embodiments, the presently disclosed microorganisms include mutation of a gene encoding a phosphatase. In certain embodiments, the presently disclosed microorganisms include a reduced expression of a gene encoding a phosphatase. In certain embodiments, the phosphatase is fructose- 1 -phosphate phosphatase (YqaB) (Entrez Gene ID: 945776; EcoCyc ID: G7408; UniProt ID: P77475). YqaB catalyzes the dephosphorylation of allulose-6-phosphate. In certain embodiments, YqaB is an E. coli YqaB. A representative nucleotide sequence of gene yqaB is set forth in SEQ ID NO: 61 or SEQ ID NO: 80, or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 61 or SEQ ID NO: 80, which are provided below.

[0149] In certain embodiments, E. coli YqaB 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: 60. in certain embodiments, E. coli YqaB comprises the amino acid sequence set forth in SEQ ID NO: 60. SEQ ID NO: 60 is provided below:

[0150] In certain embodiments, the phosphatase is a sugar phosphatase, e.g., YidA, P0A8Y5 (UniProt) or EG1 1195 (EcoCyc), and catalyzes the dephosphorylation of D-psicose 6-phosphate. In certain embodiments, YidA is an E. coli YidA. In certain embodiments, YidA 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: 77. In certain embodiments, YidA comprises the amino acid sequence set forth in SEQ ID NO: 77. In certain embodiments, YidA consists of the amino acid sequence set forth in SEQ ID NO: 77. SEQ ID NO: 77 is provided below:

[0151] In certain embodiments, the gene YidA 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: 78. In certain embodiments, yidA comprises the nucleotide sequence set forth in SEQ ID NO: 78. In certain embodiments, yidA consists of the nucleotide sequence set forth in SEQ ID NO: 78. SEQ ID NO: 78 is provided below:

[0152] In certain embodiments, the gene yidA 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: 83. In certain embodiments, yidA comprises thenucleotide sequence set forth in SEQ ID NO: S3. In certain embodiments, yidA consists of the nucleotide sequence set forth in SEQ ID NO: 83. SEQ ID NO: 83 is provided below:

[0153] In certain embodiments, the presently disclosed microorganisms include a mutation of a gene encoding an enzyme of al lose metabolism. In certain embodiments, the presently disclosed microorganisms include a reduced expression of a gene encoding an enzyme of allose metabolism.

[0154] In certain embodiments, the enzyme of allose metabolism is D-allose import ATP-binding protein (AlsA) (Entrez Gene ID: 948593; EcoCyc ID: EG11959; UniProt ID: P32721). In certain embodiments, AlsA is an E. coli AlsA. A representative nucleotide sequence of gene a / sA is set forth in SEQ ID NO: 65 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 65, which is provided below.

[0155] In certain embodiments, E. coh AlsA 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: 64. In certain embodiments, E. coli AlsA comprises the amino acid sequence set forth in SEQ ID NO: 64. SEQ ID NO: 64 is provided below:

[0156] In certain embodiments, the enzyme of allose metabolism is D-allose-binding periplasmic protein (AlsB) (Entrez Gene ID: 948604: EcoCyc ID: EG 12458; UniProt ID: P39265). In certain embodiments, AlsB is an E. coli AlsB. A representative nucleotide sequence of gene AlsB is set forth in SEQ ID NO: 67 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 67, which is provided below.

[0157] In certain embodiments, E. coh AlsB 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: 66. In certain embodiments, E. coli AlsB comprises the amino acid sequence set forth in SEQ ID NO: 66. SEQ ID NO: 66 is provided below:

[0158] In certain embodiments, the enzyme of allose metabolism is D-allose transport system permease protein (AlsC) (Entrez Gene ID: 948594; EcoCyc ID: EG11958; UniProl ID: P32720). In certain embodiments, AlsC is an E. coli AlsC. A representative nucleotide sequence of gene alsC is set forth in SEQ ID NO: 69 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 69, which is provided below.

[0159] In certain embodiments, E. coli AlsC 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: 68. In certain embodiments, E. coli AlsC comprises the amino acid sequence set forth in SEQ ID NO: 68. SEQ ID NO: 68 is provided below:

[0160] In certain embodiments, the enzyme of allose metabolism is D-allose kinase (AlsK) (Entrez Gene ID: 948596; EcoCyc ID: EG11956; UniProt ID: P32718). In certain embodiments, AlsK is an E. coli AlsK. A representative nucleotide sequence of gene alsK is set forth in SEQ ID NO: 71 or at least 90%, 95%, 95%. or 99% identical to SEQ ID NO: 71, which is provided below

[0161] In certain embodiments, E. coli AlsK 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: 70, In certain embodiments, E. coli AlsK comprises the amino acid sequence set forth in SEQ ID NO: 70. SEQ ID NO: 66 is provided below:

[0162] In certain embodiments, the enzyme of al lose metabolism is HTH-type transcriptional regulator (AlsR) (Entrez Gene ID: 948603; EcoCyc ID: G782I ; UniProl ID: P0ACS7). In certain embodiments, AlsR is an E. coli AlsR. A representative nucleotide sequence of gene alsR is set forth in SEQ ID NO: 73 or SEQ ID NO: 84, or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 73 or SEQ ID NO: 84, which is provided below.

[0163] In certain embodiments, E. coli AlsR 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: 72. In certain embodiments, E. coli AlsR comprises the amino acid sequence set forth in SEQ ID NO: 72. SEQ ID NO: 72 is provided below:

[0164] In certain embodiments, the deletion of a gene comprises a non-l'rameshift deletion, a frameshift deletion, or a combination thereof. In certain embodiments, the deletion of a gene can be achieved by an insertion (e.g., a non- frameshift insertion, a frameshift insertion, or a combination thereof)- In certain embodiments, the deletion of a gene comprises a nonsense mutation.2.3. Cells

[0165] 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 certain embodiments, the microorganism is selected from the group consisting of Aceiobacter aceti, Achromobacter, Acidiphilium. Acineiobacter. Actinomadura, Actinoplanes. Aeropyrumpemix, Agrobacierium. Alcaligenes, Ananas comosns (M),Arthrobacler, Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulars, Bacillus clausii, Bacillus lentuss, Bacillus lichenifirmis, Bacillus macerans, Bacillus stearothermophilus, Bacillus subfilis, Bifidobacterium, Brevibacillus brevis, Burkholderia cepacia,Candida cylindracea, Carica papaya (L), Cellulosimicrobium. Cephalosporium, Chaetomium erraticum, Chaetomium gracile. Clostridium, Clostridium butyricum, Clostridium acetobutylicum, Clostridium lhermocellum, Corynebacterium (giutamicum), Corynebacterium efftciens, Escherichia coll, Enterococcus, Erwina chrysanthemi, (Jliconobacter. Gluconacetobacter, Haloarcula. Humicola insolens, Kitasatospora setae. Klebsiella, Klebsiella oxytoca, Kocuria, Lacllactis, Lactobacillus. Lactobacillus fermentum, Lactobacillus sake. Lactococcus, Lactococcus laclis. Leuconostoc, Methylocystis, Methanolobus sicillae. Methanogen him organophilum. Melhanobacterium bryantil, Microbacterium imperiale, Micrococcus lysodeikiicus, Microlunaius, Mucorjavanicus, Mycobacterium, Myrothecium, Nitrobacter, Niirosomonas, Nocardia, Papaya carica, Pediococcus. Pediococcus halophilus, Paracoccus pantotrophus, Propionibacterium, Pseudomonas, Pseudomonasfluorescens, Pseudomonas denitrifcans, Pyrococcus, Pyrococcusfuriosus, Pyrococcus horikoshii, Rhizobium, Rhizomucor miehei, Rhizomucor pusillus Lindt, Rhizopus, Rhizopus delemar, Rhizopus japonicas, Rhizopus niveus. Rhizopus oryzae, Rhizopus oligospoms. Rhodococcus, Sckroiina libertina, Sphingobacterium multivorum, Sphingobium, Sphingomonas, Streptococcus. Streptococcus thermophilus Y-l, Streptomyces, Streptomyces griseus, Streptomyces lividans, Streptomyces murinus, Streptomyces ruhiginosus. Streptomyces violaceoruber, StreptoverticiUium mobaraense,Tetragenocoecus. Thermits. Thiosphaera pantotropha, Trameles, Vibrio alginolyticus, Xanthomonas, Zymomonas, and Zymomonus mobilis. In certain embodiments, the microorganism is Escherichia coli (E. coli). In certain embodiments, the microorganism is bacillus subtilis. In certain embodiments, the microorganism is Lactococcus lactis.

[0166] 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), Enterohenrorrhagic E. coli(EHEC), Uropathogenic E. coli (UPEC), Veroloxin-producing E. coli, E. coli 0157:H7, E. coli 0104: H4, E. coli 0121 , 7-7. coli O104: H21 , E. coli KI, and E. coli NC 101. In certain embodiments, the E. coli is E. coli KI 2. In certain embodiments, the E. coli is E. coli B. In certain embodiments, the E. coli is E. coli C.

[0167] In certain embodiments, the E. coli is derived from a strain selected from the group consisting of NCTC 12757, NCTC 12779, NCTC 12790, NCTC 12796, NCTC 12X11, 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.

[0168] In certain embodiments, the E. coli is derived from a strain selected from the group consisting of BC 4734 (O26:H1 1), BC 4735 (O157:H-), BC 4736 , BC 4737 (n.d.), BC 4738 (O157:H7), BC 4945 (O26:H-), BC 4946 (O157:H7), BC 4947 (O11EH-), BC 4948 (O157:H),BC 4949 (05), BC 5579 (OI57:H7), BC 5580 (O157:H7), BC 5582 (O3:H), BC 5643 (O2:H5), BC 5644 (0128), BC 5645 (055:H-), BC 5646 (069:H-), BC 5647 (01(H:H9), BC 5648 (O103:H2), BC 5850 (O22:H8), BC 5851 (O55:H-), BC 5852 (O48:H21), BC 5853 (O26:HI 1), BC 5854 (O157:H7), BC 5855 (O157:H-), BC 5856 (O26:H-), BC 5857 (O1()3:H2), BC 5858 (026: Hl 1), BC 7832, BC 7833 (O raw formiH-), BC 7834 (ONTiH-), BC 7835 (O103:H2), BC 7836 (O57:H-), BC 7837 (ONT:H-), BC 7838, BC 7839 (O128:H2), BC 7840 (O157:H-), BC 784] (O23:H-), BC 7842 (O157:H-), BC 7843, BC 7844 (O157:H-), BC 7845 (O1()3:H2), BC 7846(026:1411), BC 7847 (O145:H-), BC 7848 (0157:H-), BC 7849 (0156:H47), BC 7850, BC 7851 (O157:H-), BC 7852 (O157:H-), BC 7853 (O5:H-), BC 7854 (O1571H7), 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 raw fornrH-), BC 7865, BC 7866 (O26:H- ), BC 7867 (O raw form:H-), BC 7868, BC 7869 (ONT:H-), BC 7870 (0113:H-), BC 7871 (ONT:H-), BC 7872 (0NT:H-), BC 7873, BC 7874 (O raw form:H-). BC 7875 (O157:H-), BC 7876 (0111:H-), BC 7877 (O146:H21), BC 7878 (OI45:H-), BC 7879 (O22:H8), BC 7880 (O raw form:H-), BC 7881 (O145:H-), BC 8275 (0157:H7), BC 8318 (O55:K-:H-), BC 8325 (0157:H7), BC 8332 (ONT), and BC 8333,

[0169] In certain embodiments, the E. coli is derived from a strain selected Prom the group consisting of BC 8246 (0152:K-:H-), BC 8247 (O124:K(72):H3), BC 8248 (0124), BC 8249 (0112), BC 8250 (O136:K(78):H-), BC 8251 (O124:H-), BC 8252 (O144:K-:H-), BC 8253 (O143:K:H-), BC 8254 (0143), BC 8255 (0112), BC 8256 (O28a.e), BC 8257 (O124:H-)> BC 8258 (0143), BC 8259 (O167:K-:H5), BC 8260 (0128a. c.:H35), BC 8261 (0164), BC 8262 (0164:K-:H-), BC 8263 ( 0164), and BC 8264 (0124).

[0170] In certain embodiments, the E. coli is derived from a strain selected from the group consisting ofBC 5581 (O78:H11 ), BC 5583 (O2:K1), BC 8221 (0118), BC 8222 (O148:H-), BC 8223 (0111), BC 8224 (011():H-), BC 8225 (0148), BC 8226 (0118), BC 8227 (O25:H42), BC 8229 (06), BC 8231 (O153:H45), BC 8232 (09), BC 8233 (0148), BC 8234 (0128), BC 8235 (0118), BC 8237 (01 11), BC 8238 (01 IO:H 17), BC 8240 (0148), BC 8241 (O6H16), BC 8243 (0153), BC 8244 (015:H-), BC 8245 (020), BC 8269 (O125a.c:H-), BC 8313 (O6:H6), BC 8315 (O153:H-), BC 8329, BC 8334 (01 18:H 12), and BC 8339.

[0171] In certain embodiments, the E. coli is derived from a strain selected from the group consisting of BC 7567 (086), BC 7568 (0128), BC 7571 (0114), BC 7572 (0119), BC 7573 (0125), BC 7574 (0124), BC 7576 (O127a), BC 7577 (0126), BC 7578 (0142), BC 7579 (026), BC 7580 (OK.26), BC 7581 (0142), BC 7582 (055), BC 7583 (0158), BC 7584 (0-), BC 7585 (0-), BC 7586 (0-), BC 8330, BC 8550 (026), BC 8551 (055), BC 8552 (0158), BC 8553 (026), BC 8554 (0158), BC 8555 (086), BC 8556 (0128), BC 8557 (0K26), BC 8558 (055), BC 8560(0158), BC 856 ] (0158), BC 8562 (01 14), BC 8563 (086), BC 8564 (0128), BC 8565 (0158), BC 8566 (0158), BC 8567 (0158), BC 8568 (01 i 1), BC 8569 (0128), BC 8570 (01 14), BC 8571 (0128), BC 8572 (0128), BC 8573 (0158), BC 8574 (0158), BC 8575 (0158), BC 8576 (0158), BC 8577 (0158), BC 8578 (0158), BC 8581 (0158), BC 8583 (0128), BC 8584 (0158), BC 8585 (0128), BC 8586 (0158), BC 8588 (026), BC 8589 (086), BC 8590 (0127), BC 8591 (0128), BC 8592 (01 14), BC 8593 (O1 14), BC 8594 (0114), BC 8595 (0125), BC 8596 (0158), BC 8597 (026), BC 8598 (026), BC 8599 (0158), BC 8605 (0158), BC 8606 (0158), BC 8607 (0158), BC 8608 (0128), BC 8609 (055), BC 8610 (0114), BC 8615 (0158), BC 8616 (0128), BC 8617 (026), BC 8618 (086), BC 8619, BC 8620, BC 8621, BC 8622, BC 8623, BC 8624 (0158), and BC 8625 (0158).

[0172] In certain embodiments, the B. subtilis is derived from Strain 168.

[0173] In certain embodiments, the L. laclis is derived from Strain A12.

[0174] In certain embodiments, the microorganism is a fungal cell. In certain embodiments, the fungal cell is selected from the group consisting of Aspergillus, Aspergillus mdulans, Aspargillus niger, Aspargillus oryze, Aspergillus melleus, Aspergillus pulvenilennis, Aspergillus saiioi, Aspergillus sojea, Aspergillus ierreus, Aspergillus pseudoterreus, Aspergillus usamii, Candida rugosa, Issatchenkia orientalis, Kluyveromyces, Kluyveromycesfragilis. Kluyveromyces laclis. Kluyveromyces marxianas, Penicillium, Penicillium camembert, Penicillium cilrinum, Penicillium emersonii, Penicillium roqueforli, Penicillum lilactinum, Penicillum multicolor, Rhodosporidium toruloides, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, Trichoderma longibrachiaium, Trichoderma reesei, Trichoderma viride, Trichosporon penicillaium, Yarrowia lipolylica, and Zygosaccharomyces rouxii.

[0175] In certain embodiments, the microorganism is a yeast cell. In certain embodiments, the yeast cell is Saccharomyces cerevisiae.2.4. Exemplary microorganisms

[0176] In certain embodiments, the present disclosure provides a recombinant microorganism comprising an increased production of allose as compared to a naturally occurring microorganism. In certain embodiments, the recombinant microorganism comprises an exogenous epimerase, an exogenous isomerase and an exogenous phosphatase. In some embodiments, the exogenous epimerase is an allulose-6-phosphate 3-epimerase (AlsE). In certain embodiments, the exogenous isomerase is allose-6-phosphale isomerase (RpiB). In certain embodiments, the exogenous phosphatase is a hexitol phosphatase A (HxpA). In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is E. coll.

[0177] In certain embodiments, the present disclosure provides a recombinant microorganism comprising an increased production of allose as compared to a naturally occurring microorganism.In certain embodiments, the recombinant microorganism comprises an exogenous epimerase, an exogenous isomerase, and an exogenous phosphatase. In some embodiments, the exogenous epimerase is an allulose-6-phosphate 3-epimerase (AlsE). In certain embodiments, the exogenous isomerase is allose-6-phosphate isomerase (RpiB). In certain embodiments, the exogenous phosphatase is fructose- 1 -phosphate phosphatase (YqaB). In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is E. coli.

[0178] In certain embodiments, the present disclosure provides a recombinant microorganism comprising an increased production ofallose as compared to a naturally occurring microorganism. In certain embodiments, the recombinant microorganism comprises an exogenous epimerase, an exogenous isomerase, an exogenous phosphatase, and a deletion of one, two, three, or four (4), or more genes. In certain embodiments, the exogenous epimerase is an allulose-6-phosphate 3- epimerase (AlsE). In certain embodiments, the exogenous isomerase is an allose-6-phosphate isomerase (RpiB). In certain embodiments, the exogenous phosphatase is a hexitol phosphatase A (HxpA). In certain embodiments, the deleted genes are g]ucose-6-phosphate 1 -dehydrogenase, phospho fructokinase-1, and mannose-6-phosphate isomerase. In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is E. coli.

[0179] In certain embodiments, the present disclosure provides a recombinant microorganism comprising an increased production of allose as compared to a naturally occurring microorganism. In certain embodiments, the recombinant microorganism comprises an exogenous epimerase, an exogenous isomerase, an exogenous phosphatase, and a deletion of one, two, three, or four (4), or more genes. In certain embodiments, the exogenous epimerase is an allulose-6-phosphale 3- epimerase (AlsE). In certain embodiments, the exogenous isomerase is an allose-6-phosphate isomerase (RpiB). In certain embodiments, the exogenous phosphatase is a fructose- 1 -phosphate phosphatase (YqaB). In certain embodiments, the deleted genes are glucose-6-phosphale l- dehydrogenase, phosphofructokinase- 1, and mannose-6-phosphate isomerase. In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is E. coli.

[0180] In certain embodiments, the present disclosure provides a microorganism comprising a recombinant polynucleotide, wherein the microorganism comprises an increased production of allose as compared to a naturally occurring microorganism. In certain embodiments, the recombinant polynucleotide comprises a nucleotide sequence encoding an exogenous epimerase, an exogenous isomerase, and an exogenous phosphatase. In some embodiments, the epimerase is an allulose-6-phosphate 3-epimerase (AlsE). In certain embodiments, the exogenous isomerase is allose-6-phosphate isomerase (RpiB). In certain embodiments, the exogenous phosphatase is ahexilol phosphatase A (HxpA). In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is E. coli.

[0181] In certain embodiments, the present disclosure provides a microorganism comprising a recombinant polynucleotide, wherein the microorganism comprises an increased production of allose as compared to a naturally occurring microorganism. In certain embodiments, the recombinant polynucleotide comprises a nucleotide sequence encoding an exogenous epimerase, an exogenous isomerase, and an exogenous phosphatase. In certain embodiments, the exogenous epimerase is an allulose-6-phosphate 3-epimerase (AlsE). In certain embodiments, the exogenous isomerase is allose-6-phosphate isomerase (RpiB). In certain embodiments, the exogenous phosphatase is a fructose- 1 -phosphate phosphatase (YqaB). In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is E. coli.

[0182] In certain embodiments, the present disclosure provides a microorganism comprising a recombinant polynucleotide, wherein the microorganism comprises an increased production of allose as compared to a naturally occurring microorganism. In certain embodiments, the recombinant polynucleotide comprises a nucleotide sequence encoding an exogenous epimerase, an exogenous isomerase, and an exogenous phosphatase. In some embodiments, the exogenous epimerase is an allulose-6-phosphale 3-epimerase (AlsE), In certain embodiments, the exogenous isomerase is allose-6-phosphale isomerase (RpiB), In certain embodiments, the exogenous phosphatase is a hexitol phosphatase A (HxpA), In certain embodiments, the recombinant polynucleotide further comprises a nucleotide encoding an exogenous isomerase. In certain embodiments, the exogenous isomerase is alIose-6-phosphate isomerase (RpiB). In certain embodiments, the microorganism further comprises a deletion of a first gene. In certain embodiments, the first gene is glucose-6-phosphale 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 mannose-6- phosphale isomerase. In certain embodiments, the microorganism further comprises a deletion of a fourth gene. In certain embodiments, the fourth gene is deoxyribose-phosphale aldolase (DeoC). In certain embodiments, in addition to the four deleted genes, the microorganism further comprises a deletion of at least one gene selected from D-allose import ATP-binding protein (AlsA), D-allose-binding periplasmic protein (AlsB), D-allose transport system permease protein (AlsC), D-allose kinase (AlsK), HTH-type transcriptional regulator (AlsR), fructose- 1 -phosphate phosphatase (YqaB), sugar phosphatase YbiV, hexitol phosphatase B (HxpB), sugar phosphatase YidA, or combinations thereof. In certain embodiments, the recombinant microorganism further comprises exogenous galactose:H' symportcr (GalP) and glucokinase (Glk). In certainembodiments, the GalP is an E. coli GalP and the Glk is E. coli Glk. In certain embodiments, the recombinant microorganism further comprises exogenous glucose-6-phosphate isomerase (Gpi). In certain embodiments, the recombinant microorganism further comprises a mutation of a gene encoding for an enzyme of the phosphotransferase system (PTS). In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is E. coli. In certain embodiments, the bacterium is B. sublilis. In certain embodiments, the bacterium is L. lactis.

[0183] In certain embodiments, the present disclosure provides a microorganism comprising a recombinant polynucleotide, wherein the microorganism comprises an increased production of allose as compared to a naturally occurring microorganism. In certain embodiments, the recombinant polynucleotide comprises a nucleotide sequence encoding an exogenous epimerase, an exogenous isomerase, and an exogenous phosphatase. In some embodiments, the exogenous epimerase is an allulose-6-phosphate 3-epimerase (AlsE). In certain embodiments, the exogenous isomerase is allose-6-phosphate isomerase (RpiB). In certain embodiments, the exogenous phosphatase is a fructose-1-phosphate phosphatase (YqaB). In certain embodiments, the recombinant polynucleotide further comprises a nucleotide encoding an exogenous isomerase. In certain embodiments, the exogenous isomerase is allose-6-phosphate isomerase (RpiB). 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 mannose-6- phosphate isomerase. In certain embodiments, the microorganism further comprises a deletion of a fourth gene. In certain embodiments, the fourth gene is deoxyribose-phosphate aldolase (DeoC). In certain embodiments, in addition to the four deleted genes, the microorganism further comprises a deletion of at least one gene selected from D-allose import ATP-binding protein (AlsA), D-allose-binding periplasmic protein (AlsB), D-allose transport system permease protein (AlsC), D-allose kinase (AlsK), HTH-lype transcriptional regulator (AlsR), fructose-1-phosphate phosphatase (YqaB), sugar phosphatase YbiV, hexitol phosphatase B (HxpB), sugar phosphatase YidA, or combinations thereof. In certain embodiments, the recombinant microorganism lurther comprises exogenous galactose:H ' symporler (GalP) and glucokinase (Glk). In certain embodiments, the GalP is an E. coli GalP and the Glk is E. coli Glk. In certain embodiments, the recombinant microorganism further comprises exogenous glucose-6-phosphate isomerase (Gpi). In certain embodiments, the recombinant microorganism further comprises a mutation of a gene encoding for an enzyme of the phosphotransferase system (PTS).

[0184] In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is E. coli. In certain embodiments, the bacterium is B. subtilis. In certain embodiments, the bacterium is L. lactis.

[0185] In certain embodiments, the present disclosure provides a recombinant microorganism comprising an increased production of allose as compared to a naturally occurring microorganism. In certain embodiments, the recombinant microorganism comprises an exogenous epimerase, an exogenous isomerase, an exogenous phosphatase, an exogenous nuclease, a sgRNA, and a deletion of four (4) genes. In certain embodiments, the exogenous epimerase is an allulose-6- phosphate 3-epimerase (AlsE). In certain embodiments, the exogenous isomerase is an allose-6- phosphate isomerase (RpiB). In certain embodiments, the exogenous phosphatase is a hexitol phosphatase A (HxpA). In certain embodiments, the four deleted genes are zwf pfkA, manA, and deoC. In certain embodiments, in addition to the four deleted genes, the microorganism further comprises a deletion of at least one gene selected from alsA, alsB, alsC, alsK, alsR, yqali, yhiV hxpB, yidA, or combinations thereof. In certain embodiments, the sgRNA targets pfkB. In certain embodiments, the recombinant microorganism further comprises exogenous galactose: H1symporler (GalP) and glucokinase (Glk). In certain embodiments, the GalP is an E. coli GalP and the Glk is E. coli Glk. In certain embodiments, the recombinant microorganism further comprises exogenous glucose-6-phosphate isomerase (Gpi). In certain embodiments, the recombinant microorganism further comprises a mutation of a gene encoding for an enzyme of the phosphotransferase system (PTS).

[0186] In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is E. coli. In certain embodiments, the bacterium is B. subtilis. In certain embodiments, the bacterium is L. lactis.

[0187] In certain embodiments, the present disclosure provides a recombinant microorganism comprising an increased production of allose as compared to a naturally occurring microorganism. In certain embodiments, the recombinant microorganism comprises an exogenous epimerase, an exogenous isomerase, an exogenous phosphatase, an exogenous nuclease, a sgRNA, and a deletion of four (4) genes. In certain embodiments, the exogenous epimerase is an allulose-6- phosphate 3-epimerase (AlsE). In certain embodiments, the exogenous isomerase is an allose-6- phosphate isomerase (RpiB). In certain embodiments, the exogenous phosphatase is a fructose- 1-phosphate phosphatase (YqaB). In certain embodiments, the exogenous nuclease is dCas9. In certain embodiments, the four deleted genes are zwf, pfkA, man A, and deoC. In certain embodiments, in addition to the four deleted genes, the microorganism further comprises a deletion of at least one gene selected from alsA, alsB, alsC, alsK, alsR, ybiV, hxpB, yidA, or combinations thereof. In certain embodiments, the sgRNA targets pfkB. In certain embodiments,the recombinant microorganism further comprises exogenous galactose:H' symporter (GalP) and glucokinase (Glk). In certain embodiments, the GalP is an E. coli GalP and the Glk is E. coli Glk. In certain embodiments, the recombinant microorganism further comprises exogenous glucose-6- phosphate isomerase (Gpi). In certain embodiments, the recombinant microorganism further comprises a mutation of a gene encoding for an enzyme of the phosphotransferase system (PTS). In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is E. coli. In certain embodiments, the bacterium is B. sublilis. In certain embodiments, the bacterium is L. lactis.

[0188] In certain embodiments, the present disclosure provides a microorganism comprising a recombinant polynucleotide, wherein the microorganism comprises an increased production of allose as 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 isomerase, a nucleotide sequence encoding an exogenous phosphatase, a nucleotide sequence encoding an exogenous isomerase, and a nucleotide sequence encoding a nuclease. In certain embodiments, the exogenous epimerase is an allulose-6-phosphate 3-epimerase (AlsE). In certain embodiments, the exogenous isomerase is allose-6-phosphale isomerase ( RpiB). In certain embodiments, the exogenous phosphatase is a hexilol phosphatase A (HxpA). 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 manA. In certain embodiments, the microorganism further comprises a deletion of a fourth gene. In certain embodiments, the fourth gene is deoC. In certain embodiments, in addition to the four deleted genes, the microorganism further comprises a deletion of at least one gene selected from alsA, alsB, alsC, alsK, alsR, pgm, yqaB, ybiV, hxpB. yidA, or combinations thereof. In certain embodiments, the microorganism further comprises a sgRNA. In certain embodiments, the sgRNA targets pfkR. In certain embodiments, the recombinant microorganism further comprises exogenous galactose:H’ symporter (GalP) and glucokinase (Glk). In certain embodiments, the GalP is an E. coli GalP and the Glk is E. coli Glk. In certain embodiments, the recombinant microorganism further comprises exogenous glucose-6- phosphate isomerase (Gpi). In certain embodiments, the recombinant microorganism further comprises a mutation of a gene encoding for an enzyme of the phosphotransferase system (PTS). In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments.the bacterium is E. coll. In certain embodiments, the bacterium is B. subtilis. In certain embodiments, the bacterium is L. lactis.

[0189] In certain embodiments, the present disclosure provides a microorganism comprising a recombinant polynucleotide, wherein the microorganism comprises an increased production of allose as compared to a naturally occurring microorganism. In certain embodiments, the recombinant polynucleotide comprises a nucleotide sequence encoding an exogenous epimerase, an exogenous isomerase, a nucleotide sequence encoding an exogenous phosphatase, a nucleotide sequence encoding an exogenous isomerase, and a nucleotide sequence encoding a nuclease. In certain embodiments, the exogenous epimerase is an allulose-6-phosphate 3-epimerase (AlsE). In certain embodiments, the exogenous isomerase is allose-6-phosphate isomerase (RpiB). In certain embodiments, the exogenous phosphatase is a fructose-1-phosphate phosphatase (YqaB). 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 ptkA. In certain embodiments, the microorganism further comprises a deletion of a third gene. In certain embodiments, the third gene is manA. In certain embodiments, the microorganism further comprises a deletion of a fourth gene. In certain embodiments, the fourth gene is deoC. In certain embodiments, in addition to the four deleted genes, the microorganism further comprises a deletion of at least one gene selected from ci / sA, alsB, alsC, alsK, alsR, pgm, ybiV, hxpB, yidA, or combinations thereof. In certain embodiments, the microorganism further comprises a sgRNA. In certain embodiments, the sgRNA targets pfkB. In certain embodiments, the recombinant microorganism further comprises exogenous galactose:H1symporter (GalP) and glucokinase (Glk). In certain embodiments, the GalP is an E. coli GalP and the Glk is E. coli Glk. In certain embodiments, the recombinant microorganism further comprises exogenous glucose-6-phosphate isomerase (Gpi). In certain embodiments, the recombinant microorganism further comprises a mutation of a gene encoding for an enzyme of the phosphotransferase system (PTS). In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is E. coll. In certain embodiments, the bacterium is B. subtilis. In certain embodiments, the bacterium is L. lactis.3. _ Methods for Producing mid Generating Microorganisms Allose

[0190] The present disclosure also provides methods for preparing and / or generating any of the microorganisms disclosed herein. Many recombinant techniques commonly known in the art may be used to introduce one or more recombinant polynucleotides of the present disclosure into a microorganism, including without limitation 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. Additional infonnation on these techniques can be found in Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al,, 1989); Oligonucleotide Synthesis (Gail, 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 el al., eds., 1991).3.1. Recombinant polynucleotides

[0019] ] In certain embodiments, the recombinant polynucleotides disclosed herein can be stably integrated into a microorganism chromosome. In certain embodiments, the recombinant polynucleotides disclosed herein are stably integrated into a microorganism chromosome using homologous recombination, transposition-based chromosomal integration, recombinase-mediated cassette exchange (RMCE; e.g., using a Cre-lox system), or an integrating plasmid (e.g., a yeast integrating plasmid). A variety of integration techniques suitable for a range of microorganisms are known in the art (see, e.g., Griffiths, A.J.F., Miller, J,H,, Suzuki, D.T. et al. An Introduction to Genetic Analysis. 7thed. New York: W.H. Freeman; 2000). In certain embodiments, the recombinant polynucleotides disclosed herein are maintained in a recombinant microorganism of the present disclosure on an extra-chromosomal plasmid (e.g., an expression plasmid or vector). A variety of extra-chromosomal plasmids suitable for a range of microorganisms are known in the art, including without limitation replicating plasmids (e.g., yeast replicating plasmids that include an autonomously replicating sequence, ARS), centromere plasmids (e.g., yeast centromere plasmids that include an autonomously replicating sequence, CEN), episomal plasmids (e.g., 2- p.m plasmids), and / or artificial chromosomes (e.g., yeast artificial chromosomes, YACs, or bacterial artificial chromosomes. BACs),3.1.1. Vectors

[0192] In certain embodiments, the present disclosure provides vectors including the nucleotide sequences 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 without any limitation, 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 and / or expression vector. In certain embodiments, plasmids can be extrachromosomal self-replicating genetic elements (e g., episomal plasmids) when introduced into a microorganism. In certain embodiments, plasmids can integrate into a microorganism chromosome. In certain embodiments, vectors can direct the expression of coding regions to which they arc operatively linked, e.g. “expression vectors,” These expression vectors allow theexpression of exogenous polynucleotides and / or polypeptides in microorganisms. In certain embodiments, the vectors allow the integration of one or more polynucleotides into the genome of a microorganism.

[0193] In certain embodiments, a vector disclosed herein includes a promoter. In certain embodiments, the vector is a bacterial or prokaryotic expression vector. In certain embodiments, the vector is a yeast or fungal cell expression vector.

[0194] In certain embodiments, a vector discloses herein comprises nucleotide sequences in a single operon.3.1.2. Promoters

[0195] In certain non-limiting embodiments, the recombinant polynucleotides disclosed herein include a control sequence, an enhancer, or a promoter. For example, but without any limitation, a nucleotide sequence encoding the alsE gene, the hxpA gene, and / or vigl. gene can be operably linked to a control sequence, enhancer, or promoter.

[0196] As used herein, the term “promoter” refers to any nucleotide sequence that regulates the initiation of transcription for a particular coding sequence under its control. Biologically, promoters are not transcribed but coordinate the assembly of components that initiate the transcription of other nucleotide sequences. In addition, promoters can limit this assembly and subsequent transcription to specific prerequisite conditions. For example, but without any limitation, a promoter can allow transcription in response to one or more environmental, temporal, or developmental stimuli. Bacterial and fungal cells possess a multitude of proteins that sense external or internal conditions and initiate signaling cascades ending in the binding of proteins to specific promoters and subsequent initiation of transcription of nucleic acid(s) under the control of the promoters. 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 particular species.

[0197] 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 result in a robust amount of nucleic acid expression, and, as such, are used in many recombinant engineering applications to achieve a high level of recombinant protein and enzymatic activity. Non-limiting examples of constitutive promoters encompassed by the present disclosure include E. coli promotersfromand the lambda phage promoter (Liang, S.T. 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 phasepromoters can be found in, e.g., Shimada, el al., JOURNAL OF BACTERIOLOGY , Nov. 2004, p. 71 12-7122; Pletnev at el., ACTA NATURAE | VOL. 7 > 4 (27) 2015.

[0198] In certain embodiments, the promoter is an inducible promoter. An inducible promoter is a promoter that drives the expression of a nucleotide sequence in response to a stimulus. An inducible promoter drives sustained expression upon exposure to a specific stimulus (e.g., 1PTG). In certain embodiments, an inducible promoter drives a graded level of expression correlated with the amount of stimulus. Non-limiting examples of stimuli for inducible promoters include heat shock, exogenous compounds or a lack thereof (e.g., a sugar, metal, drug, or phosphate), salts or osmotic shock, oxygen, and biological stimuli (e.g., a growth factor or pheromone). Non-limiting examples of inducible promoters include the E coli promoters and

[0199] In certain embodiments, the recombinant polynucleotide can include multiple promoters. In certain embodiments, the multiple promoters can be the same. For example, but without any limitation, the recombinant polynucleotide can include a nucleotide sequence encoding the alsE gene operably linked to a first promoter and a nucleotide sequence encoding the yigL gene operably linked to a second promoter, wherein the first and second promoter is the same. For example, but without any limitation, the recombinant polynucleotide can include a nucleotide sequence encoding the alsE gene operably linked to a first promoter and a nucleotide sequence encoding the hxpA gene operably linked to a second promoter, wherein the first and second promoter is the same. In certain embodiments, the multiple promoters can be different. For example, but without any limitation, the recombinant polynucleotide can include a nucleotide sequence encoding the alsE gene operably linked to a first promoter and a nucleotide sequence encoding the yigL gene operably linked to a second promoter, wherein the first and second promoter are different. For example, but without any limitation, the recombinant polynucleotide can include a nucleotide sequence encoding the alsE gene operably linked to a first promoter and a nucleotide sequence encoding the hxpA gene operably linked to a second promoter, wherein the first and second promoter are different. In certain embodiments, the promoter is apromoter. In certain embodiments, the promoter comprises the nucleotide sequence setforth in SEQ ID NO: 42. In certain embodiments, the promoter consists of the nucleotidesequence set forth in SEQ ID NO: 42. The promoter is a hybrid regulatory region includingthe promoter of phage lambda with the CI binding sites replaced with The hybrid designallows for a strong promotion that can be repressed by Lad, the Lac inhibitor (i e., repressor) or induced by IPTG.

[0200] In certain embodiments, the promoter is a promoter. In certain embodiments, thepromoter comprises the nucleotide sequence set forth in SEQ ID NO: 43. In certainembodiments, the promoter consists of the nucleotide sequence set forth in SEQ ID NO:43.

[0020] ] In certain embodiments, the promoter is a promoter. In certain embodiments, thepromoter comprises the nucleotide sequence set forth in SEQ ID NO: 44. In certain embodiments, the promoter consists of the nucleotide sequence set forth in SEQ ID NO: 44.

[0202] In certain embodiments, the promoter is a promoter. In certain embodiments, the PMpromoter comprises the nucleotide sequence set forth in SEQ ID NO: 45. In certain embodiments, the PM promoter consists of the nucleotide sequence set forth in SEQ ID NO: 45.

[0203] In certain embodiments, the promoter is a promoter. In certain embodiments, thepromoter comprises the nucleotide sequence set forth in SEQ ID NO: 46. In certain embodiments, the promoter consists of the nucleotide sequence set forth in SEQ ID NO: 46.

[0204] promoter nucleotide sequence:

[0205] promoter nucleotide sequence:

[0206] promoter nucleotide sequence:

[0207] promoter nucleotide sequence:

[0208] promoter nucleotide sequence:

[0209] 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 a microorganism growth. The life cycle of an E. coli culture includes 5 distinct phases: lag, logarithmic, stationary, death, and long-term stationary phase. The lag phase occurs when cells are inoculated into media and adjust their metabolic processes according to their new environment. The cells will then rapidly grow and divide, entering the logarithmic phase. It is al this lime that enzymes related to central carbon metabolism are most important, and thetranscription of corresponding genes will be upregulated. Once the cells sense environmental stressors such as scarcity of media nutrients, their growth and division slows, and the culture enters the stationary phase. The use of a stationary phase promoter prevents the production pathway from competing with central carbon metabolism for carbon flux during the logarithmic phase of growth, a time when cells need carbon to rigorously grow and divide.

[0210] In certain embodiments, the stationary phase promoter is In certain embodiments,the promoter comprises the nucleotide sequence set forth in SEQ ID NO: 46 or SEQ ID NO:53. In certain embodiments, the P^M promoter consists of the nucleotide sequence set forth in SEQ ID NO: 53.

[0211] In certain embodiments, the stationary phase promoter is. In certain embodiments, the promoter comprises the nucleotide sequence set forth in SEQ ID NO: 54. In certainembodiments, the promoter consists of the nucleotide sequence set forth in SEQ ID NO:54.

[0212] In certain embodiments, the stationary phase promoter is . In certain embodiments,the promoter comprises the nucleotide sequence set forth in SEQ ID NO: 55. In certain embodiments, the promoter consists of the nucleotide sequence set forth in SEQ ID NO: 55.

[0213] In certain embodiments, the stationary phase promoter is . In certain embodiments,the promoter comprises the nucleotide sequence set forth in SEQ ID NO: 56. In certain embodiments, the promoter consists of the nucleotide sequence set forth in SEQ ID NO: 56.3.1.3. Genetic Markers

[0214] In certain embodiments, the presently disclosed recombinant polynucleotides include genetic markers. These genetic markers allow the selection of microorganisms that have 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, Tetracyclin resistance, Neomycin resistance, Chloramphenicol resistance, Gentamycin resistance, Erythromycin resistance, Carbenicillin resistance, Actinomycin D resistance. Neomycin resistance, Polymyxin resistance, Zeocin resistance, and Streptomycin resistance. In certain embodiments, the genetic marker includes a coding sequence of an antibiotic resistance protein (e.g., a beta-lactamase for certain Ampicillin resistance markers) and a promoter or enhancer element that drives the expression of the coding sequence in a microorganism of the present disclosure. In certain embodiments, a microorganism of the present disclosure is grown under conditions in which an antibiotic resistance marker is expressed and confers resistance to the microorganism, thereby selected for the microorganism with successful integration of 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, fatly acid synthesis, or carbohydrate metabolism. In certain embodiments, the auxotrophic marker is a gene for synthesizing 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, aspartate or glutamate. In certain embodiments, the auxotrophic marker is a gene for synthesizing adenosine, biotin, thiamine, leucine, glucose, lactose, or maltose. In certain embodiments, a microorganism of the present disclosure is grown under conditions in which an auxotrophic resistance marker is expressed in an environment or medium lacking the corresponding nutrient and confers growth to the microorganism (lacking an endogenous ability to produce the nutrient), thereby selected for the microorganism with successful integration of the marker.3.2. Deletions and Reduced Expression of Genes

[0215] In certain embodiments, the present disclosure also provides methods to introduce a deletion of any of the genes or enzymes disclosed herein. These deletions can be generated by any suitable gene-editing methods. In certain embodiments, the deletion is generated by a method comprising homologous recombination, a Zinc finger nuclease, a meganuclease, a Transcriptionactivator- 1 ike effector nuclease (TALEN), a Clustered regularly-interspaced short palindromic repeats (CRISPR) system, or a combination thereof.

[0216] In certain embodiments, the deletion is generated by a CRISPR system. Clustered regularly-interspaced short palindromic repeats (CRISPR) system is a genome-editing tool discovered in prokaryotic cells. When utilized for genome editing, the system includes Cas9 (a protein able to modify DNA utilizing crRNA as its guide), CRISPR RNA (crRNA, which contains the RNA used by Cas9 to guide it to the correct section of host DNA along with a region that binds to tracrRNA (generally in a hairpin loop form) forming an active complex with Cas9), transactivating crRNA (tracrRNA, binds to crRNA and forms an active complex with Cas9), and an optional section of DNA repair template (DNA that guides the cellular repair process allowing insertion of a specific DNA sequence). Multiple crRNA’s and the tracrRNA can be packaged together to form a single-guide RNA (sgRNA). This sgRNA can be joined together with the Cas9 gene and made into a plasmid in order to be transfected into cells. In certain embodiments, the CRISPR system comprises base editors. In certain embodiments, the CRISPR system comprises transposases / recombinases. In certain embodiments, the CRISPR system comprises prime editors. In certain embodiments, the CRISPR system comprises an epigenetic modulator. In certain embodiments, the CRISPR system comprises a CRiSPRoff system. Additional details on the CRISPR systems of the present disclosure can be found in Anzalone et al., Nature biotechnology 38.7 (2020): 824-844 and in 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 entireties.

[0217] In certain embodiments, the deletion is generated by a zinc-finger nuclease. A zinc-finger nuclease (ZFN) is an artificial restriction enzyme, which is generated by combining a zinc finger DNA-binding domain with a DNA-cleavage domain. A zinc finger domain can be engineered to target specific DNA sequences and allows a zinc-finger nuclease to target desired sequences within genomes. The DNA-binding domains of individual ZFNs typically contain a plurality of individual zinc finger repeals and can each recognize a plurality of base pairs. The most common method to generate a new zinc-finger domain is to combine smaller zinc-finger “modules” of known specificity. The most common cleavage domain in ZFNs is the non-specific cleavage domain from the Type Ils restriction endonuclease Fokl.

[0218] In certain embodiments, the deletion is generated by a TALEN system. Transcription activator- 1 ike effector nucleases (TALEN) are restriction enzymes that can be engineered to cut specific sequences of DNA. TALEN system operates on almost the same principle as ZFNs. They are generated by combining a transcription activator-like effectors DNA-binding domain with a DNA cleavage domain. Transcription activator- 1 ike effectors (TALEs) arc composed of 33-34amino acid repeating motifs with two variable positions that have a strong recognition for specific nucleotides. By assembling arrays of these TALEs, the TALE DNA-binding domain can be engineered to bind desired DNA sequence, and thereby guide the nuclease to cut at specific locations in genome.

[0219] In certain embodiments, the deletion is generated by a meganuclease. A meganuclease is an endodeoxyribonuclease that recognizes a double-stranded DNA site of approx. 12 to approx. 40 base pairs that occur only once in a genome. Meganucleases are some of the most specific naturally occurring restriction enzymes. Meganucleases are also defined as molecular DNA scissors since they can replace, eliminate or modify sequences in a highly targeted way. Protein engineering allows the modification of their recognition sequence and the targeted sequence.10220 ] In certain embodiments, the present disclosure also provides methods to reduce the expression of any of the genes or enzymes disclosed herein. In certain embodiments, the reduced expression of genes and enzymes disclosed herein comprises using oligonucleotides that have complementary sequences to the mRNA of the genes disclosed herein (e.g., manA,pfkA,pfkB, etc.). Non-limiting examples of these oligonucleotides include small interference RNA (siRNA), short hairpin RNA (shRNA), and microRNA (miRNA). In certain embodiments, these oligonucleotides can be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to at least a portion of a zwf mRNA sequence. In certain embodiments, these oligonucleotides can be identical to at least a portion of a 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%, at least about 99% identical to at least a portion of a pjkA mRNA sequence. In certain embodiments, these oligonucleotides can be identical to at least a portion of a 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%, at least about 99% identical to at least a portion of a pfkB mRNA sequence. In certain embodiments, these oligonucleotides can be identical to at least a portion of a 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%, at least about 99% identical to at least a portion of a rpiK mRNA sequence. In certain embodiments, these oligonucleotides can be identical to at least a portion of a 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%, at least about 99% identical to at least aportion of a manA mRNA sequence. In certain embodiments, these oligonucleotides can be identical to at least a portion of a manA mRNA sequence. In certain embodiments, antisense nucleic acid, shRNA, miRNA, or siRNA molecules can include DNA or atypical or non-naturally occurring residues, for example, but not limited to, phosphorothioate residues.

[0221] In some embodiments, reduction of expression of genes and enzymes disclosed herein can comprise use of CRISPR, which can mutate the coding sequence or promoter, to lower or remove expression of the gene product, or CRISPRi can be targeted to the genes disclosed herein, thereby reducing expression of one of more of the genes. See, e.g., Arroya-Olarte, ci al., Microorganisms, 2021 Apr; 9(4): 844. Zhang et al., Front. Microbiol. (31 March 2021).

[0222] In certain embodiments, the reduced expression of genes and enzymes disclosed herein comprises using a CRISPR system. In certain embodiments, the CRISPR system includes a Cas9. In certain embodiments, Cas9 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: 79. In certain embodiments, Cas9 comprises the amino acid sequence set forth in SEQ ID NO: 79. In certain embodiments, Cas9 consists of the amino acid sequence set forth in SEQ ID NO: 79. SEQ ID NO: 79 is provided below:

[0223] In certain embodiments, the reduced expression of genes and enzymes disclosed herein comprises using a CRISPRi system. CRISPRi systems silence genes at the transcriptional level and can have fewer sequence-specific off-target effects than RNAi. In certain embodiments, theCRISPRi system includes a catalytically dead Cas9 (dCas9). dCas9 is a programmable transcription factor that can be targeted to promoters through sgRNAs, where it can function as repressor. In certain embodiments, dCas9 comprises an amino acid sequence that is al 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: 57. In certain embodiments, dCas9 comprises the amino acid sequence set forth in SEQ ID NO: 57. In certain embodiments, dCas9 consists of the amino acid sequence set forth in SEQ ID NO: 57. SEQ ID NO: 57 is provided below:

[0224] In certain embodiments, the nuclease (e.g., Cas9 or dCas9) is regulated by a promoter (e.g., 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 CRISPR / CRISPRi system includes a small guide RNAs (sgRNA). In certain embodiments, the sgRNA of the CRISPR / CRISPRi system targets a gene encoding an enzyme of a competing pathway. For example, but without any limitation, the sgRNA can target the zwf gene, the pgm gene, the pfkA gene, the pfkB gene, or the ManA gene. In certain embodiments, the sgRNA can target any portion of a gene. For example, but without any limitation, the sgRNA can target a promoter, an operator, or a sequence encoding a protein.

[0225] In certain embodiments, the CRISPR / CRISPRi system includes a nuclease (e.g., Cas9 or dCas9) and a sgRNA. In certain embodiments, the nuclease (e.g., Cas9 or dCas9) is regulated by an inducible promoter. In certain embodiments, the inducible promoter is In certainembodiments, the sgRNA targets a pfkB gene. In certain embodiments, the sgRNA targets a promoter of the pfkB gene.3.3. Transformation and Gene Editing

[0226] In certain embodiments, the present disclosure provides the use of transformation of the plasmids and vectors disclosed herein. Vectors and plasmids disclosed herein can be transformed into cells through any known system in the art. For example, but without any limitation, the presently disclosed microorganisms can be transformed by particle bombardment, chemical transformation, Agrobacterium transformation, nano-spike transformation, electroporation, and virus transformation.

[0227] In certain embodiments, the vectors of the present disclosure may be introduced into the microorganisms using a variety of 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, e.g., Davis, L., Dibner, M., Battey, 1., 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 (199])). In certain embodiments, transformed microorganisms are referred to as recombinant microorganisms.

[0228] In certain embodiments, the present disclosure provides methods for introducing exogenous proteins (e.g., nuclease), RNA (e.g., gRNA), and DNA (e.g., a recombinant polynucleotide disclosed herein) into the microorganism. Various methods for achieving this have been described previously including direct transfection of protein and nucleotide sequence or DNA transformation followed by intracellular expression of RNA and protein (see, e.g., Dicarlo, J. E. et al. “Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems.” Nucleic Acids Res (2013). doi: 10.1093 / nar / gktl35; Ren, Z. J., Baumann, R. G. & Black, L. W. “Cloning of linear DNAs in vivo by overexpressed T4 DNA ligase: construction of a T4 phage hoc gene display vector.” Gene 195, 303-31 1 (1997); Lin, S., Staahl, B. T., Alla, R. K. & Doudna, J. A. “Enhanced homology-directed human genome engineering by controlled timing of CRISPR / Cas9 delivery.” Elife 3, e04766 (2014)).3.4. Recombination Systems

[0229] In certain embodiments, the present disclosure also provides homologous recombination systems for editing (e.g., insertion, deletion) in a microorganism. In certain embodiments, the homologous recombination system can be native to the host cell or introduced to the cell host. For example, but without any limitation, genes for the homologous recombination system can be introduced on a plasmid, introduced on a linear DNA fragment, introduced as and translated fromRNA or set of RNAs, or introduced as a protein or set of proteins. In certain embodiments, the methods include a recombinant polynucleotide disclosed herein. In certain embodiments, the polynucleotide includes sequence homologous (e.g. left and right homology arms) to a region in a nucleic acid (e.g., genome, plasmid, etc.) such that the left and right homology arms are separated by a designed genetic edit (e.g., promoter, insertion, substitution, SNP, terminator, degron, a sequence for a tag, sequence for a degradation signal or deletion). In certain embodiments, the recombinant polynucleotide includes a genetic marker, a counter selectable genetic marker (e.g., SacB or PheS), and an origin of replication (e.g., R6K).

[0230] In certain embodiments, the recombinant polynucleotide including the homology arms and sequence for genetic editing is introduced into the microorganism using any of the methods disclosed herein (e.g., transformation via electroporation, conjugation, etc.). In certain embodiments, following transformation, the resulting transformants can be plated on a medium to select for transformants expressing the selectable genetic markers. The recombination of a plasmid comprising homology arms with a targeted locus in a nucleic acid (e.g., genome, plasmid, etc.) can occur at one of the two homology sites targeted by (he homology arms present on the plasmid and that (lank the designed genetic edit. In certain embodiments, the resulting transformants grow as colonies on the selective medium and can be selected and plated on a second type of selective medium (e.g. counter-selectable medium). In certain embodiments, the second type of selective medium allows the selection of cells that comprise the desired genetic editing.

[0231] In certain embodiments, the methods disclosed herein include using proteins from one or more recombination systems. Said recombination systems can be endogenous to the microorganism or can be exogenous. In certain embodiments, the proteins from one or more recombination systems can be introduced as nucleic acids (e.g., as a plasmid, linear DNA or RNA, or integron) and be integrated into the genome of the host cell or be stably expressed from an extrachromosomal element. In certain embodiments, the proteins from one or more recombination systems can be introduced as RNA and be translated by the host cell. In certain embodiments, the proteins from one or more recombination systems can be introduced as proteins into the host cell. Non-limiting examples of recombination systems include lambda red recombination system, RecET recombination system, Red / ET recombination system, any homologs, orthologs, or paralogs of proteins from a lambda red recombination system, RecET recombination system, Red / ET recombination system, lambda red-mediated recombination system, or any combination thereof. Details on the recombination systems from the RecET recombination system can be any of those as described in Zhang Y., Buchholz F.. Muyrers J.P.P. and Stewart A.F. “A new logic for DNA engineering using recombination in E. eoliE Nature Genetics 20 ( 1998) 123-128; Muyrers,J.P.P., Zhang, Y„ Testa, G„ Stewart, A.F. “Rapid modification of bacterial artificial chromosomes by ET-recombination.” Nucleic Acids Res. 27 ( 1999) 1555-1557; Zhang Y,, Muyrers J. P.P., Testa G. and Slewart A.F. “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 herein incorporated by reference in their entirety.4. _ Methods for Producing Allose

[0232] The present disclosure also provides methods for producing Allose. Cell-free methods (e.g., in vitro synthesis) utilize thermodynamically unfavorable enzymatic reactions. In certain embodiments, the presently disclosed methods for producing allose include culturing microorganisms (e.g., one disclosed in Section 2) and purifying allose.4 1. Cell Culture

[0233] The present disclosure provides methods of culturing microorganisms disclosed herein. As used herein, “culturing” a cell refers to introducing an appropriate culture medium, under appropriate conditions, to promote the growth of a cell. In certain embodiments, culturing is performed using a liquid or solid growth medium. In certain embodiments, culturing occurs under aerobic or anaerobic conditions based on the requirements of the microorganism and desired metabolic state of the same. In certain embodiments, culturing includes specific conditions such as temperature, pressure, light, pH, and cell density.

[0234] In certain embodiments, the methods for producing methods of producing allose include a culture medium for culturing the recombinant bacteria. “Culture medium,” as used herein, refers to any composition or broth that supports the growth of the microorganism disclosed herein. A culture media can be liquid or solid. In certain embodiments, the culture media include nutrients, salts, buffers, elements, and other compounds that support the growth and viability of cells. Additionally, culture media can include sources of nitrogen, carbon, amino acids, carbohydrates, trace elements, vitamins, and minerals. In certain embodiments, the culture media include a complex extract (e.g., yeast extract). In certain embodiments, the culture medium is enriched in order to support rapid growth. In certain embodiments, the culture medium is modified in order to support slower growth. In certain embodiments, the culture medium includes an agent that can inhibit the growth of or kill contaminating organisms (e.g., an antibiotic). In certain embodiments, the culture medium includes an agent that can activate an inducible promoter or enzyme (e.g., IPTG). Non-limiting examples of culture media encompassed by the present disclosure include M9 medium, Lysogeny Broth (LB), Terrific Broth (TB), and YT broth. In certain embodiments, the culture medium comprises a substrate that is converted by the recombinant microorganisms to allose.

[0235] In certain embodiments, the substrate is a sugar (e.g., glucose or fructose) that can be phosphorylated by the bacteria via a kinase (e.g., hexokinase) and converted into fructose-6- phosphate. In certain embodiments, the substrate is glucose. In certain embodiments, glucose can derive from cellulose, C5 sugars, hemicellulose, and / or xylose. In certain embodiments, the substrate is a constituent of the culture medium. In certain embodiments, the substrate is supplemented with the culture medium. In certain embodiments, the substrate is continuously present in the culture medium. In certain embodiments, the substrate is supplemented during the growth phase. In certain embodiments, the substrate is supplemented during the stationary phase.4.2. Purification of Allose

[0236] In certain embodiments, the methods of the present disclosure further comprise purifying allose produced by a microorganism of the present disclosure, e.g., from cell culture or cell culture medium. A variety of methods known in the art may be used to purify a product from a microorganism or microorganism culture. In certain embodiments, one or more products may be purified continuously, e.g., from a continuous culture. In certain embodiments, one or more products may be purified separately from fennentation, e.g., from a batch or fed-batch culture. One skilled in the art will appreciate that the specific purification method(s) used may depend upon, inter alia, the microorganism, culture conditions, and / or particular product(s).

[0237] In certain embodiments, purifying allose comprises separating or filtering the microorganisms from a cell culture medium, separating the allose from the culture medium (e.g., by chromatography), concentration of water (e.g., by evaporation), and lyophilization of the allose.5. Food Products

[0238] The present disclosure also provides delivery systems methods for use in food products including the D-allose prepared and / or generated by any of the microorganisms disclosed herein.

[0239] The term “food product,” as used herein, includes any food product, for example, those set forth in 21 CFR 101.12. Non-limiting examples of such food products include frozen desserts, baked goods, fillings, nutritional drinks, beverages, salad dressing or similar dressing, 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 w hich is herein incorporated by reference in its entirety. Non-limiting examples of bakery goods include cookies, cakes, rolls, pastries, pie dough, brownies, breads, bagels, and the like. The D-allose prepared and / or generated by any of the microorganisms disclosed herein are also suitable as a component in frozen foods.

[0240] In certain embodiments, the food product is prepared by admixing the D-allose in an ingeslible vehicle, together with any optional ingredients, to form a uniform mixture. The final compositions arc readily prepared using standard methods and apparatus generally known bythose skilled in the corresponding arts, such as confectionary arts. The apparatus useful per the presently disclosed subject matter comprises mixing apparatus well known in the art, and therefore the selection of the specific apparatus will be apparent to the artisan.

[0241] As used herein “admixing,” for example, “admixing D-allose with a food product,” refers to the process where the flavor composition is mixed with or added to the completed product or mixed with some or all of the components of the product during product formation or some combination of these steps. When used in the context of admixing the term “product” refers to the product or any of its components. This admixing step can include a process selected from the step of adding D-allose to the product, spraying D-allose on the product, coating D-allose on the product, suspending the product in D-allose, painting D-allose on the product, pasting D-allose on the product, encapsulating the product with D-allose, mixing D-allose with the product and any combination thereof. The D-allose can be a liquid, dry powder, spray, paste, suspension and any combination thereof.

[0242] In certain embodiments, the present application relates to the modified edible food products produced by the methods disclosed herein. In certain embodiments, the food products can be produced by processes for producing comestible products well known to those of ordinary skill in the art.

[0243] In certain embodiments, the D-allose prepared and / or generated by any of the microorganisms disclosed herein can be dissolved in or dispersed in one of many known comestible acceptable liquids, solids, or other carriers, such as water at neutral, acidic, or basic pH, fruit or vegetable juices, vinegar, marinades, beer, wine, natural water / fat emulsions such as milk or condensed milk, whey or whey products, edible oils and shortenings, fatty acids, certain low molecular weight oligomers of propylene glycol, glyceryl esters of fatly acids, and dispersions or emulsions of such hydrophobic substances in aqueous media, salts such as sodium chloride, vegetable flours, solvents such as ethanol, solid edible diluents such as vegetable powders or flours, and the like, and then combined with precursors of the comestible or medicinal products, or applied directly to the comestible or medicinal products.

[0244] Those of ordinary skill in the art of preparing and selling food products are well aware of a large variety of classes, subclasses, and species of the comestible compositions, and utilize well- known and recognized terms of art to refer to those comestible compositions while endeavoring to prepare and sell various of those comestible compositions. Such a list of terms of art is enumerated below, and it is specifically contemplated hereby that the D-allose prepared and / or generated by any of the microorganisms disclosed herein can be used to modify or enhance the taste of the following list edible compositions, either singly or in all reasonable combinations or mixtures thereof.

[0245] In certain embodiments, the Pood products to which the D-allose prepared and / or generated by any of the microorganisms disclosed herein are admixed with comprise, by way of example, the wet soup category, the dehydrated and culinary food category, the beverage category, the frozen food category, the snack food category, and seasonings or seasoning blends, described herein.10246 ] In certain embodiments, the D-allose prepared and / or generated by any of the microorganisms disclosed herein are admixed with one or more confectioneries, chocolate confectionery, tablets, countlines, bagged sei fmies / so ftlines, boxed assortments, standard boxed assortments, twist wrapped miniatures, seasonal chocolate, chocolate with toys, allsorts, other chocolate confectionery, mints, standard mints, power mints, boiled sweets, pastilles, gums, jellies and chews, toffees, caramels and nougat, medicated confectionery, lollipops, liquorice, other sugar confectionery, gum, chewing gum, sugarised gum, sugar-free gum, functional gum, bubble gum, bread, packaged / industrial bread, unpackaged / artisanal bread, pastries, cakes, packaged / industrial cakes, unpackaged / artisanal cakes, cookies, chocolate coated biscuits, sandwich biscuits, filled biscuits, savory biscuits and crackers, bread substitutes, breakfast cereals, rte cereals, family breakfast cereals, flakes, muesli, other rte cereals, children's breakfast cereals, hot cereals, ice cream, impulse ice cream, single portion dairy ice cream, single portion water ice cream, multi-pack dairy ice cream, multi-pack water ice cream, take-home ice cream, lake-home daily ice cream, ice cream desserts, bulk ice cream, take-home water ice cream, frozen yoghurt, artisanal ice cream, dairy products, milk, fresh / pasteurized milk, full fat fresh / pasteurized milk, semi skimmed fresh / pasteurized milk, long-life / uht milk, full fat long life / uht milk, semi skimmed long life / uht milk, fat-free long life / uht milk, goat milk, condensed / evaporated milk, plain condensed / evaporaled milk, flavored, functional and other condensed milk, flavored milk drinks, dairy only flavored milk drinks, flavored milk drinks with fruit juice, soy milk, sour milk drinks, fermented dairy drinks, coffee w'hiteners, powder milk, flavored powder milk drinks, cream, cheese, processed cheese, spreadable processed cheese, unspreadable processed cheese, unprocessed cheese, spreadable unprocessed cheese, hard cheese, packaged hard cheese, unpackaged hard cheese, yoghurt, plain / nalural yoghurt, flavored yoghurt, fruited yoghurt, probiotic yoghurt, drinking yoghurt, regular drinking yoghurt, probiotic drinking yoghurt, chilled and shelf-stable desserts, dairy-based desserts, soy-based desserts, chilled snacks, fromage frais and quark, plain fromage frais and quark, flavored fromage frais and quark, savory fromage frais and quark, sweet and savoiy 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 replacement products, slimming products, convalescence drinks, ready meals, canned ready meals, frozen ready meals, dried ready meals,chilled ready meals, dinner mixes, frozen pizza, chilled pizza, soup, canned soup, dehydrated soup, instant soup, chilled soup, uht soup, frozen soup, pasta, canned pasta, dried pasta, chilled / fresh pasta, noodles, plain noodles, instant noodles, cups / bowl instant noodles, pouch instant noodles, chilled 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 foods, frozen food, frozen processed red meat, frozen processed poultry, frozen processed fish / seafood, frozen processed vegetables, frozen meal 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 soup, frozen noodles, other frozen food, dried food, dessert mixes, dried ready meals, dehydrated soup, instant soup, dried pasta, plain noodles, instant noodles, cups / bowl instant noodles, pouch instant noodles, chilled food, chilled processed meals, chilled fish / seafood products, chilled processed fish, chilled coaled fish, chilled smoked fish, chilled lunch kit, chilled ready meals, chilled pizza, chilled soup, chilled / fresh pasta, chilled noodles, oils and fats, olive oil, vegetable and Seed oil, cooking fats, butter, margarine, spreadable oils and fats, functional spreadable oils and fats, sauces, dressings and condiments, tomato pastes and purees, bouillon / stock cubes, stock cubes, gravy granules, liquid stocks and Tends, herbs and spices, fermented sauces, soy based sauces, pasta sauces, wet sauces, dry sauces / powder mixes, ketchup, mayonnaise, regular mayonnaise, mustard, salad dressings, regular salad dressings, low fat salad dressings, vinaigrettes, dips, pickled products, other sauces, dressings and condiments, baby food, milk formula, standard milk formula, follow-on milk formula, toddler milk formula, hypoallergenic milk formula, prepared baby food, dried baby food, other baby food, spreads, jams and preserves, honey, chocolate spreads, nut-based spreads, and yeast-based spreads.5.1. Chewing-gum

[0247] In certain embodiments, the D-allose prepared and / or generated 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 specifics of chewing gum compositions are disclosed in U.S. Patent No. 6,899,911, the disclosure of which is incorporated herein by reference in its entirely. The chewing gum composition of the presently disclosed subject matter follows the general pattern outlined below. In general, a chewing gum composition typically contains a chewable gum base portion that is essentially free of waler and is water-insoluble, a water-soluble bulk portion, and flavors that are typically waler-insoluble. The water-soluble portion dissipates with a portion of the flavor over a period of time during chewing. The gum base portion is retained in the mouth throughout the chew. The insoluble gum base generally comprises elastomers, elastomer solvents, plasticizers, waxes, emulsifiers, and inorganic fillers. Plastic polymers, suchas polyvinyl acetate, which behave somewhat as plasticizers, are also often included. Other plastic polymers that can be used include polyvinyl laureate, polyvinyl alcohol, and polyvinyl pyrrolidone. Elastomers can include polyisobutylene, butyl rubber, (isobutylene-isoprene 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, including tallow, hydrogenated and partially hydrogenated vegetable oils, and cocoa butter. Commonly employed waxes include paraffin, microcrystalline, and natural waxes such as beeswax and carnauba. Microcrystalline waxes, especially those with a high degree of crystallinity, can be considered bodying agents or textural modifiers.

[0248] In certain embodiments, the insoluble gum base constitutes between about 5% to about 95% by weight of the gum. More preferably the insoluble gum base comprises between 10% and 50% by weight of the gum and most preferably about 20% to 35% by weight of the gum. The gum base typically also includes a filler component. The filler component can be calcium carbonate, magnesium carbonate, talc, dicalcium phosphate, or the like. The filler can constitute between about 5% and about 60% by weight of the gum base. Preferably the filler comprises about 5% to 50% by weight of the gum base.

[0249] Gum bases typically also contain softeners including glycerol monostearate and glycerol triacetate. Gum bases can also contain optional ingredients such as antioxidants, colors, and emulsifiers. The presently disclosed subject matter contemplates employing any commercially acceptable gum base.

[0250] The water-soluble portion of the chewing gum can further comprise softeners, sweeteners, flavors, physiological cooling agents, and combinations thereof. The sweeteners often fulfill the role of bulking agents in the gum. The bulking agents typically comprise about 5% to about 95% of the gum composition.

[0025] ] Softeners are added to the chewing gum in order to optimize the chewability and mouth feel of the gum. Softeners, also known in the art as plasticizers or plasticizing agents, generally constitute between about 0.5% to about 15% of the chewing gum. Softeners contemplated by the presently disclosed subject matter include glycerin, lecithin, and combinations thereof. Further, aqueous sweetener solutions such as those containing sorbitol, hydrogenated starch hydrolysate, corn syrup, and combinations thereof can be used as softeners and binding agents in gum.

[0252] As mentioned above, the D-allose prepared and / or generated by any of the microorganisms disclosed herein can be used in low-calorie gum formulations. However, formulations containing sugar are also within the scope of the invention. Sugar sweeteners generally include saccharide- containing components commonly known in the chewing gum art which comprise, but are not limited to, sucrose, dextrose, maltose, dextrin, dried invert sugar, fructose, galactose, corn syrupsolids and the like, alone or in any combination. The D-allose prepared and / or generated by any of the microorganisms disclosed herein can also be used in combination with sugarless sweeteners. Generally, sugarless sweeteners include components with sweetening characteristics but which are devoid of the commonly known sugars and comprise, but are not limited to, sugar alcohols such as sorbitol, hydrogenated isomaltulose, mannitol, xylitol, lactitol, erythritol, hydrogenated starch hydrolysate, maltitol and the like alone or in any combination.

[0253] Depending on the particular sweetness release profile and shelf stability needed, coated or uncoated high-intensity sweeteners can be used in the chewing gum composition, or can be used in a coaling applied to centers made from those gum compositions. High-intensity sweeteners, preferably aspartame, can be used at levels from about 0.01% to about 3.0%. Encapsulated aspartame is a high-intensity sweetener with improved stability and release characteristics, as compared to free aspartame. Free aspartame can also be added, and a combination of some free and encapsulated aspartame is preferred when aspartame is used. Other high-intensity sweeteners that can be used in the gum center are saccharin, Thaumatin, alitame, saccharin salts, sucralose, Stevia, and acesulfame K. Overall, the chewing gum composition will preferably comprise about 0.5% to about 90% sweetening agents. Most typically the sweetening agents will comprise at least one bulk sweetener and at least one high-intensity sweetener. Optional ingredients such as colors, emulsifiers, and pharmaceutical agents can also be added as separate components of the chew ing gum composition, or added as part of the gum base.

[0254] Aqueous syrups, such as corn syrup and hydrogenated corn syrup can be used, particularly if their moisture content is reduced. This can preferably be done by co-evaporating the aqueous syrup with a plasticizer, such as glycerin or propylene glycol, to a moisture content of less than 10%. Preferred compositions include hydrogenated starch hydrolysate solids and glycerin. Such syrups and their methods of preparation are discussed in detail in U.S. Patent No 4,671 ,967.[02551 Methods of manufacturing chew’ing gum according to the presently disclosed subject matter include the sequential addition of the various chewing gum ingredients to any commercially available mixer known in the art. After the ingredients have been thoroughly mixed, the gum is discharged from the mixer and shaped into the desired form such as by rolling into sheets and 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 the running mixer. The base can also be melted in the mixer itself. Color or emulsifiers can also be added al this time, along with syrup and a portion of the bulking agent. Further portions of the bulking agent can then be added to the mixer. Flavor systems are typically added with the final portion of the bulking agent. If the flavor system is coaled or otherwise modified when incorporated into a delivery system to modify its release rate, it will preferably be added after the final portion of the bulking agent hasbeen added. The entire mixing procedure typically lakes from five to twenty minutes, but longer mixing times can sometimes be required. Those skilled in the art will recognize that many variations of the above-described procedures can be followed.

[0256] If formed into pellets or balls, the chewing gum composition can be coated. The coating is initially present as a liquid syrup which contains from about 30% to about 80% or 85% sugars or sugar alcohols, and from about 15% or 20% to about 70% of a solvent such as water. In general, (he coaling process is carried out in conventional panning equipment. Gum center tablets to be coaled are placed into the panning equipment to form a moving mass.

[0257] The material or syrup that will eventually form the coaling is applied or distributed over the gum center tablets. The D-allose can be added before, during, and after applying the syrup to the gum centers. Once the coating has dried to form a hard surface, additional syrup additions can be made to produce a plurality of coatings or multiple layers of coating. The D-allose can be added to any or none of the coatings and / or layers.

[0258] In the panning procedure, syrup is added to the gum center tablets at a temperature range of from about 100°F to about 240°F. Preferably, the syrup temperature is from about 14O°F to about 200°F. Most preferably, the syrup temperature should be kept constant throughout the process in order to prevent the polyol in the syrup from crystallizing. The syrup can be mixed with, sprayed upon, poured over, or added to the gum center tablets in any way known to those skilled in the art.

[0259] In certain embodiments, a soft coating is formed by adding a powder coating after a liquid coating. The powder coating can include natural carbohydrate gum hydrolysates, maltodextrin, gelatin, cellulose derivatives, starches, modified starches, sugars, sugar alcohols, natural carbohydrate gums, and fillers like laic and calcium carbonate.

[0260] Each component of the coating on the gum center can be applied in a single layer or a plurality of layers. In general, a plurality of layers is obtained by applying single coats, allowing the layers to dry, and then repealing the process. The amount of solids added by each coaling step depends chiefly on the concentration of the coating syrup. Any number of coals can be applied to the gum center tablet. Preferably, no more than about 75 coats are applied to the gum center. More preferably, less than about 60 coats are applied and most preferably, about 30 to about 60 coats are applied. In any event, the presently disclosed subject matter contemplates applying an amount of syrup sufficient to yield a coated chewing gum product containing about 10% to about 65% coating. Preferably, the final product wi 11 contain from about 20% to about 50% coating.

[0261] Those skilled in the art will recognize that in order to obtain a plurality of coated layers, a plurality of premeasured aliquots of coating syrup can be applied to the gum center. It iscontemplated, however, that the volume of aliquots of syrup applied to the gum center can vary throughout the coating procedure.

[0262] Once a coaling of syrup is applied lo the gum center, the syrup is dried in an inert medium. A preferred drying medium comprises air. Preferably, forced drying air contacts the wet syrup coating in a temperature range of from about 70°F to about 110°F. More preferably, the drying air is in the temperature range of from about 80°F to about 100°F. The invention also contemplates that the drying air possesses a relative humidity of less than about 15 percent. Preferably, the relative humidity of the drying air is less than about 8 %.

[0263] The drying air can be passed over and admixed with the syrup coated gum centers in any way commonly known in the art. Preferably, the drying air is blown over and around the syrup coated gum center at a flow rate, for large scale operations, of about 2800 cubic feet per minute. If lower quantities of material are being processed, or if smaller equipment is used, lower flow rates would be used. If a flavor is applied after a syrup coaling has been dried, the presently disclosed subject matter contemplates drying the flavor with or without the use of a drying medium.

[0264] The amount of D-allose employed herein is normally a matter of preference subject to such factors as the type of final chewing gum composition, the individual flavor, the gum base employed, and the strength of flavor desired. Thus, the amount of D-allose can be varied in order to obtain the result desired in the final product and such variations are within the capabilities of those skilled in the art without the need for undue experimentation. In gum compositions, the D- allose prepared and / or generated by any of the microorganisms disclosed herein is generally present in amounts from about 0.02% to about 5%, and preferably from about 0.1 % to about 2%, and more preferably, from about 0.8% lo about 1.8%, by weight of the chewing gum composition.5.2. Sugar confectionery

[0265] Another important aspect of the presently disclosed subject matter includes a confectionery composition incorporating the D-allose prepared and / or generated by any of the microorganisms disclosed herein and a method for preparing the confectionery compositions. The preparation of confectionery formulations is well-known in the art. Confectionery items have been classified as either “hard” confectionery or “soft” confectionery. The D-allose prepared and / or generated by any of the microorganisms disclosed herein can be incorporated into the confections by admixing the compositions of the presently disclosed subject matter into the conventional hard and soft confections.

[0266] Hard confectionery can be processed and formulated by conventional means. In general, hard confectionery has a base composed of a mixture of sugar and other carbohydrate bulking agents kept in an amorphous or glassy condition. The hard confectionery can also be sugarlcss.The hard confectionery can also be low-calorie. This form is considered a solid syrup of sugars generally having from about 0.5% to about 1.5% moisture. Such materials normally contain up to about 92% sugar, up to about 55% corn syrup, and from about 0.1% to about 5% waler, by weight of the final composition. The syrup component is generally prepared from sucrose and corn syrups but can include other materials. In certain embodiments, the syrup component includes the D- allose prepared and / or generated by any of the microorganisms disclosed herein. Further ingredients such as flavorings, sweetening agents, acidulants, colorants, and so forth can also be added.

[0267] Such confectionery can be routinely prepared by conventional methods, including but not limited to methods involving fire cookers, vacuum cookers, and scraped-surface cookers also referred to as high-speed atmospheric cookers. The apparatus useful in accordance with the presently disclosed subject matter comprises cooking and mixing apparatus well known in the confectionery manufacturing arts, and therefore the selection of the specific apparatus will be apparent to the artisan.

[0268] Fire cookers involve the traditional method of making a candy base. In this method, the desired quantity of carbohydrate bulking agent is dissolved in water by heating the agent in a kettle until the bulking agent dissolves. Additional bulking agents can then be added and cooked until a final temperature of 145° C to 156° C is achieved. The batch is then cooled and worked as a plastic-like mass to incorporate additives such as flavoring agents, colorants, and the like.

[0269] A high-speed atmospheric cooker uses a heat-exchanger surface, which involves spreading a film of candy on a heat exchange surface, the candy is heated to 165° C to 170° C within a few seconds. The candy is then rapidly cooled to 100° C to 120° C and worked as a plastic-like mass enabling incorporation of the additives, such as flavoring agents, colorants, and the like. In vacuum cookers, the carbohydrate bulking agent is boiled to 125° C to 132° C, vacuum is applied, and additional waler is boiled off without extra heating. When cooking is complete, the mass is a semi-solid and has a plastic-like consistency. Al this point, flavoring agents, colorants, and other additives are admixed in the mass by routine mechanical mixing operations.

[0270] The optimum mixing required to uniformly mix the flavoring agent, colorants, and other additives during conventional manufacturing of hard confectionery is determined by the time needed to obtain a uniform distribution of the materials. Generally, mixing times of from 2 to 10 minutes have been found to be acceptable.

[0271] Once the candy mass has been properly tempered, it can be cut into workable portions or formed into desired shapes. A variety of forming techniques can be utilized depending upon the shape and size of the final product desired. 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, N.Y. al pages 419 to 582, which disclosure is incorporated herein by reference.[02721 Compressed tablet confections contain particular materials and are formed into structures under pressure. These confections generally contain sugars in amounts up to about 95%, by weight of the composition, and typical tablet excipients such as binders and lubricants as well as flavoring agents, colorants, and so forth. These confections can also be sugarless.

[0273] Similar to hard confectionery, soft confectionery can be utilized in the embodiments of the disclosed subject matter. The preparation of soft confections, such as nougat, involves conventional methods, such as the combination of two primary components, namely ( 1) a high boiling syrup such as com syrup, or the like, and (2) a relatively light textured frappe, generally prepared from egg albumin, gum arabic, gelatin, vegetable proteins, such as soy-derived compounds, sugarless milk-derived compounds such as milk proteins, and mixtures thereof. The frappe is generally relatively light, and can, for example, range in density from about 0.5 to about 0.7 grams / cc

[0274] The high boiling syrup, or “bob syrup” of the soft confectionery, is relatively viscous, has a higher density than the frappe component, and frequently contains a substantial amount of carbohydrate bulking agent. Conventionally, the final nougat composition is prepared by the addition of the “bob syrup” to the frappe under agitation, to form the basic nougat mixture. Further ingredients such as flavoring, additional carbohydrate bulking agents, colorants, preservatives, medicaments, mixtures thereof and the like can be added thereafter also under agitation. Soft confectioneries can also be prepared sugarless. 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), al pages 576-580, which disclosure is incorporated herein by reference.

[0275] In general, the frappe component is prepared first and thereafter the syrup component is slowly added under agitation al a temperature of at least about 65° C, and preferably at least about 100° C. The mixture of components is continued to be mixed to form a uniform mixture, after which the mixture is cooled to a temperature below 80° C, al which point, the flavor can be added. The mixture is further mixed for an additional period until it is ready to be removed and formed into suitable confectionery shapes.

[0276] In accordance with the present disclosure, amounts of the D-allose prepared and / or generated by any of the microorganisms disclosed herein can be admixed into the hard and soft confections. The exact amount of D-allose employed is normally a matter of preference subject to such factors as the particular type of confection being prepared, the type of bulking agent or carrier employed, the type of flavor employed, and the intensity of breath freshening perception desired.Thus, the amount of D-allose can be varied in order to obtain the result desired in the final product and such variations are within the capabilities of those skilled in the art without the need for undue experimentation. In general, the amount of D-allose normally present in a hard or soft confection will be from about 0.001% to about 20%, preferably from about 0.01% to about 15%, more preferably from about 0.01% to about 10%, and more preferably from about 0.01% to about 5%, and more preferably 0.01% to about 0.5% by weight of the confection.

[0277] The presently disclosed subject matter extends to methods for making the improved confections. The D-allose prepared and / or generated by any of the microorganisms disclosed herein can be incorporated into an otherwise conventional hard or soft confection composition using standard techniques and equipment known to those skilled in the art. The apparatus useful in accordance with the presently disclosed subject matter comprises mixing and healing apparatus well known in the confectionery manufacturing arts, and therefore the selection of the specific apparatus will be apparent to the artisan.

[0278] In such a method, a composition is made by admixing the D-allose into the confectionery composition along with the other ingredients of the final desired composition. Other ingredients will usually be incorporated into the composition as dictated by the nature of the desired composition as well known by those having ordinary skill in the art. The ultimate confectionery compositions are readily prepared using methods generally known in the food technology and pharmaceutical arts. Thereafter the confectionery mixture can be formed into desirable confectionery shapes.

[0279] The D-allose prepared and / or generated by any of the microorganisms disclosed herein can be formulated with conventional ingredients that offer a variety of textures to suit particular applications. Such ingredients can be in the form of hard and soft confections, tablets, toffee, nougat, chewy candy, chewing gum and so forth, center filled candies, both sugar and sugarless. The acceptable ingredients can be selected from a wide range of materials. Without being limited thereto, such materials include diluents, binders and adhesives, lubricants, disinlegranls, bulking agents, humectants, buffers, and adsorbents. The preparation of such confections and chewing gum products is well known.5. J. Chocolates and Fillings

[0280] The presently disclosed subject matter is also used with and / or in chocolate products, chocolate-flavored confections, and chocolate flavored compositions. Chocolates also include those containing crumb solids or solids fully or partially made by a crumb process. 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. Minific,Chocolate, Cocoa and Confectionery: Science and Technology, 2nd edition, AVI Publishing Co., Inc., Westport, Conn. (1982), which disclosure is incorporated herein by reference.

[0281] The term “chocolate” as used herein refers to a solid or semi-plastic food and is intended to refer to all chocolate or chocolate-like compositions containing a fat-based component phase or fat-like composition. The term is intended to include standardized or nonstandardized compositions conforming to the U.S. Standards of Identity (SOI), CODEX Alimentarius and / or other international standards and compositions not conforming to the U.S. Standards of Identity or other international standards. The term includes dark chocolate, baking chocolate, sweet chocolate, bittersweet or semisweet chocolate, milk chocolate, buttermilk chocolate, skim milk chocolate, mixed dairy product 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, pastels including white chocolate or coating made with cocoa butter or vegetable fat or a combination of these, nutritionally modified chocolate-like compositions (chocolates or coatings made with reduced calorie ingredients) and low fat chocolates, aerated chocolates, compound coatings, non-slandardized chocolates and chocolate-like compositions, unless specifically identified otherwise.

[0282] Nonslandardized chocolates result when, for example, the nutritive carbohydrate sweetener is replaced partially or completely; or when the cocoa butter, cocoa butler alternative, cocoa butter equivalent, cocoa butter extender, cocoa butter replacer, cocoa butter substitute or milkfat are replaced partially or completely; or when components that have flavors that imitate milk, butter or chocolate are added or other additions or deletions in formula are made outside the FDA standards of identify of chocolate or combinations thereof. Chocolate-like compositions are those fat-based compositions that can be used as substitutes for chocolate in applications such as panning, molding, or enrobing: for example, carob.

[0283] In the United Slates, chocolate is subject to a standard of identity established by the U.S. Food and Drug Administration (FDA) under the Federal Food, Drug and Cosmetic Act. Definitions and standards for the various types of chocolate are well established in the U.S. Nonstandardized chocolates are those chocolates which have compositions that Tall outside the specified ranges of the standardized chocolates.

[0284] In certain embodiments, the chocolate can contain D-allose prepared and / or generated by any of the microorganisms disclosed herein. Additionally, the chocolate can contain a sugar syrup / solids, invert sugar, hydrolyzed lactose, maple sugar, brown sugar, molasses, honey, sugar substitute and the like. Nutritive carbohydrate sweeteners with varying degrees of sweetness intensity can be any of those typically used in the art and include, but are not limited to, sucrose, c.g., from cane or beet, dextrose, fructose, lactose, maltose, glucose syrup solids, com syrup solids,invert sugar, hydrolyzed lactose, honey, maple sugar, brown sugar, molasses and the like. Sugar substitutes can partially replace the nutritive carbohydrate sweetener. High potency sweeteners include aspartame, cyclamates, saccharin, acesulfame-K, neohesperidin dihydrochalcone, sucralose, alitame, stevia sweeteners, glycyrrhizin, thaumatin and the like and mixtures thereof. The preferred high potency sweeteners are aspartame, cyclamates, saccharin, and acesulfame-K. Examples of sugar alcohols can be any of those typically used in the art and include sorbitol, mannitol, xylitol, maltitol, isomalt, lactitol and the like,

[0285] The chocolates can also contain bulking agents. The term "bulking agents" as defined herein can be any of those typically used in the art and include polydextrose, cellulose and its derivatives, maltodextrin, gum arabic, and the like.

[0286] The chocolate products can contain emulsifiers. Examples of safe and suitable emulsifiers can be any of those typically used in the art and include lecithin derived from vegetable sources such as soybean, safflower, com, etc,, fractionated lecithins enriched in either phosphatidyl choline or phosphatidyl ethanolamine, or both, mono- and digylcerides, diacetyl tartaric acid esters of mono- and diglycerides (also referred to as DATEM), monosodium phosphate derivatives of mono- and diglycerides of edible fats or oils, sorbitan monostearale, hydroxylated lecithin, lactylated fatty acid esters of glycerol and propylene glycol, polyglycerol esters of fatty acids, propylene glycol mono- and di-esters of fats and fatty acids, or emulsifiers that can become approved for the US FDA-defined soft candy category. In addition, 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 found to be suitable in chocolate or similar fat / solid system or any blend.

[0287] The term "chocolate- flavored confection” refers to food products, excluding “chocolate”, having a chocolate flavor / aroma and comprising a cocoa fraction. These products are stable at ambient temperatures for extended periods of time (e.g., greater than 1 week) and are characterized as microbiologically shelf-stable al 18-30° C under normal atmospheric conditions. Examples include chocolate-flavored hard candies, chewables, chewing gums, etc.

[0288] The term “chocolate- flavored compositions” refers to chocolate-flavored compositions, excluding “chocolate”, containing a cocoa fraction and having a chocolate flavor / aroma. Examples include chocolate-flavored cake mixes, ice creams, syrups, baking goods, etc. The term includes chocolate-flavored compositions (e.g,, cakes, nougats, puddings, etc.), as well as compositions not having a chocolate flavor (e.g., caramels, etc.),5.4. Savory Goods and Other Food Products

[0289] In certain embodiments, the D-allose prepared and / or generated by any of the microorganisms disclosed herein is incorporated into savory goods. In certain embodiments, asavory good is a food product that has savory flavors including, for example, 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 that are added or generated during heating of a food product.

[0290] In certain embodiments, the D-allose prepared and / or generated by any of the microorganisms disclosed herein is incorporated into a wet soup category food product, which comprises wet / liquid soups regardless of concentration or container, including frozen soups. In certain embodiments, the soup food product means a food prepared from meat, poultry, fish, vegetables, grains, fruit, and / or other ingredients, cooked in a liquid which may include visible pieces of some or all of these ingredients. It may be clear (as a broth) or thick (as a chowder), smooth, pureed or chunky, ready-to-serve, semi-condensed or condensed and may be served hot or cold, as a first course or as the main course of a meal or as a between meal snack (sipped like a beverage). Soup may be used as an ingredient for preparing other meal components and may range from broths (consomme) to sauces (cream or cheese-based soups).

[0029] ] In certain embodiments, the D-allose prepared and / or generated by any of the microorganisms disclosed herein is incorporated into a dehydrated and culinary food category of food products, which comprises (i) cooking aid products such as: powders, granules, pastes, concentrated liquid products, including concentrated bouillon, bouillon and bouillon like products in pressed cubes, tablets or powder or granulated form, which are sold separately as a finished product or as an ingredient within a product, sauces and recipe mixes (regardless of technology); (ii) meal solutions products such as: dehydrated and freeze dried soups, including dehydrated soup mixes, dehydrated instant soups, dehydrated ready-to-cook soups, dehydrated or ambient preparations of ready-made dishes, meals and single serve entrees including pasta, potato and rice dishes; and (iii) meal embellishment products such as: condiments, marinades, salad dressings, salad toppings, dips, breading, batter mixes, shelf stable spreads, barbecue sauces, liquid recipe mixes, concentrates, sauces or sauce mixes, including recipe mixes for salad, sold as a finished product or as an ingredient within a product, whether dehydrated, liquid or frozen.

[0292] In certain embodiments, the D-allose prepared and / or generated by any of the microorganisms disclosed herein is incorporated into a meat food product. In certain embodiments, meat food products include food products made by processing the edible remains of any dead animal, including birds, fish, crustaceans, shellfish, and mammals. Meat food products include, without limitation, for example, prepared beef, lamb, pork, poultry, or seafood products. Examples of such meat food products include, for example, bologna, frankfurters, sausage, luncheon, deli slices, loaves, bacon, meatballs, fish sticks, chicken fingers, and ground meals, c.g., meatloaf, meatballs, and hamburgers. A meal food product may be combined with asimulated meal food product. Simulated meat food products include, without limitation, for example, a meat alternative, meat analog, soy burger, soy bologna, soy frankfurter, soy sausage, soy luncheon loaves, soy bacon, and soy meatball. A simulated meal food product may be combined with a meat food product.|0293 ] In certain embodiments, the D-allose prepared and / or generated by any of the microorganisms disclosed herein is incorporated into a snack food category food product. In certain embodiments, snack food products include any food that can be a light informal meal including, but not limited to sweet and savory snacks and snack bars. Examples of snack food include, but are not limited to fruit snacks, chips / crisps, extruded snacks, tortilla / com 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.

[0294] In certain embodiments, the D-allose prepared and / or generated by any of the microorganisms disclosed herein is incorporated into frozen food products, which comprises chilled or frozen food products, for example, but not limited to, ice cream, impulse ice cream, single portion dairy ice cream, single portion water ice cream, multi-pack dairy ice cream, multi- pack water ice cream, lake-home ice cream, take-home dairy ice cream, ice cream desserts, bulk ice cream, take-home 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 fish / seafood, frozen processed vegetables, frozen meat substitutes, frozen potatoes, frozen bakery products and frozen desserts.5.4. Pharmaceuticals

[0295] The D-allose prepared and / or generated by any of the microorganisms disclosed herein can also be in the form of a pharmaceutical. One non-limiting example of a pharmaceutical form is a suspension. Pharmaceutical suspensions can be prepared by conventional compounding methods. Suspensions can contain adjunct materials employed in formulating the suspensions of (he art. The suspensions of the presently disclosed subject mailer can comprise preservatives, buffers, suspending agents, anti foaming agents, sweetening agents, flavoring agents, coloring or decoloring agents, solubilizers, and combinations thereof.

[0296] Flavoring agents such as those flavors well known to the skilled artisan, such as natural and artificial flavors and mints, such as peppermint, menthol, citrus flavors such as orange and lemon, artificial vanilla, cinnamon, and various fruit flavors, both individual and mixed and the like can be utilized in amounts from about 0.01% to about 5%, and more preferably 0.01 % to about 0.5% by weight of the suspension.

[0297] The pharmaceutical suspensions of the presently disclosed subject matter can be prepared as follows: (i) admix the thickener with waler heated from about 40° C to about 95° C, preferablyfrom 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) admix the D-allose prepared and / or generated by any of the microorganisms disclosed herein with waler to form a solution; (iii) admix, if desired, a flavoring agent with the thickener- water admixture to form a uniform thickener-flavoring agent;(iv) combine the sweetener solution with the thickener-flavoring agent and mix until uniform; and(v) admix the optional adjunct materials such as coloring agents, flavoring agents, decolorants, solubilizers, anti-foaming agents, buffers and additional water with the mixture of step (iv) to form the suspension.

[0298] The D-allose prepared and / or generated by any of the microorganisms disclosed herein can also be in chewable form. To achieve acceptable stability and quality as well as good taste and mouth feel in a chewable formulation several considerations are important. These considerations include the amount of active substance per tablet, the flavoring agent employed, the degree of compressibility of the tablet, and additional properties of the composition. Chewable pharmaceutical candy is prepared by procedures similar to those used to make soft confectionery. A general discussion of the lozenge and chewable tablet forms of confectionery can be found in H. A. Lieberman and L. Lachman, Pharmaceutical Dosage Forms: Tablets Volume 1 , Marcel Dekker, InC, New York, N.Y. ( ] 989) at pages 367 to 418, which disclosure is incorporated herein by reference. In a typical procedure, a boiled sugar-com syrup blend is formed to which is added a frappe mixture. The boiled sugar-corn syrup blend can be prepared from sugar and corn syrup blended in parts by weight ratio of about 90: 10 to about 10:90. The sugar-corn syrup blend is heated to temperatures above about 120° C to remove water and to form a molten mass. The frappe is generally prepared from gelatin, egg albumin, milk proteins such as casein, and vegetable proteins such as soy protein, and the like, which are added to a gelatin solution and rapidly mixed at ambient temperature to form an aerated sponge-like mass. The frappe is then added to the molten candy mass and mixed until homogeneous at temperatures between about 65° C and about 120° C. The D-allose prepared and / or generated by any of the microorganisms disclosed herein can then be added to the homogeneous mixture as the temperature is lowered to about 65° C-95° C whereupon additional ingredients can then be added such as flavoring agents and coloring agents. The formulation is further cooled and formed into pieces of desired dimensions.

[0299] In other pharmaceutical embodiments, the flavoring agent is incorporated into an ingeslible topical vehicle which can be in the form of a mouthwash, rinse, ingeslible spray, suspension, dental gel, and the like. Typical non-loxic ingeslible vehicles known in the pharmaceutical arts can be used in the presently disclosed subject matter. The preferred ingeslible vehicles are water, ethanol, and water-ethanol mixtures. The waler-ethanol mixlures are generally employed in a weight ratio from about 1 : 1 to about 20: 1 , preferably from about 3: 1 to about 20: 1,and most preferably from about 3: 1 to about 10: 1, respectively. The pH value of the ingestible vehicle is generally from about 4 to about 7, and preferably from about 5 to about 6.5. An ingestible topical vehicle having a pH value below about 4 is generally irritating to the ingestible cavity and an ingestible vehicle having a pH value greater than about 7 generally results in an unpleasant mouth feel.

[0300] The ingestible topical flavoring agents can also contain conventional additives normally employed in those products. Conventional additives include a fluorine-providing compound, a sweetening agent, a flavoring agent, a coloring agent, a humectant, a buffer, and an emulsifier, providing the additives do not interfere with the flavoring properties of the composition. The coloring agents and humectants, and the amounts of these additives to be employed, set out above, can be used in the ingestible topical composition. The flavoring agents (flavors, flavorants) that can be used include those flavors known to the skilled artisan, 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 employed in the ingestible topical composition is normally a matter of preference subject to such factors as the type of final ingestible composition, the individual flavor employed, and the strength of flavor desired. Thus, the amount of flavoring can be varied in order to obtain the result desired in the final product and such variations are within the capabilities of those skilled in the art without the need for undue experimentation. The flavoring agents, when used, are generally utilized in amounts that can, for example, range in amounts from about 0.05% to about 6%, by weight of the ingestible topical composition.5.5. Pet Food Produels

[0030] ] The D-allose prepared and / or generated by any of the microorganisms disclosed herein can be used in a wide variety of pet food products.

[0302] As used herein, the terms “pet food” or “pet food product” refer to a product or composition that is intended for consumption by a companion animal, 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, e g., Canis lupus familiaris. A “pet food” or “pet food product” includes any food, feed, snack, food supplement, liquid, beverage, treat, toy (chewable and / or consumable toys), meal substitute or meal replacement.

[0303] In certain embodiments, the D-allose prepared and / or generated by any of the microorganisms disclosed herein is directly added to a pel food product. In certain embodiments, the D-allose prepared and / or generated by any of the microorganisms disclosed herein can be added prior to, during or after formulation processing or packaging of the pet food product.

[0304] Non-limiting examples of suitable pel food products include wet food products, dry food products, moist food products, pet food supplements (e.g., vitamins), pel beverage products, snack and treats and pel food categories described herein.

[0305] In certain embodiments, the pet food product is a dry food product. A dry or low moisture- containing nutritionally-complete pet food product can comprise less than about 15% moisture. In certain embodiments, the pet food product is a wet food product. A wet or high moisture- containing nutritionally-complete pel food product can comprise greater than about 50% moisture. In certain embodiments, the pet food product is a nutritionally complete moist food product. A moist, e.g., semi-moist or semi-dry or soft dry or soft moist or intermediate or medium moisture containing nutritionally-complete pet food product comprises from about 15% to about 50% moisture.

[0306] In certain embodiments, the pet food product is a pet food snack product. Non-limiting examples of pel food snack products include snack bars, pet chews, crunchy treats, cereal bars, snacks, biscuits and sweet products.5.6. Delivery Systems

[0307] In certain embodiments, the D-allose can be incorporated into a delivery system for use in edible compositions. In certain embodiments, the composition will comprise another flavor or taste modifier such as a salty, umami, bitter, astringent and / or savory tastanl. Delivery systems can be liquid or solid, aqueous or non-aqueous. Delivety systems are generally adapted to suit the needs of the flavor composition and / or the edible composition into which the D-allose will be incorporated.

[0308] The D-allose can be employed in Liquid form, dried form, and / or solid form. When used in dried form, suitable drying means such as spray drying can be used. Alternatively, D-allose can be encapsulated or absorbed onto water soluble materials, including but not limited to materials such as cellulose, starch, sugar, maltodextrin, gum arabic and so forth. The actual techniques for preparing such dried forms are well-known in the art, and can be applied to the presently disclosed subject matter.

[0309] The D-allose can be used in many distinct physical forms well known in the art to provide an initial burst of taste, flavor and / or texture; and / or a prolonged sensation of taste, flavor and / or texture. Without being limited thereto, such physical forms include free forms, such as spray dried, powdered, and beaded forms, and encapsulated forms, and mixtures thereof.

[0310] In certain embodiments, D-allose is encapsulated. Encapsulating materials and / or techniques can be selected to improve the stability of the D-allose and / or food product. In certain embodiments, the encapsulating materials and / or techniques are selected to modify the release profile of D-allose.

[0311] Suitable encapsulating materials can include, but are not limited to, hydrocolloids such as alginates, pectins, agars, guar gums, celluloses, and the like, proteins, polyvinyl acetate, polyethylene, crosslinked polyvinyl pyrrolidone, polymethylmethacrylate, polylaclidacid, polyhydroxyalkanoates, ethylcellulose, polyvinyl acetatephthalate, polyethylene glycol esters, methacrylicacid-co-methylmethacrylate, ethylene-vinylacetate (EVA) copolymer, and the like, and combinations thereof. Suitable encapsulating techniques can include, but are not limited to, spray coating, spray drying, spray chilling, absorption, adsorption, inclusion complexing (e.g., creating a flavor / cyclodextrin complex), coacervation, fluidized bed coating, or other process can be used to encapsulate an ingredient with an encapsulating material.

[0312] Encapsulated delivery systems for flavoring agents or sweetening agents (e.g., D-allose) contain a hydrophobic matrix of fat or wax surrounding a sweetening agent or flavoring agent core. The fats can be selected from any number of conventional materials such as fatly acids, glycerides or poly glycerol esters, sorbitol esters, and mixtures thereof. Examples of fatly acids include but are not limited to hydrogenated and partially hydrogenated vegetable oils such as palm oil, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, soybean oil, cottonseed oil, sunflower oil, safflower oil, and mixtures thereof. Examples of glycerides include but are not limited to monoglycerides, diglycerides, and triglycerides.

[0313] Waxes useful can be chosen from the group consisting of natural and synthetic waxes, and mixtures thereof. Non-limiting examples include paraffin wax, petrolatum, carbowax, microcrystalline wax, beeswax, carnauba wax, candellila wax, lanolin, bayberry wax, sugarcane wax, spermaceti wax, rice bran wax, and mixtures thereof.

[0314] The fats and waxes can be use individually or in combination in amounts varying from about 10 to about 70%, and alternatively in amounts from about 30 to about 60%, by weight of the encapsulated system. When used in combination, the fat and wax are preferably present in a ratio from about 70: 10 to 85: 15, respectively.

[0315] Typical encapsulated compositions, flavoring agent or sweetening agent delivery systems are disclosed in U.S. Patent Nos. 4,597,970 and 4,722,X45, the disclosures of which are incorporated herein by reference in their entireties.

[0316] Liquid delivery systems can include, but are not limited to, systems with a dispersion of D-allose, such as in carbohydrate syrups and / or emulsions. Liquid delivery systems can also include extracts where D-allose is solubilized in a solvent. Solid delivery systems can be created by spray drying, spray coating, spray chilling, fluidized bed drying, absorption, adsorption, coacervation, complexation, or any other standard technique. In some embodiments, the delivery system can be selected to be compatible with or to function in the edible composition. In some embodiments, the delivery system will include an oleaginous material such as a fat or oil. In someembodiments, the delivery system will include a confectionery fat such as cocoa butter, a cocoa butter replacer, a cocoa butter substitute, or a cocoa butler equivalent.[03171 When used in dried form, suitable drying means such as spray drying may be used. Alternatively, D-allose may be adsorbed or absorbed onto substrates such as waler soluble materials, such as cellulose, starch, sugar, maltodextrin, gum arabic and so forth or may be encapsulated. The actual techniques for preparing such dried forms are well known in the art.EXAMPLES

[0318] The presently disclosed subject matter can be better understood by reference to the following. The below examples are exemplary only and should in no way be taken as limiting.Example 1 - Biosynthesis of Allose from Glucose

[0319] E. coli is naturally capable of producing trace amounts of D-allose. In the present example, D-allose production is improved by overexpressing key genes, removing competing pathway genes, and optimizing the production conditions.

[0320] Assessing allose production capabilities in E.coli

[0321] Initially, it was tested if E. coli possesses enzymes capable of producing allose. Allose production was tested in the production strain AL3601 depicted in Table 1 below. Cultures were grown on M9P media (M9 minimum media with 5 g / L yeast extract) with 10 g / L glucose al 30 °C, with samples of the supernatant taken at 24 hours after inoculation. Glucose concentration in the supernatant was analyzed using HPLC. Allose concentration in the supernatant was analyzed using HPAE-PAD. A series of strains containing gene knockouts (KOs) were constructed in AL3601. These KOs included ΔpfkA, Δzwf AmanA, ΔdeoC, Δpgm, ΔalsABCEKR, ΔhxpB, ΔyqaB, and ΔybiV resulting in strains AL3755, AL4240, AL4290, AL4311, AL4328, AL4332, AL4373, AL4374, AL4446, AL4383, and AL4387 (see Table 1). pfkA encodes for phosphofructokinase A (EC 2.7.1.1 1), which converts fructose-6-phosphare (F6P) to fructose- 1 ,6- bisphosphate (F16BP) as part of the first committed step of glycolysis, zwf encodes for 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-laclone as the first committed step in the pentose phosphate pathway (PPP). manA encodes for the enzyme mannose-6-phosphate isomerase (ManA), which catalyzes the reversible isomerization of mannose-6-phosphate and fructose-6-phosphate. deoC encodes for the enzyme deoxyribose-phosphate aldolase, which catalyzes the reversible conversion of allose-6-phosphate to erylhrose-4-phosphate and glycolaldehyde, pgm encodes for the enzyme of glycogen biosynthesis. alsABCEKR encode for enzymes of allose metabolism: alsA encodes for D-allose import ATP-binding protein; alsB encodes for D-allose-binding periplasmic protein; alsC encodes for D-allose transport system permease protein; alsK encodes for D-allosc kinase; and alsR encodes for HTH-type transcriptional regulator. hxpA encodes forthe enzyme Hexitol phosphatase A, which catalyzes the dephosphorylation of D-allose-6- phosphate. yqaB encodes for the enzyme fructose- 1 -phosphate phosphatase (Enlrez Gene ID: 945776), which catalyzes the dephosphorylation of fructose- 1 -phosphate. ybiV encodes for a sugar phosphatase, which catalyzes the dephosphorylation of D-psicose-6-phosphate. The KO strains exhibited increased production ofallose, indicating that E. call possesses enzymes capable of producing allose.

[0322] Table 1. Strain list

[0323]

[0324] Determining allose production enzymes

[0325] Next, it was hypothesized that fructose-6-phosphate (F6P) is an intermediate of allose in E. coli, since the deletion of pjkA increased allose production. The production pathway starts with the native assimilation of glucose into E. coli via the phosphotransferase system (PTS) or GalP / Glk, which converts glucose to glucose-6-phosphate (G6P) (Figure 1 ). G6P is then isomerized to F6P via glucose-6-phosphate isomerase (Gpi) (EC 5.3.1.9). F6P could be converted to psicose-6-phosphate via allulose-6-phosphate 3-epimerase (AlsE). P6P is then isomerized toallose-6-phosphate (A6P) by allose-6-phosphate isomerase (RpiB). A phosphatase could dephosphorylate allose-6-phosphate io free allose, after which it would be excreted from the cell. An allose production system using phosphorylation and dcph.osphorylat.ion steps as system driving forces should be more efficient than the pathway currently used in industrial production of allose.

[0326] By literature search and genome mining, an isomerase candidate was identified. Allose- 6-phosphaie isomerase (RpiB) has shown acti vity towards allose. In addition, several phosphatase candidates were identified, YigL, YqaB, and HxpA have shown activity towards allose-6* phosphate.

[0327] Plasmids (Table 2) was constructed to overexpress genes and hxpA under aninducible promoter, The plasmids were introduced into AL4332, and allose production was tested. Cultures were grown on M9P media with 10 g / L glucose at 30"C and induced with 1 mM IPTG. After 24 hours, induced AL4332 / pAL2310 exhibited increased production of allose.

[0328] Table 2. Plasmid list

[0329] Table 3.

[0330] D-allose is a rare monosaccharide, found .naturally in low abundances. Due to its low- calorie profile and similar taste to sucrose, D-ailose has the potential to become an ideal sugar substitute. D-allose also displays unique properties and health benefits that can be applied to various fields, including food and medical applications. D-allose can be produced using two enzymatic steps to vitro; the epimerization of D~ fructose, then the isomerization of the resulting D-psicose. This method suffers from poor yield due to the reversible nature of both reactions. It was found that Escherichia coll possesses all of the required enzymes to convert D-glucose to D- allose with a thermodynamically favorable pathway, through a series of phosphorylation- epimerization-isomerization-depliosphorylatinn steps. To increase carbon flux toward D-allose production, the pathway genes were additionally expressed, and the competing pathways were removed. The engineered strains achieved production of D-allose, at a titer of 56.4 g a productivity of 0,65 g L-1hr5, and a yield of 41 ,4% under test tube conditions.[0331 } Rare sugars are monosaccharides that are naturally found in low abundance [ 1 [. However, they are widely used in the food and pharmaceutical industries, as they can be non-nutritive or used as precursors for anticancer and antiviral drugs [2], The rare sugar D-allose is a C-3 epimerof glucose that has 80% of the sweetness of sucrose [3] and is minimally metabolized in mammals [4], As a reducing sugar, D-allose has favorable food properties, such as being capable of participating in Maillard reactions browning [5], Indeed, Maillard reactions with α -Iactalbumin occur with a faster reaction rate with D-allose as compared to with D-fructose or D-glucose [6], D-allose also exhibits cryoprotective effects [7], which can have applications in medicine and frozen food preservation,

[0332] D-allose has also has clinically relevant health properties [5], It has been shown to have anti-cancer and anti-tumor effects [8-11], Cancer cells increase their glucose uptake to meet the energy demands of their dysregulated growth; D-allose reduces glucose uptake in cancer cells

[0011] , D-allose has also been reported to have anti -oxidative and anti-inflammatory properties, which could be medicinally applied to minimize ischemia-reperfusion (IZR) damage

[0012] , particularly in the brain

[1316] . Other health benefits of D-allose include hypertension suppression

[0017] and immunosuppression for allograft survival

[0018] , Acute and sub-chronic toxicity trials in rats demonstrate that D-allose is non-toxic

[0019] .

[0333] These sensory, metabolic, physiological, and safety properties increase the demand of D- allose. As with other rare sugars, a cost-efficient, large-scale production system has not been established for D-allose; it has been extracted .from the leaves of various plants, but with low yields [5].

[0334] Enzymatic synthetic pathways have been proposed for D-allose production, but these methods are not thermodynamically favorable causing limited yields (-10%) [20,21] D-allose can be enzymatically synthesized by isomerization of D-psicose into D-allose using L-rhamnose isomerase(L-RhI, EC 5.3.1.14)

[0022] or D-ribose-5-phosphate isomerase B (RptB, EC 5, 3, 1.6)

[0023] , The overall conversion from D-fructose to D-allose is predicted to have a AG>°of 2,4 kJ mol-1(Figure 2A) [24 j . D-psicose is relatively expensive, therefore is not an ideal feedstock. This mixed solution of D-fructose, D-psicose, and D-allose also poses purification difficulties.

[0335] Sugar phosphorylation / dephosphorylation chemistry can be leveraged to produce D- psicose in an industrially relevant microorganism,

[0025] , The sugarphosphorylationZdephosphorylation chemistry is naturally used in sugar intake and metabolism

[0026] . Glucose metabolism begins with the simultaneous phosphorylation and transmembrane transfer via the phosphotransferase system (PTS)

[0027] . The present disclosure demonstrates that this strategy can also be leveraged for D- allose production. The dephosphorylation of D-allose-6- phosphate to D-allose is predicted to be a highly favorable reaction, with a predicted of-35.7 kJ mol-1at typical physiological concentrations of I mM (Figure 2B)

[0024] , To utilize the sugar phosphorylation / dephosphorylation chemistry in vitro, a phosphate source such as ATPmust be supplied, which increases the production costs. Thus, microbial production was used as a cofactor supply is not required.

[0336] E. coli can produce D-allose from D-glucose with only its native enzymes (Figure 2C). In the D-allose pathway, D-fructose-6-phosphate (F6P) is epimerized to ’D-psicose-6-phosphate (P6P) with the D-allulose-6-phosphaie 3-epimemse (AlsE). The produced P6P is further isomerized, to D-allose-6-phosphaie (A6P) by D-allose-6-phosphate isomerase / D-ribose-5- phosphate isomerase B (RpiB), which could then be dephosphorylated to D-allose by a phosphatase. The D-allose production was improved by additionally expressing the D-allose pathway genes and removing competing pathways, including the pentose phosphate pathway (PPP), glycogen biosynthesis, glycolysis, the D-mannose biosynthesis pathway, the D-psicose biosynthesis pathway, and D-allose salvage pathways. To further improve D-allose production, we modulated the expression of key pathway genes with an inducer-free stationary -phase promoter

[0028] and by modulating the design of the pathway operon. D-glucose uptake was improved using the galactose proton symporter GalP

[0029] , then D-glucose is then phosphorylatedby glucokinase Glk

[0030] .

[0337] Results and Discussion a. Design of a thermodynamically favorable D-allose production pathway

[0338] The in vitro enzymatic method for D-allose is thermodynamically unfavorable (21 ] (Figure 2A), and the resulting mixture of monosaccharides requires costly purification techniques [31- 33]. Thus, a D-allose biosynthetic pathway was developed that leverages the phosphorylation and dephosphorylation of sugars to create driving force in E. coli (Figure 2B). A biosynthetic pathway that used the phosphorylation / dephosphorylation chemistry to produce D-psicose from D-glucose was previously established in E coli

[0025] , A branch from the D-psicose pathway was designed for D-allose. Instead of dephosphorylating PGP into D-psicose, PGP could be isomerized into A6P. The dephosphorylation of A6P would be a thermodynamic driver for D-allose production. b. Elucidating enzymes involved in D-allose production

[0339] Initially, the natural capacity of E. coli to produce D-allose was tested (Table 6), but it did not produce D-allose. Then, to increase the F6P pools, pfkA was deleted to generate Strain 1 (AL3755, Table 4). F6P is converted to D-frnctose 1,6-bisphosphate in glycolysis by the phosphofructokinase A and B (PfkA and PfkB) (Figure 2C), and PfkA accounts for of phospbofructosekinase activity [34,35 J. However, the ApJkA strain did not produce D-aliose.

[0340] AlsE was capable of its reverse reaction, converting F6P to P6P

[0025] . RpiB was previously shown to be able io isomerize A6P to P6P

[0036] . The reverse reaction of this enzyme can be used to isomerize P6P to AGP (Figure 2C). E, call possesses various phosphatase enzymes with potential activity toward A6P

[0037] , and it was assumed that the natural expression of thesephosphatases would be sufficient to dephosphorylate AGP to D-allose. The oM and the rprft genes were expressed from an expression plasmid under the Pu«cOi promoter in the strain.However, the strain did not produce D-allose (Figure 3).

[0341] Additional expression of a phosphatase gene can convert A6P to D-allose. £'. co / i phosphatases with a wide range of activity toward sugar substrates were tested: hexitol phosphatase A (HxpA), hexitol phosphatase B (HxpB), sugar phosphatase YbiV, sugar phosphatase YidA, a-D-glucose-1-phosphate phosphatase YihX, phosphosugar phosphatase YigL, and fructose- 1 -phosphate phosphatase YqaB

[0037] , To test each phosphatase for the conversion of AGP to D-allose, the gene for each phosphatase was expressed from an expression plasmid under the Puarfu promoter, along withTable 5). The plasmids were introduced into thestrain. The strain was grown in M9P supplemented with 10 g L-1glucose for 24 hours. After 24 h, the strains harboring the Ar / U and the yqali plasmids (pAL-2310 and p AL2313., Table 5) produced of D-allose, respectively (Figure 3). The strains harboring otherphosphatase plasmids did not produce D-allose (Figure 3). The strains harboring, Table 5) showed increased production of D-psicose, indicating that these phosphatases are capable of dephosphorylaliag P6P. Thus, hxpA was used for further improvement.

[0342] Table 4. Strain List.

[0343]

[0344]

[0345] Table 5. Plasmid List.

[0346] Table 6. Strains used in this example.

[0347]

[0348] Increasing D- allose production by removing competing pathways

[0349] To improve D- allose production, competing pathways were removed. The plasmid containingwas then introduced into these strains, and D-allose production was measured.

[0350] First, the produced D-allose was prevented from being reassimilated into the metabolism. The afcAttCE KR ope.ron, which is responsible for D-allose catabolism

[0038] , was deleted in Strain I to Strain 2 (Figure 4). Strain 2 improved D-allose production to 1.88 g L-1, overthe 1.15 g L-1D-allose production in Strain 1 (Figure 4.}.

[0351] G6P is converted to 6-phospho-D-glucono 1.5-lactone by glucose-6-phosphate dehydrogenase (Zwf) arid diverted to the pentose phosphate pathway

[0039] . The zwf gene encoding for glucose-6-phosphate dehydrogenase was deleted in Strain 2 to generate Strain 3 (Figure 4). Strain 3 further improved D-allose production to 2.4 g L-1(Figure 4).

[0352] D-mannose is a side product when F6P pools are increased within E. coli

[0025] , Thegene encodes for mannose-6-phosphate isomerase, which isomerizes F6P into maunose-6- phosphate (MSP)

[0040] , diverting flux from the D-allose production pathway. Thegene was deleted in Strain 3 to generate Strain 4 (Figure 4). The deletiondid not improve D-allose production (Figure 4), but Strain 4 with the production plasmid consumed less glucose than the previous three strains indicating efficient conversion of ’D-glucose.

[0353] The deoC gene encoding for deoxyribose-phosphate aldolase by which D-allose can be assimilated into central carbon metabolism was deleted in Strain 4 to generate Strain 5 (Figure 4) [4] ]. The deletion of devC did not improve D-allose production (Figure 4),

[0354] Glucose can also be diverted away from the D-allose pathway through glycogen biosynthesis. G lycogen is stored for periods of starvation

[0042] , which may be unnecessary during the D-allose production. The phosphoghicomutase (Pgm) commits glucose to glycogen biosynthesis by converting G6P to glucose-l-phosphate

[0043] , Therefore, the pgm gene was deleted in Strain 5 to generate Strain 6 (Figure 4). The deletion of pgm slightly improved D-allose production (3.0 g L-1, Figure 4).

[0355] Lastly, the promiscuity of E. colis sugar phosphatases could divert flux from the D-allose production pathway by dephosphorylating P6P

[0025] . Hexitol phosphatase B (HxpB), sugar phosphatase YbiV, sugar phosphatase YidA, and fructose- I -phosphate phosphatase YqaB can dephosphorylate P6P into .D-psicose [251 (Figure 3). The phosphatase genes,were sequentially deleted to generate Strains 7, 8, 9, and 10, respectively (Figure 4). The D-allose productions in Strains 7-10 are similar, but Strain 9 showed the highest yield (Figure 4). Therefore, Strain 9 (AL4387, Table 4) was used for further experiments.

[0356] Substrate Channeling to Reduce Nonspecific Dephosphorylation

[0357] D-psicose and D-allose production compete for P6P

[0025] . To decrease D-psicose production, AlsE and RpiB were fused into a chimeric protein using flexible peptide linkers. Substrate channeling could be used to improve flux from F6P io P6P and then to A6P [44,45], Two types of linkers were useddisclosed as SEQ ID NO: 79)anddisclosed as SEQ ID NOS 78 and 79, respectively) (pAl,2562~pAL2565, Table 5) (45,46). The effects of the linkers were tested in Strain 9. However, none of the linkers decreased D-psicose production nor improved D-allose production (figure 5).

[0358] Modulation of Pathway Enzyme Expression

[0359] The expressions of the D-allose pathway genes were modulated to improve D-allose production. When expressing multiple genes from an operon, expression of downstream genes is lower than their upstream counterparts |47], To direct flux of P6P toward A6P, we constructed a plasmid containingmaking the expression of rpiB stronger relative to

[0360] D-allose production was evaluated after increasing the expression ofp , The natural tixpA gene starts with a GTG codon, which leads to lower translation compared to genes that start with the ATG codon

[0048] , The start codon of fapA was replaced with ATGTable 5).

[0361] Production can also be improved with pathway gene expression under a stronger promoter

[0025] , The early stationary phase promoter has an expression strength 100 times stronger than IPI'G-inditced promoter PM, and it improved D-psicose production

[0025] . The three factors (promoter, gene order; and start codon) were combined (pAL258O, pAL2609, pAL2624, Table 5). The effects of those modifications on D-allose production were tested in AL4387 (Fig. 5). Strain 9 containingproduced the most D-allose (3,1 g L.'*, respectively) (Figure 6).

[0362] Supplementing Glucose Import Using GalP and Glk

[0363] Continuous import, and phosphorylation of D-glucose is vital to D-allose production. A limitation of this is imposed by the deletion of, which decreases the pools of downstream metabolites of glycolysis, including phosphoesolpyruvate (PEP). PEP is utilized by the PTS to import and phosphory late extracellular D-glucose to intracellular G6P, and the reduction of PEP pools may decrease glucose uptake [49,5t)|. Furthermore, it has been shown that in the context of a deletion, the accumulated G6P and F6P may degrade the mRNA [51 |,

[0364] G6P pools were enhanced through an alternative D-glucose import, and phosphorylation mechanism. GalP is a galactose proton symporter capable of importing glucose

[0029] , and Glk is a glucokinase that phosphorylates glucose to G6P using ATP

[0030] (Figure 2C). A plasmid was constructed containing ga / P and glk under theLWH promoter (pAL2264, Table 5)25. pAL2264 was introduced into Strain 9 along with pAL2310Anticipating that glucose import would increase

[0025] , the concentration of glucose supplementedto the presently disclosed strains was increased io 40 g L-1, then D-allose production was measured after 24 hours (Figure 7).

[0365] Expression of gin Strain 9 harboringled to production of 10,59 g L-1D-allose and a yield of 39.4% (Figure 7). in contrast. Strain 9 harboring onlyproduced 4,25 g L-1D-allose, with a 26,0 % yield (Figure 7). Similarly, Strain 9harboringalso saw improvement in D-allose production whenwas expressed (Figure 7). However, this strain produced more D-allose than Strain 9 harboringproducing 6.1 g L-1’ D-allose without expression and 11.3 g L-1D-allose withexpression (33.9% and 41 4% yield, respectively) (Figure 7) Strain 9 harboringsvas thereafter used in further production experiments.

[0366] ; Interestingly, Strain 9 harboringdid not produce more D-allose whenwas expressed (Figure 7). Expression of AlsE. RpiB, and HxpA is weaker under thepromoter than the promoter

[0025] ,

[0367] Modulation of the psicose: allose prod uction ratio

[0368] Blending different sugar substitutes in a food or beverage can improve its the sensory quality while minimizing its caloric content

[0059] . Of particular interest is D-psicose, a zero-calorie sugar substitute that is already Generally Recognized as Safe (GRAS) by the U.S. Food and Drag Administration

[0060] , Its sweetness is estimated to be 70% that of sucrose and is described to provide a cooling sensation while not being bitter [61 [

[0369] Tc> co-produce D-psicose and D-allose at different ratios, different, native sugar phosphatase genes were expressed. Additional expression of the native phosphatase genes yand hxpA from a plasmid leads to both D-psicose and D-allose production (Figure 3). The plasmidswere introduced into Strain 9 (Table 4), The strains were grown in M9P supplemented with 10 g L-1glucose for 24 h. After 24 h. Strain 9 harboring pAL2310 produced 3.1 g L-1D -allose and 0.1 g L-1D-psicose, corresponding to a 27 : I ratio of D-allose : D-psicose produced (Figure 78). Strain 9 harboring pAL2313 instead produced 1.0 g L-1D -allose and 0.8 g L-1D-psicose, corresponding to a 1 3 : 1 ratio of D-allose : D-psicose produced (Figure 7B). These results indicate that the ratio of allose to psicose can be changed by differences of substrate specificity of the phosphatases.

[9370] D-allose Production Under High Density Culture Conditions

[0371] To decouple growth and production, and minimize limitations on production by glucose availability, Strain 9 containing the pAL2609 and pAL2264 plasmids (Table 5) was cultured under high cell density conditions. The strain was grown to an OD600of -1 before being induced with 1 n M 1.PTG and grown for a further 30 min. Cells were then pelleted and resuspended to an OD600of -10 with M9P containing 40 g L "aglucose and I mM 1PTG Every 24 h, 10% of the culture x oliune was removed and replaced with MOP media containing 400 g L!glucose and I mXl IP 1 G. |03 "2 ] Spain 9 harboring pA1.2(»O9 and p.AL2264 produced 56.4 g I.!D-allow alter 120 h. a net yield of 29.7% (Figure 8A). Peak productivity of 15.5 g E*1day"1was achieved between 48 h - 72 h, with a 38.8% yield (Figure 8A). Between O h - 24 h, 24 hr- 48 h, and 48 li — 72 h, the supplemented glucose was completely consumed (Figure 8B). Glucose consumption slowed after ~2 h tFiguie SB) Depletion of glucose in the media allows for easier purification of D-allose during industrial production D-psicose production increased to 28 7 g L;after 96 hours, then decreased between 96 h and 120 h (Figure 8A). The highest ratio of g L-1D-allose : g L"1D- psicose was measured at 24 h (2.43 : 1 ), at which no D-glucose was present (Figure 8 A).10.373 i Herein, / ’. .o.' / s name metabolism was tew ired to produce D-allose tn a thermodynamically fav orable manner. I he expression of the name t / M-.. hx / >A. go / / ’. and g / A- genes in combination with eliminating competing pathways were used to direct carbon flux from D-glucose; The titer of D-allose produced after 120 hr 'was 56.4 g.L1with a peak products ity of 0.65 g l.‘!hr'1under test tube conditions. The highest yield achieved was 41.4%, surpassing even one-step etizv mane methods to produce D-allose 152.531. Utis example indicates that the presently disclosed bacteria have strong potential for producing D-allose in high abundance.103741 Methods103751 Reaeents. Ail enzymes involved in the molecular cloning experiments were purchased from New Fngland Btolabs <\1 B). All synthetic oligonucleotides were synthesized by Integrated DNA Technologies. Sanger Sequencing was provided by Genewiz. D-Psicose was purchased from Sigma-Aldrich. D-Glucose was purchased from Fisher Scientific. D- Al lose was purchased from I iiermo Scientific iO376j Sintins an^^ All strains and plasmids used in this study are listed in Tables 6 and 7. respectively. All oligonucleotides are listed in Table 8. Plasmids for D-allose production were constructed using sequence and ligation independent cloning (SLIC) |54|. The constructed plasmids were verified via sequencing. A guide to the construction of plasmids used in this study is detailed in fable 9JO3771 Genome modifications such as gene deletion and gene insertion were constructed using CRISPR-Cas9-mediated homologous recombination |55|. Linear DNA repair fragments for gene deletions aud insertions were constftictefr by amplifying genomic or plasmid 'DNA via PCM assembly |5<»|. Plasmids encoding sgRNA foi ( RISPR-Cas9-mediated homologous recombination Were constructed using Q5 site-directed mutagenesis (New England Btolabs) using pTargetF plasmid (Addgene #62226 ) as a template. All genomic modifications were verified viasequencing. A guide to the CRISPR-CasV-mediated gene modifications used in this study is detailed in Table 10.

[0378] Table 7 Plasmids used in this example.I ll

[0379] Table 8.: Oligonucleotides used in this example.[0380 ) Table 9. Plasmid Construction Guide.

[0381] Table 10 Guide for CRlSPR-Cas9-medialed gene deletions and insertions.

[0382] Culturing condition*. Oxernighl cu lunes were grown al .V C m 3 mL of Luria-Bei tani (LB) media with appropriate antibiotics. Antibiotic concentrations were as Mows: spectinomycin (50 pg mL-1). ampicillin (2<i(» pg ml; r kanainycm <50 pg ml;'' ). gentamycin (3 75 pg ml;'). M9 minimal media comiMs of 337 niM NadlPOi. 22 mM KI I PO., s.o m\f NaCI. o 4 mM \H ;O1. 2mMMgSO4:: 0.1 mMCaCb, A5trace metals mix (2.86 tng L^flsBOs, 1.81 mgL'1MnCbAHjO, 0.079 mgL“JCtSOa-SIhO, 49,4 pg L'* CofNOjJa-OHjO), varying concentrations of glucose, and appropriate antibiotics. M9P media for the D-allose production consists of M9 minimal media supplemented with 5 g L-1of yeast extract and appropriate antibiotics. No D«al lose production was delected when cultures were grown in MOP media w ithout glucose. Inducer concentrations are as follows isopropv l-(’>-D-i -lliiogalaciopc ranoside ( IP 1 < i ) i I mM). anhvdroietracs cline (a I c)(100 ng mb*1). OD600was measured with a Synergy Hl hybrid plate reader (BioTek Instruments, Inc.).101X31 D-Allose production. tor regular cell density production experiments. overnight cultures were inoculated at 1% into 3 mL of MOP media. Cells were grown at 37 °C until the OD600described, then induced with IPTG and aTc if necessary, and grown at 30;OC for 24 h.|()3X4| For high cell density production experiments in M9P media, overnight cultures were inoculated at 2°.> into >0 nil. of MVP media. Cells were grown at 37 C until OIW I Cultures were then induced with IPTG and aTc if necessary and grown for a further 30 min. Cultures were centrifuged at 5,000 gfor 15 min and resuspended in M9P media with IPTG and aTc if necessary to a target ODMW. Cultures were grown al 30 C Every 24h, 10% of the culture volume was removed and replaced with MW media, containing 400 rg glucose, with IPTG and aTc if necessary.|O3K5| HPLC analysis. Analysis of D-glucose concentrations was performed using HPLC (Shimadzfo equipped with a refractive index detector (RID) 10 A and Reyex1* RCU-USP sugar alcohol column (Phenomenex). Mobile phase was comprised of 100% MilliQ water Samples were inn w ith tin injection volume of I ul. al a flow rale of 0 5 nil mnC for 7 min. with the column ov en al S3 ‘ ( and RID cell icmperalure of4<» (|03K61 To prepare samples for HPLC analysis, 300 pL of culture was centrifuged at 17.000 g for 5 min , Supernatants were applied to a 0.2 pm PVDF hydrophi lie membrane 96 wel I filter plate and centrifuged at 17,000 g for 2 min into a polystyrene 96 well.|O3X71 Hieh-Perlbnnance Amon-F.xehan»e cl-romaloeraphv t HPAl-A Analysis. Analysis of D- psicose -and D-allose concentrations was -perfomed using, HPAE- with Pulsed Amperometric Detection (PAD) on an ICS-5000 system with a CarboPac PA 104x250mm (Thermo Fisher). The wash phase was comprised o!200 mM NaOH tn degassed MilliQ waler at a llovv rale of O X ml. mm1for ID mm 1 he equilibrium phase was comprised of |(K> mM NaOH in degassed MilliQ waler al a flow rate of U.S ml mur for 25 min Mobile phase was comprised of 100 mM NaOH in degassed MilliQ water. Samples were run with an injection volume of 10 gL at a flow rate of 0.8 mL mur* for 10 min. The column oven was maintained at 30 ’C.|03XXi To prepare samples for HPAE-PAD analysis. 300 ul of culture was centrifuged at I 7.000 g for 5 min Supernatants were diluted HlO-loId and applied to a 02 urn PVDI hy drophilic membrane 96 well filter plate and centrifuged at 17.000 g for 5 min into a polystyrene 96 well.103X91 Table 11 . Standard Dev iation of Yields and OD(>00 reported in I mure 3

[0390] Table 12. Standard Deviation of Yields and OD600 reported in Figure 4.

[0391] Table 13. StandardDeviation of Yields and OD600 reported in Figure 3.

[0392] Table 14Standard Deviation of Yields and. OD600 reported in Figure 6.

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[0394] Although the presently disclosed subject matter and its advantages have been described in detail, it should be understood that' various changes, substitutions and alterations can be made herein without departing from die spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of mater, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the presently disclosed subject mater, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized according to the presently disclosed subject mater. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

[0395] 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

WHAT IS CLAIMED IS:

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

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

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

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

5. The recombinant microorganisms of any one of claims .1-4, wherein the epimerase comprises the amino acid sequence set forth in SEQ ID NO: 1,6. The recombinant microorganisms of any one of claims 1 -5, wherein the epimerase consists of the amino acid sequence sei forth in SEQ ID NO: 1.

7. The recombinant microorganism, of claim 1, wherein the phosphatase is a hexitol phosphatase A (HxpA).

8. The recombinant microorganism of claim 1 or 7, wherein the phosphatase is an % coll HxpA,9. The recombinant, microorgan ism o f claim 17, or 8, wherein the phosphatase comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 62.

10. The recombinant microorganism of any one of claims 1 and 7-9, wherein the phosphatase comprises the amino acid sequence set forth in SEQ ID NO: 62.

11. The recombinant microorganism of any one of claims 1 and 7-10, wherein the phosphatase consists of the amino acid sequence set forth in SEQ ID NO; 62.

12. The recombinant microorganism of claim 7 or 8, wherein the phosphatase comprises an adenine-thymine-guanine (ATG) start codon.

13. The recombinant microorganism of claim 1, wherein the phosphatase is a fructose-1- phosphate phosphatase (YqaB).

14. The recombinant microorganism of claim 1 or 13, wherein the phosphatase is an E. coli YqaB.

15. The recombinant microorganism of claim 1 , 13, or 14, wherein the phosphatase comprises an amino acid sequence at least about 80% identical to the amino acid, sequence set forth in SEQ ID NO: 60.

16. The recombinant microorganism of any one of claims 1 and 13-15, wherein the phosphatase comprises the amino acid sequence set forth in SEQ ID NO; 60.

17. The recombinant microorganism of any one of claims 1 and 13-16, wherein the phosphatase consists of the amino acid sequence set forth in SEQ ID NO: 60.

18. The recombinant microorganism of any one of claims 1-17, wherein the isomerase is an allose-6-phosphale isomerase (RpiB),19. The recombinant microorganism of claim 18, wherein the RpiB is an E. coli RpiB.

20. The recombinant microorganism of claim 18 or 19, wherein the isomerase comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 24.

21. The recombinant microorganism of any one of claims 18-20, wherein the isomerase comprises the amino acid sequence set forth in SEQ ID NO: 24.

22. The recombinant microorganism of any one of claims 18-21, wherein the isomerase consists of the amino acid sequence set forth in SEQ ID NO: 24.

23. The recombinant microorganism of any one of claims 1 -22, wherein the recombinant microorganism further comprises exogenous galactose'.H* symporter (GalP) and glucokinase (Glk).

24. The recombinant microorganism of claims 23, wherein the GalP is an E. coli GalP and the Glk is E. coli Glk.

25. The recombinant microorganism of claim 24, wherein 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.

26. The recombinant microorganism of claim 25, wherein 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.

27. The recombinant microorganism of any one of claims 1-26, wherein the recombinant microorganism further comprises exogenous glucose-6-phosphate isomerase (Gpi).

28. The recombinant microorganism of any one of claims 1 -27 further comprising a mutation of a gene encoding for an enzyme of the pentose phosphate pathway compared to a naturally occurring microorganism.

29. The recombinant microorganism of claim 28, wherein enzyme of the pentose phosphate pathway is glucose-6-phosphale 1 -dehydrogenase (Zwf).

30. The recombinantmicroorganism of any one of claims .1 -29 furthercomprising a mutation of a gene encoding for an enzyme of the glycolysis compared to a naturally occurring microorganism.

31. The recombinant microorganism of claim 30, wherein the enzyme of the glycolysis is phosphofructokinase-l (PtkA), phosphofructokinase-2 (PfkB). or pyruvate kinase (PykF).

32. The recombinant microorganism of claim.30 or 31, wherein the enzyme of the glycolysis is phosphofructokinase- 1 (PfkA),33. The recombinant microorganism of any one of claims 1 -32 further comprising a mutation of a gene encoding for an aldolase.

34. The recombinant microorganism of claim 33, wherein the aldolase is deoxyribose- phosphate aldolase (DeoC).

35. The mcombinant microorganism of any one of claims 1 -34 further comprising a mutation of at least one gene encoding for a phosphatase.

36. The recombinant microorganism of claim 35, wherein the at least one gene encoding for a phosphatase is selected from fructose- 1 -phosphate phosphatase (YqaB), sugar phosphatase YbiV, hexilol phosphatase B (HxpB), sugar phosphatase YidA, or combinations thereof37. The recombinant microorganism of any one of claims 1 -36 further comprising a mutation of at: least one gene encoding for an enzyme of allose metabolism.

38. The recombinant microorganism of claim 37, wherein the at least one gene encoding for an enzyme of allose metabolism is selected from D-allose import ATP-binding protein (AlsA), D-allose-binding periplasmic protein (AlsB), D-allose transport system permease protein (AIsC), D-allose kinase (AisK), HTH-type transcriptional regulator (AlsR), or combinations thereof.

39. The recombinant microorganism of any one of claims 1 -38 further comprising a mutation of a gene encoding for an enzyme of mannose biosynthesis pathway.

40. The recombinant microorganism of claim 39, wherein the enzyme of mannose biosynthesis pathway is mannose-6-phosphate isomerase (ManA)41. The recombinant microorganism of any one of claims 1 -40 further comprising a mutation of a gene encoding for an enzyme of the phosphotransferase system (PTS).

42. The recombinant microo.rgani.sm of an y one of claims 1 -41 , further comprising a mutation of a gene encoding for an enzyme of glycogen biosynthesis selected from phosphoglucomutase (Pgm), UDP-glucose pyrophosphorylase, glycogen synthase, glycogen branching enzyme, and glycogenin.

43. The recombinant microorganism of claim 42, wherein the enzyme of glycogen biosynthesis is phosphoglucomutase (Pgm).

44. The recombinant microorganism, of any one of claims 1-43. wherein the exogenous epimerase, the exogenous isomerase, and the exogenous phosphatase are expressed by a stationary phase promoter.

45. The recombinant microorganism of any one of claims 1-43, wherein the exogenous epimerase, the exogenous isomerase, and the exogenous phosphatase are expressed by an inducible promoter.

46. A microorganism comprising a recombinant polynucleotide encoding an epimerase, an isomerase, and a phosphatase, wherein expression of the epimerase, the isomerase, and the phosphatase results in an increased production of ailose as compared to a microorganism lacking the recombinant polynucleotide.

47. The recombinant microorganism of claim 46, wherein the epimerase is an allulose-6- phosphate 3 -epimerase (AlsE).

48. The recombinant microorganism of claim 46 or 47. wherein the epimerase, is an E. coli AlsE.

49. The recombinant microorganisms of any one of claims 46-48, wherein the epimerase comprises an ammo acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 1.

50. The recombinant microorganisms of any one of claims 46-49, wherein the epimerase comprises the amino acid sequence set forth in SEQ ID NO:

1. 51, The recombinant microorganisms of any one of claims 46-50, wherein the epimerase consists of the amino acid sequence set forth in SEQ ID NO: 1 .

52. The recombinant microorganism of claim 46, wherein the phosphatase is a hexitol phosphatase A ( HxpA).

53. The recombinant microorganism of claim 46 or 52, wherein the phosphatase is an E. coli HxpA.

54. The recombinant microorganism of claim 46, 52, or 53, wherein the phosphatase comprises an amino acid sequence at least about 86% identical to the amino acid sequence set forth in SEQ ID NO; 62.

55. The recombinant microorganism of any one of claims 46 or 52-54, wherein the phosphatase comprises the amino acid sequence set forth in SEQ ID NO: 62.

56. The recombinant microorganism of any one of claims 46 or 52-55, wherein the phosphatase consists of the amino acid sequence set forth, in SEQ ID NO: 62.

57. The recombinant niicroorganism of claim 53 or 54, wherein the phosphatase comprises an adenine-thymine-guanine (ATG) start codon.

58. The recombinant microorganism of claim. 46, wherein the phosphatase is a fructose- 1 - phosphate phosphatase (YqaB).

59. The recombinant microorganism of claim 46 or 58, wherein the phosphatase is an E. coli YqaB.

60. The recombinant microorganism of claim 46, 58, or 59, wherein the phosphatase comprises an amino acid sequence ai least about 80% identical io the amino acid sequence set forth in SEQ ID NO: 60,61. The recombinant microorganism of any one of claims 46 or 58-60, wherein the phosphatase comprises the amino acid sequence set forth in SEQ ID NO: 60,62. The recombinant microorganism of any one of claims 46 or 58-61, wherein the phosphatase consists of the amino acid, sequence set forth in SEQ ID NO: 60.

63. The recombinant microorganism of any one of claims 46-62 , wherein the isomerase is an allose-6-phosphate isomerase (RpiB).

64. The recombinant microorganism of claim 63, wherein the RpiB is an E. coli RpiB.

65. The recombinant microorganism of claim 63 or 64, wherein the isomerase comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 24.

66. The recombinant microorganism of any one of claims 63-65, wherein the isomerase comprises the amino acid sequence set forth in SEQ ID NO: 24.

67. The recombinant microorganism of any one of claims 63-66, wherein the isomerase consists of the amino acid sequence set forth in SEQ ID NO: 24.

68. The recombinant microorganism of any one of claims 63-67, wherein the recombinant microorganism further comprises exogenous galactoseTT symporier (GalP) and glucokinase (Glk),69. The recombinant microorganism of claims 68, wherein the Gal? is an E. coli GalP and the Glk is E. coli Glk.

70. The recombinant microorganism of claim 69, wherein the GalP comprises an amino acid sequence at least about 80% identical to the ammo 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: 40,71. The recombinant microorganism of claim 70, wherein 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.

72. The recombinant microorganism of any one of claims 46-7!, wherein the recombinant microorganism further comprises exogenous ghicose-6-phosphate isomerase (Gpi).

73. The recombinant microorganism of any one of claims 46-72 further comprising a mutation of a gene encoding for an enzyme of the pentose phosphate pathway compared to a naturally occurring microorganism.

74. The recombinant microorganism of claim 73, wherein enzyme of the pentose phosphate pathway is glucose-6-phosphate 1 -dehydrogenase (Zwf).

75. The recombinant microorganism of any one of claims 46-74 furth er comprising a mutation of a gene encoding for an enzyme of the glycolysis compared io a naturally occurring microorganism.

76. The recombinant microorganism of claim 75, wherein the enzyme of the glycolysis is phosphofructokinase-1 (PfkA), phosphofructokmase-2 (PfkB), or pyruvate kinase (PykF).

77. The recombinant microorganism of chum 75 or 76, wherein the enzyme of the glycolysis is phosphofructokinase- 1 (PfkA).

78. The recombinant microorganism of any one of claims 46-77 further comprising a mutation of a gene encoding for an aldolase.

79. The recombinant microorganism of claim 78, wherein the aldolase is deoxyribosephosphate aldolase (DeoC).

80. The recombinant microorganism of any one of claims 46-79 further comprising a mutation of at least one gene encoding for a phosphatase,81. The recombinant microorganism of claim 80, wherein the at least one gene encoding for a phosphatase is selected from fructose- 1 -phosphate phosphatase (YqaB), sugar phosphatase YbiV, hexitol phosphatase B (H.xpB), sugar phosphatase YidA, or combinations thereof.

82. The recombinant microorganism of any one of claims 46-81 further comprising a mutation of at least one gene encoding for an enzyme of allose metabolism.

83. The recombinant microorganism of claim 82, wherein the at least one gene encoding for an enzyme of allose metabolism Is selected from D-allose import ATP-binding protein (AlsA), D-allose-binding periplasnuc protein (AIsB), D-allose transport system permease protein (AIsC), D-allose kinase (AlsK), HTH-type transcriptional regulator (AlsR), or combinations thereof.

84. The recombinant microorganism of any one of claims 46-83 further comprising a mutation of a gene encoding for an enzyme of mannose biosynthesis pathway,85. The recombinant microorganism of claim 84, wherein the enzyme of mannose biosynthesis pathway is mannose-6-phosphate isomerase (ManA)86. The recombinant microorganism of any one of claims 46-85 further comprising a mutation of a gene encoding for an enzyme of the phosphotransferase system (PTS).

87. The recombinant microorganism of any one of claims 46-86, further comprising a mutation of a gene encoding for an enzyme of glycogen biosynthesis selected from the group consisting of phosphoglucomutase (Pgm), UDP-glacose pyrophosphorylase, glycogen synthase, glycogen branching enzyme, and glycogenin.

88. The recombinant microorganism of claim 87, wherein the enzyme of glycogen biosynthesis is phosphoglucomutase (Pgm).

89. The recombinant microorganism of any One of claims 46-88, wherein the exogenous epimerase, the exogenous isomerase, and the exogenous phosphatase are expressed by a stationary phase promoter.

90. The recombinant microorganism of any one of claims 46-88, wherein the exogenous epimerase, the exogenous isomerase, and the exogenous phosphatase are expressed by an inducible promoter.

91. A microorganism comprising: a) a recombinant polynucleotide encoding an epimerase, an isomerase, and a phosphatase', b) a mutation of a gene encoding for an enzyme of the pentose phosphate pathway ; c) a mutation of a gene encoding for au enzyme of the glycolysis; d) a mutation of a gene encoding for an aldolase; e) a mutation of a gene encoding for an enzyme of mannose biosynthesis pathway: f) optionally a recombinant polynucleotide encoding GalfT Glk, or both; g) optionally a recombinant Gpi; and h) optionally a mutation of a gene encoding for an enzyme of the phosphotransferase system.

92. The microorganism of claim 91, further comprising a mutation of at least one enzyme encoding for a phosphatase.

93. The microorganism of claim 91 or 92, further comprising a mutation of at least one enzyme of a'llose metabolism.

94. A microorganism comprising: a) a recombinant polynucleotide encoding an allulose-6-phosphate 3-epimerase (AlsE), an allose-6-phosphaie isomerase (RpiB), and a hexilol phosphatase A (HxpA);b) a mutation of glucose-6-phosphale 1 -dehydrogenase (Zwf); c) a mutat ion of phosphofructokinase- 1 (Pile A); d) a mutation of deoxyribose-phosphate aldolase (DeoC); e) a mutation of mannose-6-phosphate isomerase (Man A); f) optionally a recombinant polynucleotide encoding GalP, Glk. or both; g) optionally a recombinant polynucleotide encoding Gpi; and h) optionally a mutation of a gene encoding for an enzyme of the phosphotransferase system.

95. The microorganism of claim 94, further comprising a mutation of at least one gene selected from fructose- 1 -phosphate phosphatase (YqaB), sugar phosphatase YbiV, hexitol phos- phatase B (HxpB), sugar phosphatase (YidA), ATP-binding protein (AlsA), D-allose- binding periplasmic protein (AIsB), D-aHose transport system permease protein (AlsC), D- allose kinase (AlsK), HTH-type transcriptional regulator (AlsR), or combinations thereof.

96. .A microorganism comprising: a) a recombinant polynucleotide encoding an. allulose~6~phosphate 3-epimerase (AlsE), an allose-6-phosphate isomerase (RpiB), and a fructose- 1 -phosphate phosphatase (YqaB); b) a mutation of glucose-6-phosphate 1 -dehydrogenase (Zwf); c) a mutation of phosphofructakinase-l (PikA). d ) a mutation of deoxyribose-phosphate aldolase (DeoC); e) a mutation of mannose-6-phosphate isomerase (ManA); f) optionally a recombinant polynucleotide encoding GalP, Glk, or both; g) optionally a recombinant polynucleotide encoding Gpi; and h) optionally a mutation of a gene encoding for an enzyme of the phosphotransferase system.

97. The microorganism of claim 96, further comprising a mutation of at least one gene selected from, sugar phosphatase YbiV, hexitol phos-phatase B (HxpB), sugar phosphatase (YidA), ATP-binding protein (AlsA), D-allose-binding .periplasmic protein (AlsB), D-allose transport system permease protein (AlsC), D-allose kinase (AlsK), HTH-type transcriptional, regulator (AlsR), or combinations thereof;9B. A microorganism comprising: a) a recombinant polynucleotide encoding an allulose-6-phosphaie 3-epimerase ( AlsS);b) a recombinant polynucleotide encoding an allose-6-phosphate isomerase (RpiB); c) a recombinant polynucleotide encoding a hexitol phosphatase A (HxpA); d) a mutation of glucose-6-phosphate 1 -dehydrogenase (Zwf); e) a mutation ofphosphofructokinase-1 (PlkA); f) a mutation, of deoxyribose-phosphate aldolase (DeoC); g) a mutation of mannose-6-phosphate isomerase (Man A); fa) optionally a recombinant.polynucleotide encoding GalP, Glk, or both; i) optionally a recombinant polynucleotide encoding Gpi; and j) optionally a mutation of a gene encoding for an enzyme of the phosphotransferase system. )9. The microorganism of claim 5)8, further comprising a mutation of at. least one gene selected from fructose*. I -phosphate phosphatase (YqaB), sugar phosphatase YbiV, hexitol phosphatase B (HxpB)» sugar phosphatase (YidA), ATP-binding protein (AlsA), D-allose- binding periplasmic protein (AlsB), D-allose transport system permease protein (AlsC), D-allose kinase (AIsK), HTH-type transcriptional regulator (AlsR), or combinations thereof.

100. A microorganism comprising; a) a recombinant polynucleotide encoding an allulose-6-phosphate 3- epimerase ( AlsE); b) a recombinant polynucleotide encoding an allose-6-phosphaie isomerase (RpiB); c) a recombinant polynucleotide encoding a fructose- 1 -phosphate phosphatase (YqaB); d) a mutation of glucose-6-phosphate 1 -dehydrogenase (Zwf); e) a mutation of phosphofructokinase- 1 (PfkA); f) a mutation of deoxyribose-phosphate aldolase (DeoC); g) a mutation of mannose-6-phosphate isomerase (ManA); h) optionally a recombinant polynucleotide encoding GalP, Glk, or both; i) optionally a recombinant polynucleotide encoding Gpi; and j ) optional ly a mutation of a gene encoding for an enzyme of the phosphotransferase system.

191. The microorganism of claim 109, further comprising a mutation of at least one gene selected from sugar phosphatase YbiV, hexitol phosphatase B (HxpB), sugar phosphatase (YidA), ATP-binding protein (AlsA), D-allose-binding periplasmic protein (AlsB), D-aliose transport system permease protein (AlsC), D-allose kinase (AlsKx HTH-type transcriptional regulator (AlsR), or combinations thereof.

102. The microorganism of any one of claims 28-45 and 73-101. wherein the mutation is a deletion.

103. The microorganism of any one of claims 28-45 and 73-102, wherein the mutation reduces or eliminates expression or activity of the enzyme.

104. The microorganism of any one of claims 1-103, wherein the microorganism is E. coli,105. A method for producing aliose. comprising culturing the microorganism of any one of claims 1. - 104 under conditions suitable for converting a substrate to aliose.

106. The method of claim 105, wherein the substrate comprises glucose.

107. A method of making a food product comprising allose comprising a) culturing the microorganism of any one of claims 1-104 under conditions suitable for converting a substrate to allose: and b) admixing the allose with one or more foods to form a food comprising allose.

108. The method of claim 107, wherein the food product is a beverage, yogurt, ice cream, a baked good, or a nutritional bar.

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