Microorganisms for the production of l-sorbose

Abstract

The presently disclosed subject matter relates to compositions and methods for producing L-sorbose in microorganisms.

Description

MICROORGANISMS FOR THE PRODUCTION OF L-SORBOSECROSS-REFERENCE, TO RELATED PAlENlfAPPLlCATIONS

[0001] The present application claims benefit of priority to U.S. Provisional Patent Application Nos. 63 / 692,582, filed September 9, 2024 and 63 / 773,631, filed, March 18, 2025, each of which are incorporated by reference in their entireties for all purposes.TECHNICAL FIELD

[0002] The presently disclosed subject mater relates to composi tions and methods for producing L-sorbose in microorganisms.BACKGROUND

[0003] It has been known that D-sugars (e.g., D-sorbose, etc.) can be used in preparation of compositions as sweeteners. These sugars can lead to increased caloric intake and increased risk of developing certain metabolic diseases (e.g., insulin resistance, type 2 diabetes, obesity, etc,). To overcome the drawback of D-sugars, L-sugars (e.g., L-sorbose) could be used as they do not lead to increased caloric intake. However, the synthesis of L-sugars is too expensive to be viable. Thus, there is a need for producing L-sugars, in particular L-sorbose, in an economic and efficient manner.SUMMARY

[0004] In certain embodiments, the presently disclosed subject matter relates to a recombinant microorganism that produces an increased amount of an L-sorbose (and / or in some embodiments D-fructose, D-sorbitol and / or D-sedoheptulose) as compared to a naturally occurring microorganism, wherein the recombinant microorganism includes at least one exogenous oxidoreductase.

[0005] In one aspect, the presently disclosed subject matter relates to a recombinant microorganism that produces an increased amount of an L-sorbose (and / or in some embodiments D-fiuctose, D-sorbitol and / or D-sedoheptulose) as compared to a naturally occurring microorganism, wherein the recombinant microorganism includes at least one exogenous oxidoreductase and at least one exogenous phosphatase. In some embodiments, the at least one exogenous oxidoreductase is a sorbitol-6-phosphate dehydrogenase (SorD / SrlD), a L-sorbose 1 - phosphate reductase (SorE), or a combination thereof.

[0006] In some embodiments, the SorD / SrlD includes an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: I , SEQ ID NO: 2. or SEQ ID NO: 4. In some embodiments, the SorD / SrlD includes or consists of the amino acid sequence setforth in SEQ ID NO: I , SEQ ID NO: 2, or SEQ ID NO; 4. In some embodiment, the SorD / SrID includes or consists of the amino acid sequence set forth in SEQ ID NO: 2.

[0007] In some embodiments, the SorE includes an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 1 .1 , In some embodiments, the SorE includes or consists of the amino acid sequence set forth in SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 1 1. In some embodiments, the SorE includes or consists of the amino acid sequence set forth in SEQ I D NO: 9.

[0008] In some embodiments, the phosphatase is a hexitol phosphatase B (HxpB), a fructose- 1 phosphate phosphatase (YqaB), a sugar phosphatase YbiV (YbiV), Hexitol phosphatase A (HxpA), sugar phosphatase YidA, phosphosugar phosphatase Y'igL, sugar phosphatase YihX or a combination thereof. In some embodiments, the HxpB includes an amino acid sequence that is at least about 80% identical io the amino acid sequence set forth in SEQ ID NO; 15. In some embodiments, the HxpB includes or consists of the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the YqaB includes an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ) ID NO: 17. In some embodiments, the YqaB includes or consists of die amino acid sequence sei forth in SEQ ID NO: 17. In some embodiments, the YbiV includes an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO; 3. In some embodiments, the YbiV includes or consists of the amino acid sequence set forth in SEQ ID NO: 3 In some embodiments, the HxpA includes an amino acid sequence that is at least about 80% identical to the amino add sequence set forth in SEQ ID NO: 64. In some embodiments, the HxpA includes or consists of the amino acid sequence set forth in SEQ ID NO: 64. In some embodiments, the YidA includes an amino acid sequence that Is at least about 80% identical to the amino acid sequence set forth in SEQ) ID NO: 66, In some embodiments, the YidA includes or consists of the amino acid sequence set forth in SEQ ID NO; 66. In some embodiments, the Y'igL includes an amino acid sequence that is at. least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 69. In some embodi men ts, the YigL includes or consists of the amino acid sequence set forth in SEQ ID NO: 69, In some embodiments, the YihX includes an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 71 , In some embodiments, the YihX includes or consists of the amino acid sequence set forth in SEQ ID NO: 7.1.

[0009] In some embodiments, the presently disclosed microorganism further includes a mutation of a gene encoding an enzyme of the pentose phosphate pathway. In some embodiments, the enzyme of the pentose phosphate pathway is gIncose-6-phosphate dehydrogenase (Zwf).

[0010] hi some embodiments, the presently disclosed microorganism further includes a mutation of a gene encoding an enzyme of glycogen biosynthesis. In some embodiments, the enzyme of glycogen biosynthesis is phosphoglucomutase (Pgm).

[0011] in some embodiments, the presently disclosed microorganism further includes a mutation of a gene encoding an enzyme of the mannose biosynthesis pathway. In some embodiments, the enzy me of the mannose biosynthesis pathway is mannose-6-phosphate i somerase (ManA).

[0012] In some embodiments, the presently disclosed microorganism further includes a mutation of a gene encoding an enzyme of glycolysis, In some embodiments, the enzyme of glycolysis is selected from phosphofructokinase A (PfkA), phosphofructokinase B (PffcB), fructose- biphosphate aldolase, triosephosphate isomerase, glyceraldehyde-3 -phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, or a combination thereof. In some embodiments, the enzyme of glycolysis is phosphofructokinase A (PlkA), phosphofnictokinase B (PfkB), or a combination thereof.

[0013] In some embodiments, the presently disclosed microorganism further includes a mutation of a gene encoding an enzyme of D-tagatose biosynthesis. In some embodiments, the enzyme of D-tagatose biosynthesis is D-tagatose- 1 ,6-bisphosphate aldolase subunit GatZ (GatZ).

[0014] In some embodiments, the presently disclosed microorganism further includes a mutation of a gene encoding an enzyme of allulose biosynthesis. In some embodiments, the enzyme of allulose biosynthesis is D-allulose-6-phosphate 3 -epimerase (AlsE).

[0015] In some embodiments, the presently disclosed microorganism further includes a mutation of one or more gene encoding a transaldolase. In some embodiments, the transaldolase is TalA or TalB or both TalA and TalB.

[0016] In some embodiments, the presently disclosed microorganism further includes an exogenous galactose:H-r symporter (GalP) and glucokinase (Glk).

[0017] In some embodiments, at least one exogenous oxidoreductase and / or at least one exogenous phosphatase are expressed by a stationary phase promoter or an inducible promoter. In some embodiments, at least one exogenous oxidoreductase and / or at least one exogenous phosphatase are expressed by a stationary phase promoter. In some embodiments, the stationary phase promoter i

[0018] In another aspect, the presently disclosed subject matter relates to a recombinant microorganism that produces an increased amount of an L-sorbose as compared to a naturally occurring microorganism, wherein the recombinant microorganism includes a recombinant polynucleotide encoding at least one exogenous oxidoreductase and at least one exogenous phosphatase. In some embodiments, the at least one exogenous oxidoreductase is a sorbitol-6-phosphate dehydrogenase (SorD / SrlD), a L-sorbose I -phosphate reduc tase (SorE), or a combination thereof. In some embodiments, the phosphatase is a hexitol phosphatase B (HxpB), a fructose- 1 -phosphate phosphatase (YqaB), a sugar phosphatase YbiV (YbiV), Hexitol phosphatase A (HxpA), sugar phosphatase YidA, phosphosugar phosphatase YigL, sugar phosphatase Y.ihX or a combination thereof.

[0019] In some embodiments, the presently disclosed microorganism further includes a mutation of a gene encoding an enzyme of the pen tose phosphate pathway, a mutation of a gene encoding an enzyme of glycogen biosynthesis, a mutation of a gene encoding an enzyme of the mannose biosynthesis pathway, a mutation of a gene encoding an enzyme of glycolysis, a mutation of a gene encoding an enzyme of D-tagato.se biosynthesis, a mutation of a gene encoding an enzyme of allulose biosynthesis, or a combination thereof. In some embodiments, the enzyme of the pentose phosphate pathway is glucose-6-phosphate dehydrogenase (Zwf). In some embodiments, the enzyme of glycogen biosynthesis is phosphoglucomutase (Pgm). In some embodiments, the enzyme of the mannose biosynthesis pathway is matmose-6-phosphate isomerase (Man A). In some embodiments, the enzyme of glycolysis is phosphofractokinase A (PfkA), phosphofructokinase B (PfkB), or a combination thereof. In some embodiments, the enzyme of D-tagatose biosynthesis is D-tagatose- 1,6-bisphosphate aldolase subunit GatZ (GatZ). In some embodiments, the enzyme of allulose (also known as D-psicose) biosynthesis is D-allulose-6- phosphate 3 -epimerase (AlsE),

[0020] In some embodiments, the presently disclosed recombinant microorganism further includes a mutation of a gene encoding transaldolase. In some embodiments, the transaldolase is TalA or TalB or both TalA and TalB.In some embodiments, the recombinant microorganism further includes exogenous gaIactose:H+ symporter (Gall*) and glucokinase (Glk). In some embodiments, the recombinant polynucleotide includes a stationary phase promoter or an inducible promoter. In some embodiments, the stationary phase promoter is 1^®.

[0021] 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 i£ , B i , or Z s',£0022] In a further aspect, the presently disclosed subject matter relates to a method for producing L-sorbose including culturing the microorganism under conditions suitable for converting a substrate to L-sorbose. In some embodiments, the substrate includes D-glucose.

[0023] In one aspect, the presently di scl osed subject matter relates to a method of making a food product comprising an L-sorbose comprising: a) culturing the microorganism disclosed hereinunder conditions suitable for converting a substrate to L-sorbose; and b) admixing the L-sorbose with one or more foods to form a food including the L-sorbose. In some embodiments, the substrate includes D-glucose. In some embodiments, the food product is a beverage, yogurt, ice cream, a baked good, or a nutritional bar.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 depicts L-sorbose pathway. A novel pathway to L-sorbose is proposed relying on dephosphorylation to enable yields. The L-sorbose pathway uses three steps to synthesize L- sorbose from D-fructose 6P: two oxidoreduclase steps (SorD / SorE) to synthesize L-sorbose IP and a final dephosphorylation. Abbreviations: PTS: Phosphotransferase System, PGI: phosphoglucoisotnerase, SorD: D-sorbitol 6-phosphate 2-dehydrogenase. SorE: L-sorbose 1- phosphate reductase

[0025] Figure 2 depicts Step 1 in L-sorbose pathway uses SorD enzyme to convert D-fructose 6P to D-sorbitol 6P, SorD (D-sorbitol 6-phosphate 2-dehydrogenase) is an enzyme that reversibly converts D-fructose 6P (F6P) to D-sorbitol 6P (SbtlbP, also abbreviated as S6P) in the presence of the redox cofactor NAD(H). This conversion leads to a change in mass between substrate and product (D-sorbitol 6P has 2 more hydrogen atoms), which allows us to detect conversion by monitoring for the specific mass of the product on HPLC-MS. The chemical change between a carbonyl (F6P) and hydroxyl (S6P) is highlighted in red. Three SorD enzymes were chosen to characterize their ability to perform this step (Table 1 ).

[0026] Figure 3 depicts pathway for the biosynthetic production of L-sorbose.

[0027] Figures 4A-4E depict MS traces showing positive and negative controls for SorD activity. Synthetic substrate and synthetic product were separately prepared by combining equal volumes 8 mM of the synthetic compound (aqj and elution buffer (80 rnM HEPES, 40 mM NaCl, 200 rnM imidazole). Using an HPLC-MS to monitor for the mass of the product (S6P, 262 g / mol), the substrate (F6P) produces no peak (Fig. 4A), while the product does (Fig. 4B). Reaction controls were prepared by combining equal volumes of water and purified SorD enzyme (>0.1 mg / mL SorD, 80 mM HEPES, 40 mM NaCl, 200 mM imidazole). Water acted as a negative control due to not including the substrate (-F6P). No signal was observed for SorD enzymes C3SXZ2 (Fig. 4C), P05707 (Fig. 4D), or P37079 (Fig, 4E). Reaction assays with substrate added (+F6P) are shown in Figures 5A-5E.

[0028] Figures 5A-5E depict MS traces showing successful SorD Activity to produce D-sorbitol 6P product. Synthetic substrate (Fig. 5A) and synthetic product (Fig. 5B) are shown using an H PLC-M S to monitor for the mass of the product ( S6P, 262 g / mol). Reaction mixes were prepared by combining equal volumes of substrate (8 rnM F6P, 20 mM NADH) and purified SorD enzyme(>0,1 mg / mL SorD, 80 niM HEPES, 40 ntM NaCi, 200 inM imidazole), active enzymes produced a peak corresponding to the S6P product, which is shown for SorD enzymes C3SXZ2 (Fig, 5C), P05707 (Fig, 5D), and P37079 (Fig, 5E). Overall, all 3 SorD enzymes were shown to successfully produce D-sorbitol 6P from D- fructose 6P m vi / ro.

[0029] Figure 6 depicts combined HPLC-MS trace of the 3 active SorD enzymes. Revisualization of Figures 5C-5E. HPLC-MS was configured to detect the mass of the product. D-sorbitol 6- phospliate 262 g / mol. SorD enzymes C3SXZ2, PO57O7, and P37079 all successfully produce peaks corresponding to the proper retention time of the D-sorbitol 6P product, indicating successful conversion from D-fiuctose 6P substrate. All traces were cropped to 30 minutes,

[0030] Figure 7 depicts combined HPLC-MS trace comparing the active SorD C3SXZ2 enzyme and the synthetic product D-sorbitol 6P, Re visualization of Figures 5B-5C. HPLC-MS was configured to delect the ma ss of the product, D-sorbitol 6-phosphate:::262 g / 'mol. SorD C3SXZ2 (green) successfully produces a peak corresponding to the proper retention time of the D-sorbitol 6F product (purple), indicating successful conversion from D-fiuctose 6P substrate,

[0031] Figure 8 depicts combined HPLC-MS trace comparing the active SorD PO57O7 enzyme and the synthetic product D-sorbitol 6P. Revisualization of Figure 5B and 5D. HPLC-MS was configured to detect the mass of the product, D-sorbitol 6-phosphate ~ 262 gZrnol. SorD P05707 (green) successfully produces a peak corresponding to the proper retention time of the D-sorbitol 6P product (purple), indicating successful conversion from D-fiuctose 6P substrate.

[0032] Figure 9 depicts combined HPLC-MS trace comparing the active SorD P37079 enzyme and the synthetic product D-sorbitol 6P. Revisualization of Figures 58 and 5E. HPLC-MS was configured to detect the mass of the product, D-sorbitol 6-phosphate - 262 g / mol. SorD P37O79 (green) successfully produces a peak corresponding to the proper retention time of the D-sorbitol 6P product (purple), indicating successful conversion from D- fructose 6P substrate,

[0033] Figure 10 depicts Step 2 in L-sorbose pathway uses SorE enzyme to convert D-sorbitol 6P to L-sorbose I P. SorE (L-sorbose 1 -phosphate reductase) is an enzyme that reversibly converts D-sorbitol 6P to L-sorbose IP (SIP) in the presence of the redox cofactor NAD(H). This conversion leads to a change in mass between substrate and product (L-sorbose IP has 2 fewer hydrogen atoms), which allows us to detect conversion by monitoring for the specific mass of the product on HPLC-MS. The chemical change between a hydroxyl (S6P) and a carbonyl (SIP) is highlighted in red. Three SorE enzymes were chosen to characterize their ability to perform this step (Table 1).

[0034] Figures 1 1 A- 1 IE depict MS traces showing positive and negative controls for SorE Activity. Synthetic substrate and synthetic product were separately prepared by combining equalvolumes 8 mM of the synthetic compound (aq. ) and elution buffer (80 mM HEPES, 40 mM NaCI, 200 mM imidazole). Using an HPLC-MS to monitor for the mass of the product (Si P, 260 g / mol), the substrate (S6P) produces no peak (Fig, 11 A), while the product does (Fig. 1 IB), Reaction controls were prepared by combining equal volumes of water and purified SorE enzyme (>0.1 mgZmL SorE, 80 mM HEPES, 40 mM NaCI, 200 mM imidazole). Water acted as a negative control due to not including the substrate (~S6P). .No signal was observed for SorE enzymes P37084 (Fig, 11C), A0A2K9PGB6 (Fig. HD), and A0A066SY94 (Fig. HE). Reaction assays with substrate added (4-S6P) are shown in Figures 12A-12E.

[0035] Figures 12A-12E depict MS traces showing successful SorE Activity to produce L-sorbose 1 P product. Synthetic substrate (Fig. 12A) and synthetic product ( Fig. 12B) are shown using an HPLC-MS to monitor for the mass of the product (S i P, 260 g / mol). Reaction mixes were prepared by combining equal volumes of substrate (8 mM S6P, 20 mM NAD-t ) and purified SorE enzyme (>0.I mg / mL SorE, 80 mM HEPES, 40 mM NaCl, 200 mM imidazole), active enzymes produced a peak corresponding to the SI P product, which was not shown for SorE P37084 (Fig. 12C) but was successfully shown for SorE enzymes A0A2K9PGB6 (Fig. 12D) and A0A066SY94 (Fig. 12E). Overall, SorE enzymes A0A2K9PGB6 and A0A066SY94 were shown to successfully produce L-sorbose IP from D-sorbitol 6P m vitro.

[0036] Figure 13 depicts combined HPLC-MS Trace of the 1 inactive, 2 active SorE enzymes. Revisualization of Figures 12C- I2E. HPLC-MS was configured to detect the mass of the product, L-sorbose 1 -phosphate ~ 260 g / mol. SorE P37084 does not produce a significant peak, whereas SorE enzymes A0A2K9PGB6 and A0A066SY94 successfully produce peaks corresponding to the proper retention time of the L-sorbose IP product, indicating successful conversion from D- sorbitol 6P substrate. All traces were cropped to 30 minutes.

[0037] Figure 14 depicts combined HPLC-MS trace comparing the active SorE A0A2K9PGB6 enzyme and the synthetic product L-sorbose 1 P. Revisualization of Figures 12B and 12D. HPLC- MS was configured to detect the mass of the product, L-sorbose 1 -phosphate ~ 260 g / mol. SorE A0A2K9PGB6 (red) successfully produces a peak corresponding to the proper retention time of the L-sorbose I P product (green), indicating successful conversion from D-sorbitol 6P substrate.

[0038] Figure 15 depicts combined HPLC-MS trace comparing the active SorE A.0A066SY94 enzyme and the synthetic product L-sorbose I P. Revisualization of Figures 12B and 12E. HPLC- MS was configured to detect the mass of the product, L-sorbose 1 -phosphate ~ 260 g / mol. SorE A0A066SY94 (red) successfully produces a peak corresponding to the proper retention time of the L-sorbose I P product (green), indicating successful conversion from D-sorbitol 6P substrate.

[0039] Figure 16 depicts Step 3 in L-sorbose pathway uses phosphatase enzyme to convert I.,~ sorbose IP to L-sorbose. Phosphatases are enzymes that irreversibly removes the phosphate group of L-sorbose I P. This con version leads to a change in mass between substrate (L-sorbose IP) and product (L-sorbose), which allows us to detect conversion by monitoring for the specific mass of the substrate on HPLC-MS. Note: during experimental proceeding HPLC-MS was configured to detect only phosphorylated compounds, thus depletion of substrate in this step was monitored. The chemical change between a phosphate ester (SIP) and a hydroxyl (L-sorbose) is highlighted in red. Three phosphatase enzymes were chosen to characterize their ability to perform this step (Table 1 ).

[0040] Figures 17A- 17D depict MS traces showing positive and negative controls for phosphatase activity. Synthetic substrate was prepared by combining equal volumes 4 mM of L-sorbose IP (aq.) and elution buffer (80 mM HEPES, 40 mM NaCl, 200 mM imidazole). Using an HPLC-MS to monitor for the mass of the substrate (SIP, 260 g / mol), the substrate (SI P) produces a peak (Fig. 17 A), as expected. Reaction controls were prepared by combining equal volumes of water and purified phosphatase enzyme (>0.1 mg / mL phosphatase, 80 mM HEPES, 40 mM NaCl, 200 mM imidazole). Water acted as a negative control due to not including the substrate (-S1P). No signal was observed for phosphatase enzymes A0A2S8E3A7 (Fig. 17B), A0A0L6Y216 (Fig. 17C), and A0A4P5R.0U6 (Fig, 17D). Reaction assays with substrate added (+S1P) are shown in Figures 18A-18D.

[0041] Figures 1 SA- 18D depict MS traces showing successful phosphatase activity to produce L- sorbose product. Synthetic substrate (Fig. 1.8 A) is shown using an HPLC-MS to monitor for the mass of the substrate (SIP, 260 g / mol). Reaction mixes were prepared by combining equal volumes of substrate (4 mM S IP) and purified phosphatase enzyme (>0.1 mg / mL phosphatase, 80 mM HEPES, 40 mM NaCl, 200 mM imidazole). Active enzymes were expected to lack a peak, indicating depletion of the substrate by the enzyme. This was not shown for Phosphatase enzymes A0A2S8E3A7 (Fig. 18B) and A0A4P5R0U6 (Fig. 18D) but was successfully shown for Phosphatase enzyme A0A0L6Y216 (Fig. I8C). Overall, Phosphatase A0A0L6Y216 was shown to successfully produce L-sorbose from L-sorbose 1 P m vitro.

[0042] Figure 19 depicts combined HPLC-MS Trace of the 2 inactive, 1 active phosphatase enzymes. Revisualization of Figures 18B-18D. HPLC-MS was configured to detect the mass of the substrate, L-sorbose 1 -phosphate - 260 g / mol. Phosphatase enzymes A0A2S8E3A7 and A0A4P5R0U6 maintain significant peaks, indicating that substrate was not depleted and that the enzymes were inactive. Phosphatase A0A0L6Y216 successfully depletes the peak correspondingto the proper retention time of the L-sorbose IP substrate, indicating successful conversion to the L-sorbose product. All traces were croppedto 30 minutes.

[0043] Figure 20 depicts combined HPLC-MS Trace comparing the active phosphatase A0A0L6Y216 enzyme and the synthetic substrate L-sorbose IP. Revisualization of Figures 18A and 18C. HPLC-MS was configured to detect the mass of the substrate, L-sorbose 1 -phosphate:= 260 g / mol. Phosphatase enzyme AOAOL6Y216 (red) successfully reduces a peak corresponding to the proper retention time of the L-sorbose IP substrate (green), indicating successful conversion to L-sorbose product.

[0044] Figure 21 depicts D-sorbitol production capability of E. coli. Plasmids containing the IPTG- induciblpromoter expressing either a sorbitol -6-phosphate dehydrogenase (sar / J' or 5‘FZZ>) gene or a sorD / srlD and the phosphatase hxpB were tested for D-sorbitol production (Table 3). Production experiments were carried out using strain AL4386, which is MG 1655 with a Z1 fragment (kicP fez / ? ynec55) and genetic knockouts ApfkA AzwfAmafiA AaEE dpgm AgafZ ('fable 2). When grown on M9P media with 5 g L'1glucose at 30 "C for 24 h, significant D-sorbitol was detected in induced cultures containing .sr / D P05707 and hxpB (0.73 g L'1) or xarD P37079 and hxpB (0.50 g L'1). Error bars indicate s.d, (n™ 3 biological replicates).

[0045] Figure 22 depicts L-sorbose production capability of E. coH. Plasmids containing the IPTG-inducible Piiaeos promoter expressing either sr / 1) PO5707 or sorD P37079, with either sorE A0A2K9PGB6 or sorE AOA006Y94, and phosphatase yt / nE were tested for L-sorbose production (Table 3). Production experiments were carried out using strain AL4386. which is MG 1655 with a Z1 fragment (kwE ZefR spec®) and genetic knockoutsEzwfEmanA AaEE Apgm EgafZ (Table 2). When grown on M9P media with 15 g L‘sglucose at 30 *C, L-sorbose was detected in cultures containing induced srZD P057O7 with either xo / E A0A2K9PGB6 (1.7 g L*1) or sorE A0A006Y94 (0.8 g L'1) and yqaB. Error bars indicate s.d. (n - 3 biological replicates).

[0046] Figure 23 depicts inducer-free [.-sorbose production in E. coll, L-sorbose production genes wZZi) ECG 707, wrE A0A2K9PGE6 or AOABMSWJ, and phosphataswere cloned under the stationary phase promoter Pgas / s (Table 3). Resulting plasmids pALl 989 and pAL l 993 were tested for production in strain AL4386, which is MG 1655 with a Z1 fragment (k / cE zelB sped5) and genetic knockouts zip / E4AalxE Apgm AgatZ (Table 2). When grown on M9P media with 15 g L*!glucose at 30 'C for 48 h, cultures containing pAL1989 production 1.60 g L'!of L- sorbose, while cultures containing p AL 1993 produced an average of 1 .05 g L‘!. Error bars indicate s.d. (n ~ 3 biological replicates).

[0047] Figure 24A-C. The biosynthetic production of L-sorbose. (A) Pathway schematic showing sugar structures in their favored intracellular forms. Key Intermediates.’ G6P (glucose-6-phosphate), F6P (ftactose-b-phosphate), Sbtl6P (D-sorbitot 6-phosphate), and SIP (L-sorbose l- phosphate). Major competing pathways and genetic knockouts are labeled in blue. (B) F6P is converted to Sbtl6P by sorbitol -6-phosphate dehydrogenase (SorD), then to SI P by L-sorbose 1- phosphate reductase (SorE), and dephosphorylated to L-sorbose by phosphatase (YbiV). L- sorbose diffuses into the supernatant (C) In vitro and in vivo screening of SorD and SorE enzymes revealed candidates for L-sorbose production. HPLC-MS analysis for in vitro activity is shown in Figs. 27 & 28. For in vivo production, codon-optimized SorD2 was ineffective; the native gene sequence (SorD2*) was used instead.

[0048] Figure 25A.-C. Optimization of L-sorbose production. (A) Expression of .swZ)2, sarD2, sorD2* or sorD 3 rtnd hxpB under Frtai-oi in AL4386 (A / j / fcl AxH / A^tonJ AnZsE A / .yyn AgazZ) grown on M9P media with 5 g L"* glucose tor 24 h. (B) L-sorbose production using sorD2\ swEI or sorE3, andyqaE expressed under Z^a or JVfacOz in AL4386 grown on M9P media with 15 g L1glucose for 48 h. (C) Screening of phosphatases (YqaB, HxpA, HxpB, YbiV, YidA, YigL, YihX) for L-sorbose production using Pg«<& along with SorD2* and SorEl in AL4386 grown on M9P media with 15 g L ’ glucose for 24 h. Table S3 reports the AODoi values, which indicate growth differences over the experimental period. Error bars represent s.d. (n::::3 biological replicates).

[0049] Figure 26, High, density L-sorbose production, Plasmids containing production operon .Z^ws:<sorD2*-wrEZ -y&F and supplemental glucose import operon PriMOVgoiP-g^ were introduced to strain AL4386 (ApfEl tot / Amanff AtrfeE Apgm AgnrZ. Table I). Cultures were concentrated to an ODSM of -10, and then grown for 96 h, with 10% replacement of culture volume and the addition of 15 g L"!of glucose every 24 h. Error bars indicate s.d. (n - 3 biological repHcat.es).

[0050] Figure 27. Comparison of Activities for SorD-Catalyzed Conversion of F6P to Sbtl6P, HPLC-MS traces showing production of Sbtl6P using SorD enzymes in vitrotSorD enzymes C3SXZ2 (SorD I), P05707 (SarD2), and P37079 (SorD3) were individually incubated with excess F6P (4 mM) and NADH (10 mM) for 24 h, and the reactions were analyzed using HPLC-MS, alongside Sbtl6P and F6P standards. HPLC-.MS was configured to only detect the mass of D- sorbitol 6-phosphate. Traces for C3SXZ2 (A), P05707 (B), and P37079 (C) demonstrate >1000 mAU peaks corresponding to Sbtl6P (D). F6P (E) shows no signal >1000 mAU. An overlay of the five traces (F) demonstrates that the product peaks of the SorD reactions align with the SbtlbP peak. Three biological replicates were performed for each trace, and a representative trace is shown for each.

[0051] Figure 28A-E. Comparison of Activities for SorE-Catalyzed Conversion of Sbt.16P to SIP. FIPLC-MS traces showing production of S IP using SorE enzymes m vitro. SorE enzymes P37084 (SorE2), A0A2K9PGB6 (SorEl), and A0A066SY94 (SorE3) (Fig. 24C) were individually incubated with excess SbtlbP (4 mM) and NAD' (10 tnM) for 24 h, and the reactions were analyzed using HPLC-MS, alongside SIP and Sbtl6P standards. HPLC-MS was configured to only detect the mass of SIP. The trace for P37084 (A) does not demonstrate a >1000 m AU peak, whereas traces for A0A2K9PGB6 (B) and A0A066SY94 (C) demonstrate >1000 rnAJJ peaks corresponding to SIP (D). SbtlbP (E) shows no signal >1000 mAU. An overlay of the five traces (F) demonstrates that the product peaks of the SorE reactions align with the SI P peak. Three biological replicates were performed for each trace., and a representative trace is shown, for each.

[0052] Figure 29, L-sorbose production in E. coli using the IPTG-inducible promoterafter 24 h. Genes for L-sorbose production, including sorD2*, sorE / orand yrpffi, were expressed using the IPTG-inducible promoter Ffexxn (Table 4). Resulting plasmids were tested for L-sorbose production in strain AL4386 (4 / yO A^wfAmonA AubE Apgm zigu / Z, 'fable 1). Cells were grown in M9P medium with 15 g L glucose for 24 h. For comparison, 48 h production data are presented in Fig. 2B. AODw indicates the difference in ODroo values between 0 h and 24 h. Error bars represent standard deviation (s.d.) from three biological replicates (n::::3).

[0053] Figure 30. Impact of fold and Zt.? / E deletions on L-sorbose production. The talA and talB genes, encoding the transaldolases TaLA and TalB, respectively , were deleted in AL4386 (Table 4). Strains were transformed with the production plasmid containing Pg^s:sorD2*-sorEl-y^^ (pAL2571, Table 1 ). Cultures were grown on M9P media with 15 g L*‘ glucose at 30*C for 48 h. (n ~ 3 biological replicates, except for AtaiB data where n - 6).

[0054] Figure 31. Supplementation of gl ucose import using PijiWof :galP~glk. To increase carbon flux through tire L-sorbose production pathway, a supplemental glucose import operon Pu^r-galP-g / k was introduced to AL4386 (Table 4) along with the production plasmid containing PgMi):sorD2*~sofEi~ybiV. Cultures were grown with and without induction of the glucose import pathway by 1 mM IPTG. Cells were grown in M9P media with 15 g L‘!glucose at 37aC to ODoxt "0.4, then grown at 30 °C for 24 h. AOD«x> indicates the difference in ODsoo values between 0 h and 24 h. Error bars indicate s.d, (n ~ 3 or more biological replicates).

[0055] DETAILED DESCRIPTION

[0056] The market for rare sugars as a foodstuff, dietary supplement, and health aid is expanding. Among rare sugars, L-sorbose has attracted particular attention. However, the current methods of producing L-sorbose are costly, inefficient, and thermodynamically unfavorable, limiting theirpotential for wide-spread use. Significantly, the present disclosure addressed various obstacles in L-sorbose production including thermodynamic barriers, limited yield, the requirement for purified enzymes and the addition of cofactors. The present disclosed subject matter facilitates the industrial-scale production of L-sorbose without the need for costly enzyme purification or difficult feedstock and product separation. In some embodiments, the microorganisms described herein also produce D-fiuctose, D-sorbitol and / or D~sedoheptuiose.

[0057] 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 L-sorbose, a low- calorie sugar. For clarity and not by way of limitation, the detailed description of the presently disclosed subject matter is divided into the following subsections:

[0058] 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.

[0059] 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.”

[0060] 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.

[0061] The terms “comprisg(s),” “include(s),” “having,” “has,” “can,” “contam(s),” and variants thereof as used herein, are intended to be open-ended transitional phrases, terms, or words thatdo 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.

[0062] 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 eubacteria 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.

[0063] As used herein, the terms “bacterium “bacteria,” or “eubacteria ” refers to a domain of prokaryotic organisms. In certain embodiments, bacteria include gram-negative bacteria, grampositive bacteria, proteobacteria, cyanobacteria, spirochetes, and related species, planctomyces, bacteroides, chlamydia, green sulfur bacteria, green non-sulfur bacteria, radioresistant micrococci, and thermotoga and Thermosipho thermophiles.

[0064] 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,Pasteurella, Brucella, Yersinia, Francisella, Haemophilus,Bordetella, Fscherichia,- Salmonella, Shigella, Kleb.sfoh'a, Proteus, Vibrio, Pseudomonas, Bacteroides, Acetobacter, Aerobacter, Agrobacterium, Azotobacter, Spin / hi, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium.

[0065] As used herein, the term “gram-positive bacteria” includes cocci, nonsporulating rods, and sporulating rods. The genera of gram-positive bacteria include, for example, and without any limitation, Actinomyces, Bacillus, Clostridium, Corynebacterium, .Erysipelothrix, Lactobacillus, Listeria, Mycobacterium, Myxococctis, Nocardia, Staphylococcus, Streptococcus, and Streplomyces.

[0066] As used herein, the term “recombinant microorganism” refers to a microorganism that contains one or more recombinant polynucleotides.

[0067] 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 ceil in nature.

[0068] 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 deoxyribonucleotide bases or ribonucleotide bases and can be manufactured synthetically fo vifro or isolated from natural sources,

[0069] As used herein, "'recombinant polynucleotide” refers to a polynucleotide wherein the exact nucleotide sequence of the polynucleotide is foreign to (be., not naturally found in) a given host. In certain embodiments, a recombinant polynucleotide sequence is naturally found in a given host, but in an unna tural (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 nticroorgamsrn. With reference to the microorganism’s genome, then, the polynucleotide sequence that encodes the polypeptide is recombinant or heterologous,

[0070] 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.

[0071] The terms '‘polypeptide,” “peptide,” “amino acid sequence” and "protein;5used 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.

[0072] 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 are those encoded by the genetic code, as well as those amino acids that are later modified, e,g., hydroxyproline, gamma- carboxyglutafflate, 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, z, e. , a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, «?.$., homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. Such analogs can have modified R groups (e.g., norleacine) 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

[0073] 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.

[0074] 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 .

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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).

[0079] 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%.

[0080] 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 I O-fold, about 100-fold, or about 1000-fold or more. In certain embodiments, the increase can be about 100- fold or more, about 1000-fold or more, or about 10,000- fold, or more.

[0081] 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 mRN A 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 nucleotide-to- nucleotide or amino acid-to-ami.no 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 mul tiplied 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.nIm.nih.gov / BLAST. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code-standard; filter:::none; strand-both; cutoff:::60; expectMO; Matrix~BLOSUM62; Description$~50 sequences; sort by=HIGH SCORE; Databases-non-reduadaat, GenBank+EMBH DDBJdPDB + GenBaak CDS translations-^ Swiss proteinTSpupdate+PIR. Details of these programs can be found on the GenBank website.

[0082] 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.X Microorganisms Prffd / ieins L -Sorbose

[0083] The present disclosure provides genetically engineered microorganisms, la certain embodiments, the presently disclosed microorganisms can produce an increased amount of L- sorbose, e.g.. an increased amount compared to a control microorganism that is natural ly- occurring, In certain embodiments, the presently disclosed microorganisms can produce, or also produce, an increased amount of D-fructose, D-sorbitol and / or D-sedoheptulose, e.g., an increased amount compared to a control microorganism that is naturally-occurring.

[0084] The present disclosure provides genetically engineered microorganisms that have increased production of "L-sugars as compared, to naturally-occurring microorganisms.

[0085] In certain embodiments, the L-sugar is L-sorbose. Figure 3 illustrates the biochemical pathways modulated in an exemplary microorganism of the present disclosure. In this exemplary microorganism, glucose is imported and phosphorylated to glucose-b-phosphate (G6P) by the phosphotransferase system (PTS). G6P is then isomerized to fructose-6-phosphate (F6.P) by glucose-6-phosphate isomerase. F6P is then converted to D-sorbitol 6-phosphate (SbtfoP, also abbreviated as S6P) by a sorbitol -6-phosphate dehydrogenase SorD / SrID. From there, Sbtl6P is converted to L-sorbose 1 -phosphate (SIP) by L-sorbose 1 -phosphate reductase SorE and dephosphorylated to free 'L-sorbose by phosphatase YqaB. Finally, free L-sorbose can diffuse across the cell membrane into the supernatant. Competing pathways include the pentose phosphate pathway (catalyzed by glucose-6-phosphate dehydrogenase (Zwf)), glycogen biosynthesis (catalyzed by phosphoglucomutase Pgm), D-mannose biosynthesis (catalyzed by D-mannose 6- phsophate isomerase MauA), glycolysis (catalyzed by phosphofructokinases PfkA and PfkB), D- tagafose biosynthesis (catalyzed by putative tagatose-l,6-bisphosphaie aldolase 2 chaperone GatZ), and D-psicose (also known as allulose) biosynthesis (catalyzed by D-allulose 6-phospbate 3 -epimerase AlsE). D-sorbitol biosynthesis (catalyzed by hexitol phosphatase B HxpB) serves as a checkpoint to evaluate the effectiveness of SorD / SrID within the presently disclosed pathway.

[0086] In certain embodiments, the presently disclosed microorganisms include overexpression of at least one gene encoding an enzyme catalyzing reactions for the production of L-sorbose andin some embodiments, also producing D~fractose, D~sorbitol and / or D-sedoheptulose. In certain embodiments, the presently disclosed microorganisms include a recombinant polynucleotide encoding at least one enzyme catalyzing reactions for the production of L-sorbose, D-fructosefD- sorbitol and / or D-sedoheptulose.

[0087] In certain embodiments, the enzyme is a dehydrogenase. In certain embodiments, SorD is an E. coli SorD. In certain embodiments, the dehydrogenase is a sorbitoI-6-phosphafe dehydrogenase (SorD) having UniProt No. C3SXZ2. In certain embodiments, SorD 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, SorD comprises the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, SorD consists of the amino acid sequence set forth in SEQ ID NO: I . SEQ ID NO: I is provided below:

[0088] In certain embodiments, the dehydrogenase is a sorbitol-6-phosphate dehydrogenase (SrlD) having UniProt No. P05707. In certain embodiments, SrlD 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: 2. In certain embodiments, SrlD comprises the amino acid sequence set forth in SEQ ID NO; 2, In certain embodiments, SrlD consists of the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 2 is provided below:

[0089] In certain embodiments, SorD is an Al pneumoniae SorD. In certain embodiments, the dehydrogenase is a sorbitol-6-phosphate dehydrogenase (SorD) having UniProt No. P37079. In certain embodiments, SorD 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: 4. In certain embodiments, SorD comprises the amino acid sequence set.forth in SEQ ID NO: 4. In certain embodiments, SorD consists of the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO: 4 is provided below:

[0090] In certain embodiments, the SorD / SrlD 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, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. In certain embodiments, SorD / SrlD is encoded by a nucleotide sequence comprising the nucleotide sequence set forth in SEQ ID NO: 5, SEQ ID NO: 6, SEQ I D NO: 7, or SEQ ID NO: 8. In certain embodiments, SorD / SrlD is encoded by a nucleotide sequence consisting of the nucleotide sequence set forth in SEQ ID NO; 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8. SEQ ID NOs: 5-8 are provided below:

[0091] In certain embodiments, the dehydrogenase is a L-sorbose 1 -phosphate reductase (SorE). In certain embodiments, SorE is a C amakmaiieus SorE. In certain embodiments, the dehydrogenase is a L-sorbose 1 -phosphate reductase (SorE) having UniiProt No. A0A2K9PGB6. In certain embodiments, SorE 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: 9, In certain embodiments, SorE comprises the amino acid sequence set forth in SEQ ID NO: 9. In certain embodiments, SorE consists of the amino acid sequence set forth in SEQ ID NO: 9. SEQ ID NO: 9 is provided below:

[0092] In certain embodiments, the dehydrogenase is a L-sorbose I -phosphate reductase (SorE). In certain embodiments, SorE is a X. pneumoniae SorE. In certain embodiments, thedehydrogenase is a L-sorbose 1 -phosphate reductase (SorE) having UniProt No. P37084. In certain embodiments, SorE 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: 10, In certain embodiments, SorE comprises the amino acid sequence set forth in SEQ ID NO: 10, In certain embodiments, SorE consists of the amino acid sequence set forth in SEQ ID NO: 10. SEQ ID NO: 10 is provided below:

[0093] In certain embodiments, the dehydrogenase is a L-sorbose I -phosphate reductase (SorE). In certain embodiments, SorE is a £. co / i SorE. In certain embodiments, the dehydrogenase is a L-sorbose 1 -phosphate reductase (SorE) having UniProt No, A0A066SY94, In certain embodiments, SorE 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: 11 , In certain embodiments, SorE comprises the amino acid sequence set forth in SEQ ID NO: 11 . In certain embodiments, SorE consists of the amino acid sequence set forth in SEQ ID NO: I I. SEQ ID NO: 1 1 is provided below:

[0094] In certain embodiments, the SorE 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: 12, SEQ ID NO; 13, or SEQ ID NO; 14. In certain embodiments, SorE is encoded by a nucleotide sequence comprising the nucleotide sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14. hi certain embodiments, SorE is encoded by a nucleotide sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14. SEQ ID NOs: 12-14 are provided below:

[0095] In certain embodiments, the enzyme is a phosphatase. Selection of the phosphatase can in some embodiments be used to control production of I.-sorbose, D-fruetose, D-sorbitol and / or D- sedoheptulose. See, e.g. , FIG. 25C. In certain embodiments, phosphatase is a E. co / Z phosphatase. In certain embodiments, the phosphatase is a Hexitol. phosphatase B (HxpB) having UniProt No. P77247 or UniProt No. Q7ADF8. 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: 15, In certain embodiments, HxpB comprises the amino acid sequence set forth in SEQ ID NO; 15. In certain embodiments, HxpB consists of the amino acid sequence set forth in SEQ ID NO: 15, SEQ ID NO: 15 is provided below:

[0096] In certain embodiments, the 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: 16, In certain embodiments, HxpB is encoded by a nucleotide sequence comprising the nucleotide sequence set forth in SEQ ID NO: 16* I n certain embodiments, HxpB is encoded by a nucleotide sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 16. SEQ ID NO: 16 is provided below:

[0097] hi certain embodiments, the enzyme is a phosphatase. In certain embodiments, phosphatase is a £ coll phosphatase, in certain embodiments, the phosphatase is a fructose- 1 - phosphate phosphatase (YqaB) having UniProt No. P77475. In certain embodiments, 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%s identical to the amino acid sequence set forth in SEQ ID NO: 17. In certain embodiments, YqaB comprises the amino acid sequence set forth in SEQ ID NO: 1 7. In certain embodiments, YqaB consists of the amino acid sequence set forth in SEQ ID NO: 17. SEQ ID NO: 17 is provided below:[(>098] In certain embodiments, the YqaB 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; 18. In certain embodiments, YqaB is encoded by a nucleotide sequence comprising the nucleotide sequence set forth in SEQ ID NO: 18. In certain embodiments, YqaB is encoded by a nucleotide sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 18. SEQ ID NO: 18 is provided below:

[0099] In certain embodiments, the enzyme is a phosphatase. In certain embodiments, phosphatase is a £ coli phosphatase. In certain embodiments, the phosphatase is a sugar phosphatase YbiV (YbiV) having UniProt No. P75792. 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: 3, In certain embodiments, YbiV comprises the amino acid sequence set forth in SEQ ID NO: 3. In certainembodiments, YbiV consists of the amino acid sequence set forth in SEQ ID NO: 3. SEQ ID NO: 3 is provided below:

[0100] In certain embodiments, the YbiV is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%. at least about 96%, at least about 97%, at least about 98%. at least about 99%, or at least about 100% identical to the nucleotide sequence set forth in SEQ ID NO: 63. In certain embodiments. YbiV is encoded by a nucleotide sequence comprising the nucleotide sequence set forth in SEQ ID NO: 63, In certain embodiments, YbiV is encoded by a nucleotide sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 63. SEQ ID NO: 63 is provided below:

[0101] In certain embodiments, the phosphatase is a Hexitol phosphatase A (HxpA) (UniProt No. P77625). HxpA catalyzes the dephosphorylation of D~sedoheptulose-7P. In certain embodiments, HxpA is an £ call 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: 64. In certain embodiments, HxpA comprises the amino acid sequence set forth in SEQ ID NO: 64. In certain embodiments, HxpA consists of the amino acid sequence set forth in SEQ ID NO: 64. SEQ ID NO: 64 is provided below:

[0102] In certain embodiments, the gene hxpA is encoded by a nuc leotide 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: 65. In certain embodiments, / nyzd comprises the nucleotide sequence set forth in SEQ ID NO: 65. In certain embodiments, hxpA consists of the nucleotide sequence set forth in SEQ ID NO: 65. SEQ ID NO: 65 is provided below:

[0103] In certain embodiments, the phosphatase is a sugar phosphatase, e.g., YidA, P0A8Y5 (UniProt) or EG 11195 (EcoCyc), and catalyzes the dephosphorylation of D-sedoheptuIose-7P. In certain embodiments. YidA is an £. coZI 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: 66. In certain embodiments, YidA comprises the amino acid sequence set forth in SEQ ID NO: 66. In certain embodiments, YidA consists of the amino acid sequence set forth in SEQ ID NO: 66, SEQ ID NO: 66 is provided below:

[0104] 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: 68. In certain embodiments, yidA comprises the nucleotide sequence sei forth in SEQ ID NO: 68. In certain embodiments, yidA consists of the nucleotide sequence set forth in SEQ ID NO: 68. SEQ ID NO: 68 is provided below.

[0105] In certain embodiments, the phosphatase is a sugar phosphatase, e.g., phosphosugar phosphatase (YigL), (UniProt K7848) that dephosphorylates D-sedoheptulose-7P. 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 the amino acid sequence set forth in SEQ ID NO: 69. In certain embodiments, YigL comprises the amino acid sequence set forth in SEQ ID NO: 69. In certain embodiments, YigL. consists of the amino acid sequence set forth in SEQ ID NO: 69. SEQ ID NO: 69 is provided below:

[0106] In certain embodiments, the geneyzgl 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: 70. In certain embodiments, wgZ. comprises the nucleotide sequence set forth in SEQ ID NO: 70. in certain embodiments, %gZ. consists of the nucleotide sequence set forth in SEQ ID NO: 70. SEQ ID NO: 70 is provided below:

[0107] in certain embodiments, the phosphatase is a sugar phosphatase, e.g., YihX, (UniProt P0A8Y3), and catalyzes the dephosphorylaticn of D-sedoheptuiose-7P. In certain embodiments, YihX is an £ call YihX. In certain embodiments, YihX 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: 71. In certain embodiments, YihX comprises the amino acid sequence set forth in SEQ ID NO: 71. In certain embodiments, YihX consists of the amino acid sequence set forth in SEQ ID NO: 71. SEQ ID NO: 71 is provided below:

[0108] In certain embodiments, the gene j’Z / zY 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: 72. In certain embodiments,comprises the nucleotide sequence set forth in SEQ ID NO: 72, In certain embodiments, .w’ / rY consists of the nucleotide sequence set forth in SEQ ID NO: 22. SEQ ID NO: 72 is provided below:

[0109] 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: IF symporter (GalP) and gl ucokinase (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.

[0110] In certain embodiments, the galactose:H7 syntporter (GalP) is an .£ coll GalP. In certain embodiments, GalP has EcoCyc No. 'EG 12148 or UniProt No, POAEPl . In certain embodiments,GalP comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 19. In certain embodiments, GalP comprises the amino acid sequence set forth in SEQ ID NO: 19, In certain embodiments, GalP consists of the amino acid sequence set forth in SEQ ID NO: 19. SEQ ID NO: 19 is provided below:

[0111] In certain embodiments, the 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: 20, In certain embodiments, GalP is encoded by a nucleotide sequence comprising the nucleotide sequence set forth in SEQ ID NO: 20. In certain embodiments, GalP is encoded by a nucleotide sequence consisting of the nucleotide sequence set forth in SEQ ID NO; 20. SEQ ID NO: 20 is provided below:[0 i 12] In certain embodiments, the glucokinase (Glk) is an E. coll GIL In certain embodiments, Glk has EcoCyc No. EG12957 or UniProt No. P0A6V8, In certain embodiments, Glk comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, Glk comprises the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, Glk consists of the amino acid sequence set forth in SEQ ID NO: 21. SEQ ID NO: 21 is provided below

[0113] In certain embodiments, the 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 about97%, at least about 98%, at least about 99%, or at least about 100% identical to the nucleotide sequence set forth in SEQ ID NO: 22, In certain embodiments, Glk is encoded by a. nucleotide sequence comprising the nucleotide sequence set forth in SEQ ID NO: 22. In certain embodiments, Glk is encoded by a n ucleotide sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 22. SEQ) ID NO: 22 is provided below:

[0114] 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 without any limitation, the presentlydisclosed microorganism can include any enzyme that catalyzes the conversion of D-sorbitol 6- phosphate to L-sorbose 1 -phosphate. In another non-limiting example, the presently disclosed microorganism can include any enzyme that dephosphorylates L-sorbose 1 -phosphate to free L- sorbose.2.2. Competing Pathways(0115] In certain embodiments, the presently disclosed microorganisms include a mutation of one or more gene encoding one or more enzymes regulating biochemical path ways that can reduce the production of L-sorbose. hi certain embodiments, the presently disclosed microorganisms include a reduced expression of a. gene encoding enzymes regulating biochemical pathways that can reduce the production of L-sorbose. 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 genes encoding enzymes of certain metabolic pathways results in increased L-sorbose production.

[0116] 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 m icroorganisms incl ude 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~phosphate dehydrogenase, 6- phosphogluconolactonase, phosphogluconate dehydrogenase, phosphopentose isomerase, phosphopentose epimerase, transketolase, and transaldolase. In certain embodiments, the enzyme of the pentose phosphate path way is glucose-6-phosphate dehydrogenase (Zwf) (Entrez Gene ID: 946370). Zwf catalyzes the oxidation of glucose 6- phosphate to 6-phosphogIuconoIactone. In certain embodiments, Zwf is an E. colt Zwf. A representative nucleotide sequence of the gene XM / is set forth in SEQ ID NO: 23 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 23. SEQ ID NO: 23 is provided below:

[0117] In certain embodiments, K co / 7 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: 24. In certain embodiments, £ co / z Zwf comprises the amino acid sequence set forth in SEQ ID NO: 24, SEQ ID NO: 24 is provided below:

[0118] In certain embodiments, Zwf is a ffoO / fos .szfotzfe Zwf. A representative amino acid sequence of Bad / hzs stfoti / A Zwf is found as P54547 (Uniprot) / BSU2385O (KEGG) or as set forth in SEQ ID NO; 25 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO; 25. SEQ ID NO; 25 is provided below;

[0119] 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:

[0120] In certain embodiments, Zwf is a factoc&cew ktctis Zwf. A representative amino acid sequence of l.&cft)coccMS lacfis Zwf is found as LLAI2__RSl2225i glucose-6-phosphate dehydrogenase, EC L I J .49. or as set forth in SEQ ID NO: 1 1 or at least 90%, 95%. 95%, or 99% identical to SEQ ID NO: 1 L SEQ ID NO: 1 1 is provided below;[012.1 ] 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:

[0122] In certain embodiments; the presently disclosed microorganisms include a mutation of a gene encoding an enzyme of glycogen biosynthesis. In certain embodiments, the presently disclosed microorganisms include a reduced expression of a gene encoding an enzyme of glycogen biosynthesis. In certain embodiments, the enzyme of 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 glycogen biosynthesis is phosphoglucomutase (Pgm) (Entrez Gene ID: 946370; EcoCyc ID: EG! 2144; UniProt ID: P36938), 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.

[0123] In certain embodiments, Pgm is an fo coli Pgm. In certain embodiments, E. coll 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; 29. In certain embodinients, / i. coli Pern comprises the amino acid sequence set forth in SEQ ID NO; 29. SEQ ID NO: 29 is provided below:

[0124] A representative nucleotide sequence of gene / ?g / n 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:

[0125] In certain embodiments, Pgm is ii / kickfks' sikkk / s Pgm. In certain embodiments, Uacz / to wbti / ix 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: 35. In certain embodiments, Bacikus wfofife Pgm comprises the amino acid sequence set forth in SEQ ID NO: 31 . SEQ ID NO: 31 is provided below:

[0126] A representative nucleotide sequence of gene pg / n 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, Pgm is a Zacfococcie laciix Pgm. In certain embodiments, Lactococcus lactis Pgm comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 33. In certain embodiments, Lactococcus lactis Pgm comprises the amino acid sequence set forth in SEQ ID NO; 33. SEQ ID NO: 33 is provided below:

[0129] 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 inchide 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) (Entrez Gene ID: 944840). ManA is involved in the synthesis of the GDP-mannose and dolicliol-phosphate-mannose required for a number of critical mannosyl transfer reactions. ManA also catalyzes the interconversion of fmctose-6-phosphate and mannose-6-phosphate.

[0130] In certain embodiments, ManA is an E. coll ManA. In certain embodiments, E. coll 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: 35, In certain embodiments, E. coE ManA comprises the amino acid sequence set forth in SEQ ID NO: 35. SEQ ID NO: 35 is provided below:

[0131] A representative nucleotide sequence of gene man„4 is set forth in SEQ ID NO: 36 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 36, which is provided below.

[0136] A representative nucleotide sequence of Buci / 'fas xubfitis gene vw / is set forth in SEQ I D NO: 40 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 40, SEQ ID NO: 40 is provided below.

[0137] A representative amino acid sequence of GmuF is found at 005511 (Uniprot) / BSU0587O (KEGG) or is set forth in SEQ ID NO: 41 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 41. SEQ ID NO: 41 is provided below:

[0139] In certain embodiments, ManA is a LaciMwci / s factis ManA. A representative amino acid sequence of lacfococctw Zactfis ManA is found as LLA12 RS03920: mannosc-6-pbosphate isomerase, EC5.3. 1.8 or is set forth in SBQ ID NO: 43 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 43. SEQ ID NO: 43 is provided below:

[0140] A representative nucleotide sequence of a 7.,a<4ococcw faetis gene manA is sei forth in SEQ ID NO: 44 or at least 90%s95%, 95%, or 99% identical to SEQ ID NO: 44. SEQ ID NO: 44 is provided below:

[0141] 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 glycolysis. In certain embodiments, the enzyme of glycolysis is selected from the group consisting of phosphofructokinase A, phosphofructokinase B, iructose-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 A (PfkA). In certain embodiments, the enzyme of glycolysis is phosphofimctoktuase B (PfkB). In certain embodiments, the enzyme of glycolysis is pyruvate kinase.

[0142] In certain embodiments, the enzyme of glycolysis is phosphofructokinase A (PfkA) (Entrez Gene ID: 948412). PfkA catalyzes the phosphorylation of D-fructosefo-phosphate to fructose l ,6~bisphosphate by ATP, the first committing step of glycolysis. In certain embodiments, PfkA is an E. co ft PfkA.

[0143] In certain embodiments, E. <?# / / PfkA comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about. 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 45. In certain embodiments, E. coll PfkA comprises the amino acid sequence set forth in SEQ ID NO: 45. SEQ ID NO: 45 is provided below:

[0144] A representative nucleotide sequence of gene / t / W is set forth in SEQ ID NO: 46, SEQ ID NO: 46 is provided below;

[0145] In certain embodiments, PfkA is a Bacillus siibolis PfkA. A representative amino acid sequence of BacillusPfkA is found as 034529 (Uniprot) / BSU29190 (KEGG) or as set forth in SEQ ID NO: 12 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 47, SEQID NO: 47 is provided below:

[0146] A representative nucleotide sequence of a Bad / to su&tits gene pfkA is set forth in SEQ ID NO: 48 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 48. SEQ ID NO: 48 is provided below:

[0147] In certain embodiments, PtkA is a Lacfococcus lactLs PtkA. A representative amino acid sequence of Lactococcus lactis PtkA is set forth in SEQ ID NO: 49 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 49, which is provided below:

[0148] A representati ve nucleotide sequence of a f^ctococcus tactis gene pflcA is set forth in SEQ ID NO: 50 or at least 90%, 95%s95%, ar 99% identical to SEQ ID NO: 50. SEQ ID NO: 50 is provided below:

[0149] In certain embodiments, PfkB is an ft. cu / 7 PfkB. In certain embodiments, S'. coZz 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: 51, In certain embodiments, % coll PfkB comprises the amino acid sequence set forth in SEQ ID NO: 51. S EQ ID NO: 51 is provided below.

[0150] A representative nucleotide sequence of geneis set forth in SEQ ID NO: 52. SEQID NO: 52 is provided below:

[0151] In certain embodiments, the presently disclosed microorganisms include a mutation of a gene encoding an enzyme of D-tagatose biosynthesis. In certain embodiments, the presently disclosed microorganisms include a reduced expression of a gene encoding an enzyme of D- tagatose biosynthesis. In certain embodiments, the enzyme of D-tagatose biosynthesis is D- tagatose~l,6~bisphosphate aldolase subunit GatZ (GatZ) (Entrez Gene ID: 916601 ). In certain embodiments, GatZ is an K. coli GatZ. A representative nucleotide sequence of gene gatZ is setforth in SEQ ID NO: 53 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 53. SEQID NO: 53 is provided below:

[0152] In certain embodiments, the presently disclosed microorganisms include a mutation of a gene encoding an enzyme of allulose biosynthesis. In certain embodiments, the presently disclosed microorganisms include a reduced expression of a gene encodi ng an enzyme of allulose biosynthesis. In certain embodiments, the enzyme of allulose biosynthesis is D-alluIose-6- phosphate 3-epimerase (AlsE) (Entrez Gene ID 948595). AlsE is an enzyme that catalyzes the reversible epimerization of D-allulose 6-phosphate to D-fractose 6-phosphate. AlsE can also catalyze with lower efficiency the reversible epimerization of D-ribulose 5-phosphate to D- xyltdose 5-phosphate. In certain embodiments, AlsE is ait K co / z AlsE. A representative nucleotide sequence of gene o&E is set forth in SEQ ID NO: 54 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 54. SEQ ID NO: 54 is provided below:

[0153] In certain embodiment, the presently disclosed microorganisms include a mutation of a gene encoding a transaldolase. In some embodiments, reduction in traasladolase activity increases fructose-6-phosphate which can in turn be converted to other sugars as described herein. In some embodiments, the transaldolase is TalA or TalB or the genes of both Tai A and TalB are mutated.

[0154] In certain embodiments, TalA is an E. co / i TalA. A representative nucleotide sequence of gene TalA is set forth in SEQ ID NO: 73 or at least 90%. 95%, 95%, or 99% identical to SEQ ID NO: 73. SEQ ID NO: 73 is provided below:

[0155] In certain embodiments, TalB is an E- call TalB. A representative nucleotide sequence of gene EalE is set forth in SEQ ID NO: 74 or at least 90%. 95%, 95%, or 99% identical to SEQ IDNO: 74. SEQ ID NO: 74 is provided below:i SEQ :w NO * 74 ]

[0156] In certain embodiments, the deletion of a gene comprises a non-frameshift 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.LLGih

[0157] 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 microorgamsm is selected from the group consisting of Jceto&ctez' aceti, Acbromobacter, Acidipbilitmi Acmetobdcter. Actinomadura, Actinoplanes,.Aeropyrumpemix, Agrobacterium. Alcaligenes, Ananas comosus (M), Arthrobacter, Bacillus alcalophilus, Bacilhisamyioliquefadens, Bacillus brevis, Bacd / ws circulars, Bacillus dausii, Bacillus kmtus, Bacillus licbeui / irmis, Bacillus macerans, .BacillusBacillus subiilis, Bifidobacterium, Zhwzbncz / M brevis, Burkbnlderia cepacia, Candida cylindracea, Carica papaya (L), Cellulosi'mi'crobtum. Cephalosporium, Chaet-omnim erraiicum, Chaetomium gracile. Clostridium, Clostridium buiyricum, Clostridium acelobutylicum. Clostridium thermocellum, Cotyitebacierium Ig' lufamicum), Caqmeifaterium Escherichia coll, Enterococcus, Erwja chryxanfhemi, ( Biconobacter. Gltxiouaceiobacter, Haloarcula. Humicola insole} is, Kifasataspora sc toe, Klebsiella, Klebsiella oxytoca, Kocuria, Lactlaclis, LacMbacillus. Lactabaciib{S fermcHtum, Lactobacilhis sake, Laetococcus, Lactococcus lactis, Leucouasioc, Methylacyslis, Methanahbus sic / Hae. Afefto / zogezhzzm o / gaHapbidm. bfeihambaderiiM biyanlil, Microbacterium imperiale, Micrococcus fysodeikticus, Microlumtus, Mtcorjavanicus, Mycobacierium, Mv'ozhechz / z?, Ab / rohac / er, AbBjosomonas, Nocardia, Papaya carica, kbdifx:occus, Pediococctts halophilus, Idiracoccus pantotrophus, Propionibacierium. Pseudomonas, .P.setdt^ Psznjdomonas z7z?z?iZrt / K.wzs, ffrroczzcczzy Ib'fx^oeinzsfi / riosi / x, Pyrtx.xK.'c?z.s' horikushii, Rfez&iwm, Phizaumior miehei, Rhizainucor pusillus Lindl, jRMizo / wrs, Kbizopus delemar, Khiziiptisjapmicas, K / tfrtyjja wvm. Rhizopus o / ysae, Bhizopus oligosporizs. P / zztobcut’czzy Sckmma liberiina, Sphiagoba^ multivorum, Sphingobhari, Sphingomonas, Sirepfoeoccus. Sfrepioeoeczzx ibermophihis ¥- / , Strep6?m)?ces, S&vpzomj’ces griseus, Sinpwnyces lividiws, mumms,.ShiyjtoworG ndiginosi / s, Slnpionyees vio / rK'eorzfZzez',1S?rtyJtovej7toj. / .li?^ mobaraense,Teirag&ioaiccas. Item®. m^phaefa pantotmpha, Trarneley Fibria a / gowfytkw, Xdrrtftomorm, Zymomanas, and Zymomaws' mobilis. In certain embodiments, the microorganism is Escherichia coll (E. call). In certain embodiments, the microorganism is 8aci!fas subiilis. In certain embodiments, the .microorganism is Laciococcas laais.

[0158] In certain embodiments, the E call is selected from die group consisting of Enterotoxigenic E. call (ETEC), Enteropathogenic E. call (EPEC), Enteroinvasive E. coll (EIEC), Enierohem.orfhagic E. coli (EHEC), Uropathogenic E coir (UPEC), Verotoxin-pixidiicing £. coli, E. call 0157 :H7, £. coli 0104:H4, K coli 0121 , £. coli 0104:H21 , £. coli Kt, and £. coli NC 101. In certain embodiments, the £ coli is E. coll K 12. In certain embodiments, the E. coli is .£ coli B. In certain embodiments, the E. coll is E. call C.

[0159] 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 1281 1, 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.[016'0] In certain embodiments, the £ coll is derived from a strain selected from the group consisting of BC 4734 (026: HU), BC 4735 (O157:H-), BC 4736 , BC 4737 (n.d.), BC 4738 (O157:H7), BC 4945 (O26:H-), BC 4946 (OI57:H7), BC 4947 (Oi l 1 :H-), BC 4948 (0157:11), BC 4949 (05), BC 5579 (O157:H7), BC 5580 (O157:H7), BC 5582 (03 :H), BC 5643 (O2:H5), BC 5644 (0128), BC 5645 (055:11-), BC 5646 (O69:H-), BC 5647 (O10I:H9), BC 5648 (0103:112), BC 5850 (022:148), BC 5851 (O55:H-), BC 5852 (048:1121), BC 5853 (O26:H11), BC 5854 (0157:117), BC 5855 (0157:11-), BC 5856 (026:11-), BC 5857 (0103:112), BC 5858 (O26:HH), BC 7832, BC 7833 (O raw foriwH-), BC 7834 (ONT:H-), BC 7835 (O103:H2), BC 7836 (057:11-), BC 7837 (ONT.’H-), BC 7838, BC 7839 (0128:112), BC 7840 (O1571H-), BC 7841 (O23:H-), BC 7842 (0157:11-), BC 7843, BC 7844 (0157:110, BC 7845 (0103:112), BC 7846 (026:1111), BC 7847 (0145:0-), BC 7848 (0157:11-), BC 7849 (0156:1147), BC 7850, BC 7851 (0157:11-), BC 7852 (0157:11-), BC 7853 (O5:H-), BC 7854 (0157:117), BC 7855 (0157-H7), BC 7856 (026:H~), BC 7857, BC 7858, BC 7859 (0NT:ll-), BC 7860 (O129:H-), BC 7861, BC 7862 (0103:112), BC 7863, BC 7864 (0 raw form:Il-), BC 7865, BC 7866 (O26:H- ), BC 7867 (O raw form:H-), BC 7868, BC 7869 (ONT:H-), BC 7870 (OH3:H-), BC 7871 (ONT:H-), BC 7872 (ONT:H-), BC 7873, BC 7874 (O raw form:!!-), BC 7875 (OI57:H-), BC 7876 (OU 1:H-), BC 7877 (0146:1121), BC 7878 (O145:H-), BC 7879 (O22:H8)JBC 7880 (Oraw fornr.H-), BC 7881 (O145:H-), BC 8275 (0157:I-I7), SC 8318 (O55;K-:H-)> BC 8325 (O157:H7), BC 8332 (ONT), and BC 8333.

[0161] In certain embodiments, the £ co / f is derived from a strain selected iron's the group consisting of BC 8246 (O152: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 (O1 12), BC 8256 (028a. e), BC 8257 (OI24:H~), BC 8258 (0143), BC 8259 (O167:K~:H5), BC 8260 (O128a. C / H35), BC 8261 (0164), BC 8262 (O164:K-:H«), BC 8263 (0164), and BC 8264 (0124).

[0162] la certain embodiments, the £ coii is derived from a. strain selected from the group consisting of BC 5581 (078: HI 1), BC 5583 (O2:K1), BC 8221 (0118), BC 8222 (O148:H-), BC 8223 (O1 11), BC 8224 (0110:H-), BC 8225 (0148), BC 8226 (01 18), BC 8227 (025:H42), BC 8229 (06), BC 8231 (O153:H45), BC 8232 (09), BC 8233 (0148), BC 8234 (0128), BC 8235 (0118), BC 8237 (0111), BC 8238 (0110:1317), BC 8240 (0148), BC 8241 (O6H16), BC 8243 (0153), BC 8244 (015:H-), BC 8245 (020), BC 8269 (0125a.c:H-), BC 8313 (O6:H6), BC 8315 (O153:H-), BC 8329, BC 8334 (0118:H12), and BC 8339.

[0163] 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 (0127a), BC 7577 (0126), BC 7578 (0142), BC 7579 (026), BC 7580 (OK26), 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 (OK26), BC 8558 (055), BC 8560 (0158), BC 8561 (0158), BC 8562 (0114), BC 8563 (086), BC 8564 (0128), BC 8565 (0158), BC 8566 (0158), BC 8567 (0158), BC 8568 (01 11), BC 8569 (0128), BC 8570 (0114), 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 (O1 14), BC 8593 (0114), 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).

[0164] In certain embodiments, the B. swhr / fe is derived .tram Strain 168.

[0165] In certain embodiments, the L / acifc is derived from Strain A 12.

[0166] In certain embodiments, the microorganism is a fungal cell. In certain embodiments, the fungal cell is selected from the group consisting of Aspergd / us. Aspergdlas n&hdans, Aspargdlns ntger, Aspargilfas o?yze, Aspwgidus we&w. Aspergillus pulyendenftts, Aspergillus .wtof Aspetgillits xojea, Aspergillus terreus, Aspergillus pseadoterretts, Aspe / gidus usumii, Candida rngosa, Issalchenkia orientalis, Kluyvemmyces, Kiiy^rumycesfmgilis. .Kluyveromyces lacfix. ffluyveromyces i»w»fis, Penic / llh / m, Penicilimm camembert?, Petticillmm cilrinum, Penicillium emersonii, Pemcillinm roquefbrti, Penieillmrt lilactinwn, Penicillum multicolor, Ilhodasiroridmm londoides, Sacchammyces cerevisiue, Schizas'aceharomyx'es pomhe, Triehoderma, rndtotfenna langibrachiatum, Trichoderma reesei, Trichoderma viride, Trichosporon petticflldium, Yarrawia

[0167] In certain embodiments, the microorganism is a yeast cell. In certain embodiments* the yeast cell is Saeeharomycex cerevisiae.2.4. Exemplary microorganisms

[0168] In certain embodiments, the present disclosure provides a recombinant microorganism comprising an increased production of L-sorbose as compared to a naturally occurring microorganism. In certain embodiments, the recombinant microorganism comprises an exogenous sorbitoi-6-phosphate dehydrogenase (SorD) and an exogenous L-sorbose- 1 -phosphate by L-sorbose 1 -phosphate reductase (SorE). In certain embodiments, the recombinant microorgani sm further comprises an exogenous phosphatase. In certain embodiments, any of the recombinant microorganism disclosed herein can include one or more mutations of at least one gene. In certain embodiments, the at least one gene is selected from CM / gene, pgm gene, pftA gene. pfkB gene. manA gene, alsE gene, go.tZ gene, or a combination thereof. In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is E. coll.3, Methods for Producing and Generating Microorganisms E-sngars

[0169] 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 fog,, recombinant techniques), microbiology, cell biology, and biochemistry', which are within the skill of the art. Additional information on these techniques can be found in Molecular Cloning: A Laboratory' Manual, second edition (Sambrook et al., 1989); Oligonucleotide Synthesis (Gait, ed., 1984); Animal Cell Culture (Freshney, ed„ 1987); Gene Transfer Vectors for hfemmalian Cfells (Miller& Calos, eds., 1987); Current Protocols in Molecular Biology (Ansubel el al, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al, eds., 1994); and Current Protocols in Immunology (Coligan et al., eds., 1991 ),3 / / . liee&mbmanf po / wzdemfcfev

[0170] 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 homo I ogous recombinati on, transposition-based chromosomal integration, recontbinase-tnedi ated cassette exchange (RMCE; e.g.fusing 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. & 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 6?.g.(yeast replicating plasmids that include an autonomously replicating sequence, ARS), centromere plasmidsyeast centromere plasmids that include an autonomously replicating sequence, CEN), episomal plasmids (e.g., 2- p.m plasmids), and / or artificial chromosomes A,g., yeast artificial chromosomes, YACs, or bacterial artificial chromosomes. BACs).3.7,7. Fecfors

[0171] 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 are operatively linked, e.g>, “expression vectors.” These expression vectors allow the expression 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.

[0172] 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.

[0173] In certain embodiments, a vector discloses herein comprises nucleotide sequences in a single operon.x

[0174] 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 smT.) gene and / or genes can be operably linked to a control sequence, enhancer, or promoter.

[0175] As used herein, the term ^promoter” refers to any nucleotide sequence that regulates the initiation of transcription for a particular ceding 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.

[0176] 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 £ eoll promoter, , A and A from mrZl, and the lambda phage promoter Id, (Liang, S.T. et al. Jlfoi Zito / , 292(1): 19-37 (1999)). In some embodiments, the promoter is active in the stationary phase of the microorganism. Exemplary stationary phase promoters can be found in, e.g., Shimada, ef al.sJOf AAl-IZ. OF BACTEKIOLOGT} Nov. 2004, p. 7112-7122; Pletnev at el., ACTA NATURAE I VOL. 7 M 4 (27) 2015.

[0177] 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., IPTG), 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 a sugar, metal, drug, or phosphate), salts orosmotic shock, oxygen, and biological stimuli fo.g., a growth factor or pheromone). Nou-limiting examples of inducible promoters include the / r promotersD, and

[0178] 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 adj ust their metabolic processes according to their new environment. The cells will then rapidly grow and divide, entering the logarithmic phase. It is at this time that enzymes related to central carbon metabolism are most important, and the transcription of corresponding genes will be upreguiated. 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.

[0179] 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 sw'D gene operably linked to a first promoter and a nucleotide sequence encoding the xorE 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 sori) gene operably linked to a first promoter and a nucleotide sequence encoding the sorE gene operably linked to a second promoter, wherein the first and second promoter are different.

[0180] In certain embodiments, the promoter is a PI.IMVI promoter. The few; promoter is a hybrid regulatory region including the promoter ft. of phage lambda with the CI binding sites replaced with lacOi. The hybrid design allows for a strong promotion that can be repressed by Lad, the Lac inhibitor (i.e.. repressor) or induced by IPTG. In certain embodiments, the Ptiscoipromoter comprises the nucleotide sequence sei forth in SEQ ID NO: 55. In certain embodiments, the promoter consists of the nucleotide sequence set forth in SEQ ID NO: 55. SEQ ID NO:55 is provided below:

[0181] In certain embodiments, the promoter is apromoter. In certain embodiments, the ftfeiO / promoter comprises the nucleotide sequence set forth in SEQ ID NO; 56. In certain embodiments, the Pamm promoter consists of the nucleotide sequence set forth in SEQ ID NO: 56. SEQ ID NO: 56 is provided below: A

[0182] In certain embodiments, the promoter is a Pn promoter. In certain embodiments, thepromoter comprises the nucleotide sequence set forth in SEQ ID NO: 57. In certain embodiments, the Pr? promoter consists of the nucleotide sequence set forth in SEQ ID NO: 57. SEQ ID NO:57 is provided below: T

[0183] In certain embodiments, the promoter is a P!&promoter. In certain embodiments, the 7G.< promoter comprises the nucleotide sequence set forth in SEQ ID NO: 58. In certain embodiments, the Pmt promoter consists of the nucleotide sequence set forth in SEQ ID NO: 58. SEQ ID NO:58 is provided below:

[0184] In certain embodiments, the promoter is a PgM promoter. In certain embodiments, the Pgidii promoter comprises the nucleotide sequence set forth in SEQ ID NO: 59. In certain embodiments, thepromoter consists of the nucleotide sequence set forth in SEQ ID NO: 59.SEQ ID NO: 59 is provided below:

[0185] In certain embodiments, the stationary phase promoter is P^2. In certain embodiments, the promoter comprises the nucleotide sequence set forth in SEQ ID NO: 60. In certainembodiments, the Pc^i? promoter consists of the nucleotide sequence set forth in SEQ ID NO:60. SEQ ID NO: 60 is provided below:

[0186] In certain embodiments, the stationary phase promoter is / G / M. In certain embodiments, the promoter comprises the nucleotide sequence set forth in SEQ ID NO: 61 . In certain embodiments, the / W-r promoter consists of the nucleotide sequence set forth in SEQ ID NO: 61. SEQ ID NO: 61 is provided below:

[0187] In certain embodiments, the stationary phase promoter is / Ata. In certain embodiments, the fys promoter comprises the nucleotide sequence set forth in SEQ ID NO: 62. In certain embodiments, thepromoter consists of the nucleotide sequence set forth in SEQ ID NO: 62. SEQ ID NO' 62 is provided below:3X3, Genedc Afartort

[0188] 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 Apramyciu 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, Zeochi 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 underconditions 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, fatty 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.

[0189] 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 Transcription activator-like effector nuclease (TALEN), a Clustered regularly interspaced short palindromic repeats (CRISPR) system, or a combination thereof.

[0190] 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 acti ve complex with Cas9), transactivating crRNA (tracrRNA, binds to crRNA and forms an active complex with Cas9), and an optional section of DNA repair template (DN A that guides the cellular repair process allowing insertion of a specific DNA sequence). Multiple crRNAN and the tracrRN A 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 comprisestransposases / 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 biatec / mo / ogy 38.7 (2020): 824-844 and in Nunez et aL, Ceil 184.9 (2021): 2503-251$), and Jiang et ah, Appl Environ Microbiol 2015 Apr:81(7):2506-14, the contents of each of which are incorporated by reference in their entireties.

[0191] In certain embodiments, the deletion is generated by a zinc-finger nuclease. A zine-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 D.NA 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 repeats and can each recognize a plurality of base pairs. The most common method to generate a new zinc-finger domain is to combine smaller zine-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.

[0192] In certain embodiments, the deletion is generated by a TALEN system. Transcription activator-like 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-like effectors (TALEs) are composed of 33-34 amino 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.

[0193] 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.

[0194] In certain embodiments, the present disclosure also provides methods to reduce the expression of any of the genes or enzymes disc losed herein. In certain embodiments, the reduced expression of genes and enzymes disclosed herein comprises using oligonucleotides that havecomplementary sequences to the mRNA of the genes disclosed herein (e.g., described in Section 2.2; ma^A. pfkA, pflc-B, 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 nt / mRNA sequence. In certain embodiments, these oligonucleotides can be identical to at least a portion of a zw / ’ 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 pgm mRNA sequence. In certain embodiments, these oligonucleotides Can be identical to at least a portion of a / / gm mRN A sequence. In c ertain embodiments, these o l igonucleotides 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 pjkA 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 pflcB mRNA sequence. In certain embodiments, these oligonucleotides can be identical to at least a portion of a pflcB mRNA sequence, hi 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 an alsE mRNA sequence. In certain embodiments, these oli gonucleotides can be identical to at least a portion of an aisE 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 manA mRNA sequence. In certain embodiments, these oligonucleotides can be iden tical to at least a portion of a mttnA mRN A 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 abou t 99% identical to at least a portion of a gatZ mRN A sequence. In certain embodiments, these oligonucleotides can be identical to at least a portion of a gat'Z mRNA sequence. In certain embodiments, antisense nucleic acid, shRNA, miRN A, orsiRN A molecules can include DNA or atypical or non-naturally occurring residues, for example, but not limited to, phosphorothioate residues.

[0195] 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, er a / .2021 Apr; 9(4): 844. Zhang ez ciLyFr<mL Microbi()L (31 March 2021 ).

[0196] 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 transform ation.

[0197] 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, I., 1986 “Basic Methods in Molecular Biology”; Gietz et al., Nucleic Acids Res. 27:69-74 (1992); Ito et al., J. Bacterol. 153: 163-168 ( 1983); and Becker and Guarente, Methods in Enzymology 194: 182-187 ( 1991 )). In certain embodiments, transformed microorganisms are referred to as recombinant m icroorgani sms .

[0198] In certain embodiments, the present disclosure provides methods for introducing exogenous proteins (e.g., nuclease), RNA (e.g. , gRNA)tand 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 Saccbaromyces cerevisiae using CRISPR-Cas systems.” Nucleic Acids Res (2013). doi: 10.1093foax / g:ktl 35; Ren, Z. J., Baumann, R. G. & Black, L. W. “Cloning of linear DN As 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, .1. A. “Enhanced homology-directed human genome engineering by controlled timing of CRISPfo'Cas9 delivery.” Elite 3, e04766 (2014)).3.4. Recombination Systems

[0199] In certain embodiments, the present disclosure also provides homologous recombination systems for editing (e.g., insertion, deletion) in a microorganism. In certain embodiments, foe 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 DN A fragment introduced as and translated from RNA 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, foe 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 foe 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).

[0200] In certain embodiments, the recombinant polynucleotide including foe homology arms and sequence for genetic editing is introduced into the microorganism using any of foe 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 foe two homology sites targeted by the homology arms present on the plasmid and that flank foe designed genetic edit In certain embodiments, foe 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, foe second type of selective medium allows foe selection of cells that comprise the desired genetic editing.

[0201] 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, foe 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 foe genome of foe 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 foe host cell. In certain embodiments, foe proteins from one or more recombination systems can be introduced as proteins into the host cell.59Non-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. “Anew logic for DNA engineering using recombination in E. col / E 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 Stewart A.F. “DNA cloning by homologous recombination in £. eo / z.” 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 far Pmlaans L-sorbose

[0202] The present disclosure also provides methods for producing L-sorbose. In certain embodiments, the presently disclosed methods for producing L-sorbose include culturing microorganisms (e.g., one disclosed in Section 2) and purifying L-sorbose.4, / . Cell Culture

[0203] 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.[0204'1 In certain embodiments, the methods for producing methods of producing L-sorbose 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 modifiedin 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 L-sugars.

[0205] 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, Cs 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..2. / hcfy / Lu / h w of f~$orfwse

[0206] In certain embodiments, the methods of the present disclosure further comprise purifying L-sorbose 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 fermentation, 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, nrter u / m, the microorganism, culture conditions, and / or particular product(s).

[0207] In certain embodiments, purifying L-sorbose comprises separating or filtering the microorganisms from a cell culture medium, separating the L-sorbose from the culture medium fy.g., by chromatography), concentration of water (kg., by evaporation), and lyophilization of the L-sorbose.5. Food Pmduefa

[0208] The present disclosure also provides delivery systems methods for use in food products including the L-sorbose prepared and / or generated by any of the microorganisms disclosed herein.

[0209] 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, baters, and the like. Various baked goods are disclosed in U.S. Patent No, 6,536,599, the disclosure of which 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 L-sorbose prepared and / or generated by any of the microorganisms disclosed herein are also suitable as a component in frozen foods.

[0210] In certain embodiments, the food product is prepared by admixing the L-sorbose in an ingestible vehicle, together with any optional ingredients, to form a uniform mixture. The final compositions are readily prepared using standard methods and apparatus generally known by those 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.

[0211] As used herein “admixing,” for example, ^admixing L-sorbose 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~ ribose to the product, spraying D- ribose on the product, coating D~ ribose on the product, suspending the product in D- ribose, painting D- ribose on the product, pasting D- ribose on the product, encapsulating the product with D- ribose, mixing D- ribose with the product and any combination thereof. The L-sorbose can be a liquid, dry' powder, spray, paste, suspension and any combination thereof

[0212] 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.

[0213] In certain embodiments, the L-sorbose 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 fatty 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 orflours, and die like, and then combined with precursors of the comestible or medicinal products, or applied directly to the comestible or medicinal products.

[0214] 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 L-sorbose 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

[0215] In certain embodiments, the food products to which the L-sorbose 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,

[0216] In certain embodiments, the L-sorbose prepared and / or generated by any of the microorganisms disclosed herein are admixed with one or more confectioneries, chocolate confectionery, tablets, coantlines, bagged selfmies / softlines, 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 / lndustrial bread, unpackaged / 'artisanal bread, pastries, cakes, packaged / industrial cakes, unpackaged / artisaual 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, take-home dairy ice cream, ice cream desserts, bulk ice cream, take-home water ice cream, frozen yoghurt, artisanal ice cream, dairy products, milk, fresh / pasteurized milk, fell fat fresh / pasteurized milk, semi skimmed fresh / pasteurized milk, longdife / 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 / evaporated 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 whiteners, 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 / natural 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, fiomage frais and quark, . plain fromage frais and quark, flavored frontage frais and quark, savory frontage frais and quark, sweet and savory snacks, fruit snacks, chips / crisps, extruded snacks, tortilla / corn chips, popcorn, pretzels, nuts, other sweet and savory snacks, snack bars, granola bars, breakfast bars, energy bars, fruit bars, other snack bars, meal 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 meat substitutes, frozen potatoes, oven baked potato chips, other oven baked potato products, non-oven frozen potatoes, frozen bakery products, frozen desserts, frozen ready meals, frozen pizza, frozen 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 meats, chilled ftsh / seafbod products, chilled processed fish, chilled coated 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 fonds, 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.

[0217] in certain embodiments, the L-sorbose 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. 6,899,9.1 1, the disclosure of which is incorporated herein by reference in its entirety. 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 water and is water-insoluble, a water-soluble bulk portion, and flavors that are typically water-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, such as 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 poly isobutylene, butyl rubber, ( isobutyl ene-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 wdth a high degree of crystallinity, can be considered bodying agents or textural modifiers.

[0218] 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 1.0% 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.

[0219] 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.

[0220] The water-soluble portion of the chewing gum can further comprise softeners, sweeteners, flavors, physiological cooling agents, and combinations thereof The sweeteners often fulfill therole of bulking agents in the gum. The balking agents typically comprise about 5% to about 95% of the gum composition.

[0221] 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.

[0222] As mentioned above, the L-sorbose 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-contaming 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 syrup solids and the like, alone or in any combination. The L-sorbose 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 isomalnilose, mannitol, xylitol, lactitol, erythritol, hydrogenated starch hydrolysate, maltitol and the like alone or in any combination.

[0223] 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 coating 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, T haumatin. 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 chewing gum composition, or added as part of the gum base.

[0224] Aqueous syrups, such as corn syrup and hydrogenated con? 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.

[0225] Methods of manufecturing chewing 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 at 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 coated or otherwise modified when incorporated into a delivery system to modify its release rate, it will preferably be added alter the final portion of the bulking agent has been added. The entire mixing procedure typically takes 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.[022.6] 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, the coating process is carried out in conventional panning equipment. Gum center tablets to be coated are placed into the panning equipment to form a moving mass.

[0227] The material or syrup that will eventually form the coating is applied or distributed over the gum center tablets. The L-sorbose 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 L-sorbose can be added to any or none of the coatings and / or layers,

[0228] In the panning procedure, syrup is added to the gum center tablets at a temperature range of from about 100°F to about 240’3F. Preferably, the syrup temperature is from about I40°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 mixedwith, sprayed upon, poured over, or added to the gum center tablets in any way known to those skilled in the art,

[0229] 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 talc and calcium carbonate,

[0230] 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 repeating the process. The amount of solids added by each coating step depends chiefly on the concentration of the coating syrup. Any number of coats 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 will contain from about 20% to about 50% coating,

[0231] Those skilled in the art will recognize tha t 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 is contemplated, however, that the volume of aliquots of syrup applied to the gum center can vary throughout the coating procedure.

[0232] Once a coating of syrup is applied to 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'T to about 1.00°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 %.

[0233] 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 coating has been dried, the presently disclosed subject matter contemplates drying the flavor with or without the use of a drying medium.

[0234] The amount of L-sorbose 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 baseemployed, and the strength of flavor desired. Thus, the amount of L-sorbose 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 L- sorbose 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% to about 1.8%, by weight of the chewing gum composition.5.2, eonfermnen:5

[0235] Another important aspect of the presently disclosed subject matter includes a confectionety composition incorporating the L -sorbose 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 eitheruhard” confectionery or “soft” confectionery. The L-sofbose 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.

[0236] 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 sugatiess. 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% com syrup, and from about 0. 1% to about 5% water, by weight of the final composition. The syrup component is generally prepared from sucrose and corn syrups bit can include other materials. In certain embodiments, the syrup component includes the L- sorbose 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.

[0237] 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.

[0238] 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 kettleuntil 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.

[0239] 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 water is boiled off without extra heating. When cooking is complete, the mass is a semi-solid and has a plastic-like consistency. At this point, flavoring agents, colorants, and other additives are admixed in the mass by routine mechanical mixing operations.

[0240] 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.

[0241] 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 coniposition 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. at pages 419 to 582, which disclosure is incorporated herein by reference.

[0242] Compressed tablet confections contain particular materials and are formed into structures under pressure. These confections generally contain sugars in amoun ts 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.

[0243] 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 corn syrup, or the like, and (2) a relatively light textured frappe, generally prepared from egg albumin, gum arable, 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.

[0244] 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. Minifte, Chocolate, Cocoa and Confectionery. Science and Technology, 2nd edition, AVI Publishing Co., Inc., Westport, Conn. (1983), at pages 576-580, which disclosure is incorporated herein by reference.

[0245] In general, the frappe component is prepared first and thereafter the syrup component is slowly added under agitation at 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 tempera ture below 80° C, at 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.

[0246] In accordance with the present disclosure, amounts of the L-sorbose prepared and / or generated by any of the microorganisms disclosed herein can be admixed into the hard and soft confections. The exact amount of L-sorbose emp loyed is normally a matter of preference subj ect 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 L-sorbose 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 L-sorbose 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.

[0247] The presently disclosed subject matter extends to methods for making the improved confections. The L-sorbose 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 heating apparatus well known in the confectionery manufacturing arts, and therefore the selection of the specific apparatus will be apparent to the artisan.

[0248] In such a method, a composition is made by admixing the L-sorbose 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.

[0249] The L-sorbose 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, clisintegraats, bulking agents, humectants,, buffers, and adsorbents. The preparation of such confections and chewing gum products is well known.5, .3, Oocofofex and Fdlmgs

[0250] The presently disclosed subject matter is also used with and / or in chocolate products, chocolate-fiavored 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. Minifie, Chocolate, Cocoa and Confectionery ' Science and Technology, 2nd edition, AVI Publishing Co., Inc,, Westport, Conn, (1982), which disclosure is incorporated herein by reference.

[0251] 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 vegetablefat 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-standardized chocolates and chocolate-like compositions, unless specifically identified otherwise.[02 ^2 j N'onstandardized chocolates result when, for example, the nutritive carbohydrate sweetener is replaced partially or completely; or when the cocoa butter, cocoa butter alternative, cocoa butter equivalent, cocoa butter extender, cocoa butter replaces', cocoa butter substitute os' 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 theFDA 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.

[0253] In the United States, 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 fell outside the specified ranges of the standardized chocolates.

[0254] In certain embodiments, the chocolate can contain L -sorbose prepared andfor 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, e.g„ from cane or beet, dextrose, fructose, lactose, maltose, glucose syrup solids, corn 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 acesuIfame-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.

[0255] 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, nialtodextrin. gum arable, and the like.

[0256] 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 monostearate, 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 p'hosphatidic acid, (e.g., YN) sucrose esters, oat extract, etc,, any emulsifier found to be suitable in chocolate or similar fai / solid system or any blend.

[0257] The term “chocolate -flavored confection” refers to food products, excluding “chocolate”, having a chocolate flavor / aroma and coinprising 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 at 18-30° C under normal atmospheric conditions. Examples include chocolate-flavored hard candies, chewables, chewing gums, etc.

[0258] 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, Sdt-wy GocWs nW Other Food Products

[0259] in certain embodiments, the L-sorbose prepared and / or generated by any of the microorganisms disclosed herein is incorporated into savory goods. In certain embodiments, a savory 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 d uring heating of a food product,

[0260] In certain embodiments, the L-sorbose 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 visiblepieces of some or ail of these ingredients. It may be clear (as a broth) or thick (as a chowder), smooth, pareed 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 (si pped 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).

[0261] In certain embodiments, the L-sorbose 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, inc luding 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 (ni) 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.

[0262] In certain embodiments, the L-sorbose 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, meatbails, fish sticks, chicken fingers, and ground meats, e.g... meatloaf, meatballs, and hamburgers. A meat food product may be combined with a simulated meat 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 meatbail, A simulated meat food product may be combined with a meat food product.

[0263] In certain embodiments, the L-sorbose 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 foodinclude, but are not limited to fruit snacks, chips / crisps, extruded snacks, tortilla / corn chips, popcorn, pretzels, nuts, and other sweet and savory snacks. Examples of snack bars include, but are not limited to granolafoiuesli bars, breakfast bars, energy bars, fruit bars, and other snack bars,

[0264] in certain embodiments, the L-sorbose 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, multipack water ice cream, take-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.X4. Pharmaceuticals

[0265] The L-sorbose 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 the art. The suspensions of the presently disclosed subject matter can comprise preservatives, buffers, suspending agents, antifoaming agents, sweetening agents, flavoring agents, coloring or decoloring agents, solubilizers, and combinations thereof.

[0266] 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.

[0267] The pharmaceutical suspensions of the presently disclosed subject mater can be prepared as follows: (1) admix the thickener with water heated from about 40* C to about 95° C, preferably from 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 L-sorbose prepared and / or generated by any of the microorganisms disclosed herein with water to form a solution; (hi) 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 die mixture of step (iv) to form the suspension.

[0268] The L~sorbose 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 c hewable tablet forms of confectionery can be found in H. A. Lieberman and L. Lachman, Pharmaceutical Dosage Forms: Tablets Volume I, Marcel Dekker, InC, New York, N.Y. (1989) at pages 367 to 418, which disclosure is incorporated herein by reference. In a typical procedure, a boiled sugar-corn syrup blend is formed to which is added a frappe mixture. The boiled sugar-com 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 L-sorbose 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 form ulation is further cooled and formed into pieces of desired dimensions.

[0269] In other pharmaceutical embodiments, the flavoring agent is incorporated into an ingestible topical vehicle which can be in the form of a mouthwash, rinse, ingestible spray, suspension, dental gel, and the like. 'Typical non-toxic ingestible vehicles known in the pharmaceutical arts can be used in the presently disclosed subject matter. The preferred ingestible vehicles are water, ethanol, and water-ethanol mixtures. The water-ethanol mixtures 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.

[0270] 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 cornposition. 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 fla vors. 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. 'Hie 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. Pe / fiWIfoxto

[0271] The L-sorbose prepared and / or generated by any of the microorganisms disclosed herein can be used in a wide variety of pet food products.

[0272] 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 Inmiliaris. 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.

[0273] In certain embodiments, the L-sorbose prepared and / or generated by any of the microorganisms disclosed herein is directly added to a pet food product. In certain embodiments, the L-sorbose prepared and / or generated by any of the microorganisms disclosed herein can be added prior to, during or after form ulation processing or packaging of the pet food product.

[0274] Non-limiting examples of suitable pet food products include wet food products, dry food products, moist food products, pet food supplements (e.g., vitamins), pet beverage products, snack and treats and pet food categories described herein.

[0275] In certain embodiments, the pet food product is a dry food product. A dry or low moisturecontaining nutri tlonally-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 tnoistare- containing nutritionally-complete pet food product can comprise greater than about50% 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.

[0276] In certain embodiments, the pet food product is a pet food snack product. Non-limiting examples of pet food snack products include snack bars, pet chews, crunchy treats, cereal bars, snacks, biscuits and sweet products.

[0277] In certain embodiments, the L-sorbose 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 tastant Delivery systems can be liquid or solid, aqueous or non-aqueous. Delivery systems are generally adapted to suit the needs of the flavor composition and / or the edible composition into which the D- ribose will be incorporated.

[0278] The D- ribose 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- ribose 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.

[0279] The D- ribose 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.

[0280] In certain embodiments, D- ribose is encapsulated. Encapsulating materials and / or techniques can be selected to improve the stability of the D- ribose and / or food product. In certain embodiments, the encapsulating materials and / or techniques are selected to modify the release profile of D- ribose,

[0281] 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, polylactidacid, polyhydroxyalkanoates, ethylcellulose , polyvinyl acetatephthalate, polyethylene glycol esters,nietwciylicacid-co-methyhnethacrylate, eihylene-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

[0282] Encapsulated delivery systems for flavoring agents or sweetening agents (e.g., D~ ribose) 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 fatty acids, glycerides or poly glycerol esters, sorbitol esters, and mixtures thereof. Examples of fatty acids include but are not limited to hydrogenated and partial ly 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.

[0283] 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, candelilla wax, lanolin, bayberry wax, sugarcane wax, spermaceti wax, rice bran wax, and mixtures thereof

[0284] 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.

[0285] Typical encapsulated compositions, flavoring agent or sweetening agent delivery systems are disclosed in U,S, Patent Nos, 4,597,970 and 4,722,845, the disclosures of which are Incorporated herein by reference in their entireties.

[0286] Liquid delivery systems can include, but are not limited to, systems with a dispersion of D- ribose, such as in carbohydrate syrups and / or emulsions. Liquid delivery systems can also include extracts where D- ribose 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 some embodiments, the delivery system will include a confectionery fat such as cocoa butter, a cocoa butter replacer. a cocoa butter substitute, or a cocoa butter equivalent.

[0287] When used in dried form, suitable drying means such as spray drying may be used. Alternatively, D- ribose may be adsorbed or absorbed onto substrates such as water 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

[0288] 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 Prothution ofL-sorhose in Escherichia coli

[0289] Rare sugars are monosaccharides with low natural abundance and unique biological properties’. Many rare sugars display a sweet taste but are metabolically inactive, showing potential as artificial sweeteners with novel bulking properties’ \ The rare sugar D-psicose, for example, displays 70% sweetness, while only providing 0.3% of the energy of sucrose4. Rare sugars also show antitumor, anti-inflammatory, and crop-protective properties, among others12 5Despite these promising applications, a complete understanding of the application of rare sugars remains limited by a lack of high-yield, high-purity synthesis routes from cheap feedstocks’’s. Using in vivo enzyme cascades, the rare sugar D-psicose was recently biosynthesized in E. coli from the common sugar D-glucose". This approach overcame low yields using in vivo enzymatic conversions between phosphorylated intermediates, followed by a dephosphorylation step to prox ide drix ing force". Once dephusphorx laied. the rare sugar is exported into the production medium, enabling straightforward purification once the feedstock is fully consumed. Ultimately. D-psicose was produced in 62% yield to >95% purity in unoptimized test-tube conditions, demonstrating the potential for this system to synthesize rare sugars efficiently7.10290] L-sugars represent half of all sugars, yet synthesis has proven especially challenging due to the need to inv ert the stereochemistry at the C-5 position. L-sugars have previously been synthesized from abundant D-sugars (e.g., D-fructose) using oxidoreductases. This strategy, initially developed as part of the Izumoring route to synthesize rare sugars, uses two successive oxidoreductases to first reduce the D-sugar into a sugar alcohol, and subsequently oxidize the polyol into an L-sugar9. These redox reactions have the chemical consequence of flipping the C-5 stereochemistry, yielding an L-configuration. A limitation of this strategy in vitro is the need for expensive redox cofactors, which constrains its wide-scale use. In vivo systems have been developed wherein polyols are fed to microbes overexpressing a single oxidoreductase. conxerting the substrate to an L-sugar of interest"1 1’. While obviating the need for exogenous cofactors, this system relies on a more expensive feedstock, polyols, than the in vitro system.which can begin with an abundant D-sugar such as D-fructose. Thus, an ideal system to synthesize L-sugars would utilize in vivo production alongside a cheap feedstock

[0291] L-sorbose is an attractive target for expansion of die D-psicose system [Figure 1]. At a stereochemical level, it has high similarity to D-fructose, an abundant cellular metabolite. Additionally, catabolic oxidoreductases for the conversion of L-sorbose 1 -phosphate (IP) to D- fructose 6-phosphate (6P) have been previously demonstrated12 13. L-sorbose IP reductase (SorE) and D-sorbitol 6P2-dehydrogenase (SorD) act successively to convert L-sorbose IPto D-sorbitol 6P and then to D-fructose tiP, respectively, where it enters central metabolism. These enzymes have yet to be evaluated for their use in synthesizing L-sorbose IP in vitro or in vivo. Beyond its feasibility, L-sorbose offers several potential applications. As an alternative sweetener, L-sorbose displays 60-75% tire sweetness of sucrose, while only 25% caloric utilization14,15. L-sorbose is also used as a substrate in tire industrial synthesis of vitamin C (L-ascorbic acid), and it has demonstrated potential anti-cancer properties in several human cancer lines and in mouse models6,11

[0292] Here, the present disclosure demonstrates a novel system to synthesize L-sorbose from D- glucose in vivo. First, bacterial oxidoreductases and phosphatases were identified and characterised for their capacity to produce L-sorbose from D-fructose tiP in vitro. These enzymes were that introduced into an engineered E. coli host and successfolly achieved biosynthesis of L- sorbose.Designing an L-sorbose production pathway

[0293] It was theorized that the thermodynamically favorable process of phosphorylating and dephosphorylating sugars could be used to produce L-sorbose from D-glucose [Figure 3]7. Within E. coli, D-glucose is simultaneously phosphorylated and transferred across die cell rane by the phosphotransferase system (PTS)18. D-glucose 6-phosphate (G6P) is then isomerized to D-fructose 6-phosphate (F6P) by glucose 6-phosphate isomerase Pgi18. F6P is typically directed into glycolysis, but from tire literature we leaned that F6P and D-sorbitol 6- phosphate (SbtltiP, also abbreviated as S6P) can be interconverted by a sorbitol-6 phosphate dehydrogenase SorD / SrlD13,19,20. Although tins reaction usually runs from SbtltiP to F6P as part of the sorbitol degradation pathway, ah accumulation of intracellular F6P could cause the reaction to run in reverse19. From there, SbtltiP can be converted to L-sorbose l-phosphate (SlP)by anL- sorbose 1 -phosphate reductase SorE and dephosphorylated to free L-sorbose by a phosphatase211" 22

[0294] Directing carbon flux towards L-sorbose production requires eliminating competing pathways. In the following experiments a previously established strain, AL4386, containinggenetic knockouts meant to remove enzymes at key branch points where these pathways diverge was used. These genetic knockouts include Arn; / ' (encoding D-glucose 6-phosphate dehydrogenase, w hich div erts G6P into the Pentose Phosphate Pathway (PPP)). \pfkA (encoding phosphofructokinase A. which diverts FOP into glycolysis). AnianA (encoding D-mannose 6- phsophate isomerase, which diverts F6P into the D-mannose biosynthesis pathway). &a / \E (encoding D-allulose 6-phosphate 3-epimerase, which diverts F6P into the D-psicose biosynthesis pathway), A / <gw (encoding phosphoglucomutase. which diverts F6P into glycogen biosynthesis), and Aga / Z (encoding a putative tagatose- 1 ,6-bisphosphate aldolase 2 chaperone, which diverts F6P into D-tagatose biosynthesis)’ls 25klentdvms potential xenex encoding for Sori), SorE, and phosphatases

[0295] SorD'SrlD (D-sorbitol 6-phosphate 2-dehydrogenase) and SorE (L-sorbose I -phosphate 2-reductase) enzymes were identified as having the potential to synthesize L-sorbose 1 -phosphate from D-fructose 6-phosphate. Past research had shown that these enzymes belong to the Sor operon, which is involved in catabolism of L -sorbosel i l 3Given this hypothesis and the D- psicose system being hosted in E. eoli, 3 microbial representatives of each enzyme type were selected based on annotation in the UniProt database as SorD / SrlD or SorE [Table 1 ]. At least one representative within each class had been previously characterized for L-sorbose catabolism in wW21”0Phosphatase enzyme representatives were selected in a similar fashion, based on UniProt annotation as being a microbial phosphatase capable of dephosphorx lating sugar 1- phosphates. At least one phosphatase representative had been previously demonstrated to dephosphorylate sugar I -phosphates in vitro''1.

[0296] Table I . Enzymes Identified from UniProt for Potential L-sorbose ProductionValidating SorD / SrlD activity in vitro

[0297] Three microbial SorD / SrlD representatives were selected for in vitro characterization: SorD C3SXZ2 (E. coll), SrlD P05707 (E. co / / , strain K12), and SorD P37079 (Klebsiella pneumoniae). Genes for tach enzyme were transformed into an expression strain of E coli, purified, and assayed on HPLC-MS to determine successfill product formation. The desired reaction was conversion of D-fructose 6-phosphate (F6P) to D-sorbitol 6-phosphate (S6P), and die change in mass was leveraged to detect successful conversion [Figure 2]. Enzymes were incubated with an excess of substrate (F6P) and cofactor (NADH) for 24 hours. Controls showed the expected peaks or lack of peaks [Figures 4A-4E]. C3SXZ2, P05707, and P37079 all successfully produced peaks corresponding to the D-sorbitol 6-phosphate product [Figures 5A- 9], These enzymes were selected for further characterization / zrvzvo.Validating SorE activity in vitro

[0298] Three microbial SorE representatives were selected fin* in vitro characterization: P37084 (K. pneumoniae), AQA3XISPGB6 (Citrobacterjreundii complex sp. CFNIH2), and A0A066SY94 (E colt). Genes for each enzyme were transformed into an expression strata of E. coli, purified, and assayed on HPLC-MS to determine successfill product formation. The desired reaction was conversion of D-sorbitol 6-phosphate (S6P) to L-sorbose 1 -phosphate (SIP), and the change in mass was leveraged to detect successfill conversion [Figure 10]. Enzymes were incubated with an excess of substrate (S6P) and cofactor (NAD4-) for 24 hours. Controls showed the expected peaks or lack of peaks [Figures 11A-11E]. P37084 was found to be inactive, while A0A2K9PGB6 and A0A066SY94 both successfully produced peaks corrding to the L-sorbose I -phosphate product [Figures 12A-15]. These enzymes were selected for further characterization in vivo.Validating phosphatase activity in vitro

[0299] Three microbial phosphatase representatives were selected for in vitro characterization: A0A2S8E3A7 (Shigella dysenteriae), A0A0L6Y216 (E. coli), and A0A4P5R0U6 (B. sp. Alli). Each Phosphatase enzyme was transformed into an expression strata of E coli, purified, and assayed on HPLC-MS to determine successful product formation. The desired reaction was conversion of L-soibose IP (SIP) to L-sorbose ., and the change in mass was leveraged to detect successful conversion [Figure 16]. Enzymes were incubated with an excess of substrate (SIP) for 24 hours. Controls showed the expected peaks or lack of peaks [Figures 17A-17D]. A0A2S8E3A7 and A0A4P5R0U6 were found to be inactive, due to maintaining die substrate peak, while A0A0L6Y216 significantly reduced the peak corresponding to the L-sorbose 1- phosphate substrate [Figures 18A-20].Establish D-sorbitol production to identify SorD.

[0300] Within the presently disclosed production pathway, a sorbitol-6-phosphate dehydrogenase (SorD.'SrlD) converts F6P to Sbtl6P. which can then be dephosphorylated by a phosphatase to produce D-sorbitol. Measuring D-sorbitol production provided a ’ checkpoint ” at which the efficacy of different SorD SrlD enzymes was evaluated. Four sorD srll) genes encoding for various SorD SrlD enzymes were additionally expressed under an IPTG-inducible PnMoi promoter from a plasmid and evaluated for production’2. The four genes included sori) from E. coli 0157 (Uniprot <'3SXZ2f ST / / ) from E. coli MG 1655 (Uniprot APO57O7), a codon optimized version of srll) from E. coli MG 1655 (Uniprot 7P05707). and sori) from K. pneumoniae (Uniprot 7P37079). The resulting plasmids were pAL2526. pAI.2553, pAL2527. pAL2528. and respectively. Preliminary screening of these plasmids showed no production from the codon optimized version of .v / 7 / ) PO57O7, so pAL2527 was excluded from further testing. Cultures were grown on M9P media with 5 g L’’ glucose at 30V. Supernatant samples were taken 24 h after inoculation and analyzed using High-Performance Anion-Exchange chromatography with Pulsed Amperometric Detection ( HPAE-PAD). When expressed in strain AL4386. minimal D-sorbitol production was detected [Figure 21 1.

[0301] It was theorized that the additional expression of a phosphatase would be necessary to produce D-sorbitol. From the literature hexitol phosphatase B (HxpB) was identified as a potential phosphatase that could convert Sbtl6P to D-sorbitol ”. The gene hxpH was cloned into the production plasmids containing sori) C3SXZ2, srll) PO57O7, and sori) P3707<X resulting in plasmids pAl.2540.PAI 2532. and pAL2542.

[0302] Production was tested again in AL4386. Cultures were grown on M9P media with 5 g L'!glucose at 30V. Supernatant samples were taken 24 h after inoculation and analyzed using HPAE- PAD. This time, increased D-sorbitol was detected in cultures containing the additional expression of .ST / / ) POSl’O7and sorL) P37O79 along with hxpB [Figure 21 ]. Cultures expressing srll) PO57O7 - hxpB produced an average of 0.73 g L’!D-sorbitol, while cultures expressing sori) P37O79 - hxpB produced an average of 0.50 g L’1. The following L-sorbose production experiments were therefore carried out using srll) PO57O7 and sori) P37O79.Esiahlish L-sorlmse production to idemifr SorE.

[0303] After confirming the functionality of SorD PO57O7 and SorD P37079. the next step was to additionally express a SorE enzyme to convert Sbtl6P to S I P and a phosphatase to dephosphorylate S I P to free L-sorbose . Three sorE genes encoding for a SorE from C. amalonaiicus (Uniprot # A0A2K9PGB6), a SorE from A’, pneumoniae (Uniprot 7P37084) and a SorE from E. coli (Uniprot #A0A066SY94) were tested for activity. The two enzymes yieldingpositive results from initial in vitro testing, SorE A0A2K9PGB6 and SorE A0A066SY94, were tested in vivo for production.

[0304] To test for L-sorbose production, the genes sorE A0A2K9PGB6 and sorE A0A066SY94 were added to production plasmids downstream of either srll) P05707 or sori) P37079. The gene encoding for E. coli phosphatase YqaB was also cloned in after sorE on each plasmid. YqaB is a promiscuous phosphatase that can be active on L-sorbose 6-phsophate as it has been shown to be active on L-sorbose 1 -phsophate74. The resulting plasmids are pAL2538 (Ptuoi: srll) PO57O7 - sorE AOA2K9PGB6 - yquBY pAL2539 (PnM-or. srll) PO57O7 - sorE A0A066SY94 ~~ yquBY pAL2554 (Piu on sori) P37()7q . A0A2K9PGB6 - yquli), and pAL2555 {Ptuor. sori) P37079 - sorE A0A066SY94 - yqaB)

[0305] These plasmids were introduced into AL4386 and tested for L-sorbose production. Cultures were grown on MVP media w ith 15 g L ' glucose at 30 C. Supernatant samples were taken 24 h after inoculation and analyzed using HPAE-PAD. It was difficult to explicitly quantify L-sorbose production due to the presence of D-fructose as a side product, but chromatographic analysis showed cultures containing pAL2538 and pAL2539 produced more L-sorbose relative to those containing pAL2254 and pAL2255 [Figure 22], Therefore, the combinations of srll) PO57O7 - sorE A0A2K9PGB6 - yqoB and srll) PO571E - sorE A0A066SY94 - yquB were used for further testing.Improving L-sorhose produclion using I GOH

[0306] Previously, it was described the use of E. coli promoter P^.m to strongly express genes during the stationary' phase of growth without the need for chemical induction7. Success with using P^,IH to produce D-psicose prompted the inventors of the present disclosure to try this strategy again with L-sorbose. Plasmids pAL I989 and pAL l993 were constructed to host IGo / t: srll) PO57O7 - sorE A0A2K9PGB6 - yquB and P^ur. srll) PO57O7 - sorE MA066SY94 - yquB respectively. These plasmids were introduced into AL4386 and tested for L-sorbose production. Cultures were grown on M9P media with 15 g L'1glucose at 30T. Samples for this experiment were taken after 48 h instead of 24 h in hopes that the cells would reassimilate excess D-fructose, making chromatographic analysis easier. When analyzed using HPAE-PAD. cultures containing pAL1989 produced the most L-sorbose , at 1.60 g L'1[Figure 23]. a titer almost equivalent to previous experiments using the IPTG-inducible promoter Pi,iM-oi. Cultures containing pAL 1993 produced slightly less L-sorbose , at 1.05 g L*', further supporting srll) P()57(H and sorE A0A2E9PGB6 as the superior L-sorbose enzyme combination. These results confirm P^nt as an inducer-free option for effective gene expression.

[0307] The elimination of competing pathways and additional expression of genes of the present example along with the use of static and dynamic gene regulation strategies disclosed herein can lead to a strain capable of producing L-sorbose using a thei modvnamicallv faxorable biosynthetic pathway from a readily available feedstock. Overall, the engineered strain can represent a helpful step in producing L-sorbose in a cost-effective manner, offering the food industry a viable source for creating the low-glycemic index products desired by consumers.

[0308] Enzyme IdeiuificaEion. The UniProt database was used to identify enzymes potentially capable of the following transformations: D-fructose 6-phosphate to D-sorbitol 6-phosphate, D- sorbitol 6-phosphate to L-sorbose I -phosphate, and L-sorbose 1 -phosphate to L-sorbose. Soi l) Si ll). Soi l', and Phosphatase cn / .\ mes w ere identified as candidates foi each, chemical transformation, respectively. Three enzymes of each class were selected for characterization [Table 1 ], and were chemically synthesized by Twist Bioscienccs, encoded in a pet29b+ plasmid.

[0309] Enzyme Expression and Purificalion. BL21(DE3)pLysS cells were transformed with a pet29fo plasmid (encoding polypeptides of interest with a C-terminal His-tag ). Transformed cells suspended in a 10 mL volume of Terrific Broth with 50 pg mL'1kanamycin. 1 mM MgSO4 were grown at 37 °C for 24 hours. Cultures were pelleted down at 5,000 G for 10 minutes and resuspended in 10 mL of auto-induction media (Terrific Broth, 50 pg mL'1kanamycin, I mM MgSO4, I *NPS and 1 *5052) for induction at 18 °C for 27 hours. After the induction period, cells were pelleted by centrifugation, the supernatant was removed, and cells were resuspended in 500 pL wash buffer ( 100 mM HEPES, 50 mM NaCl, 5 mM imidazole) and added to 500 pL lysis buffer (wash buffer. 1.6 mg ml.’1lysozyme. 0.2 mg mL’1PMSF, 0.2 mg mL’1DNase). The lysis mixture was rocked for 30 minutes, followed by centrifugation at 5,000 G for 30 minutes. The supernatant was loaded onto a gravity flow column with 300 pL Cobalt bead slurry and was washed with 500 pL of wash buffer five times. Proteins were eluted with 300 pL of elution buffer (80 mM HEPES, 40 mM NaCL 200 mM imidazole). Protein concentrations were determined using a Synergy H l spectrophotometer (Biotek) by measuring absorbance at 280 nm using calculated extinction coefficients. Enzymes containing a concentration of at least 0.1 mg mL’1were then as>a\ ed

[0310] Enzyme Assay. For each enzyme (SorD / SrID, SorE. Phosphatase), 50 pL of substrate mix was combined with 50 pL of purified enzyme, incubated at room temperature (21 °C) for 24 hours, and then quenched in 300 pL of methanol. For SorD / SrID. substrate mix was an aqueous solution of 8 mM D-fructose 6-phosphate . 20 mM NADH. For SorE. substrate mix was an aqueous solution of S mM D-sorhiiol 6-phosphate. 20 mM NAD - For Phosphatase, substrate mix was anaqueous solution of 4 mM L-sorbose 1 -phosphate. Controls were included wherein enzyme solution was replaced with elution buffer, or the substrate mix was replaced with water.[03 1 1 ] HPLC-MS Analysis of Enzyme Assay Results. After the 24 h incubation at 21 °C. the quenched samples were prepared for High Performance Liquid Chromatography and Mass Spectrometry ( HPLC-MS) analysis by centrifuging for 10 minutes at 5,000 g. Supernatant was collected to be used in I IPI C-MS analysis I ke column. Shodex HlLICpak \ 1 -50 2D ( 2.0 mm LD. x 1 50 mm), was used to separate the analytes in the reaction. The analysis was run at 0.3 ml. min'1flow rate with Mobile Phase A (25 mM Ammonium Formate, aqueous) and Mobile Phase B ( 100° o ACN). The analysis was started at 80% MPA. 20% MPB until minute 13. At minute 13, a gradient process altered the concentrations to 5%MPA / 95% MPB by minute 1 5. The concentration was held constant until minute 16.5, when a gradient process altered the concentrations to 80% MPA / 20% MPB by minute 17. The concentration was then held constant until minute 50. when the run ended. To detect the conx ersion between sugar-phosphates ( D- fructose 6-phosphate , L-sorbose 1 -phosphate) and sugar alcohol-phosphates (D-sorbitol 6- phosphate). Single Ion Mode w as used to scan for the unique mass of the sugars (260 g mol’1) or sugar alcohols (262 g mol"1) [Figure 2],

[0312] ( 'liming and standard reagents. All enzymes involved in molecular cloning were purchased from New England Biolabs, except for repliQa HiFi ToughMix, which was purchased from Quantabio. All synthetic oligonucleotides were synthesized by Integrated DNA Technologies. Sanger Sequencing was provided by Azenta Genewiz. L-sorbose was purchased from TCI America. D-sorbitol and D-sedoheptulose were purchased from Sigma Aldrich. D- fructose was purchased from Fisher Scientific. D-glucose was purchased from Thermo Scientific.

[0313] Strains and plasmids. The relevant strains and plasmids used for L-sorbose production in this study are listed in Table 2 and 3. respectix ely. Genome modifications such as gene deletion and gene insertion were constructed using CRISPR-CasO-mediated homologous recombination1'’. Linear DNA repair fragments for gene deletions and insertions were constructed by amplifying genomic or plasmid DN A \ ia PGR assembly' Plasmids encoding sgRNA for CR ISPR-Gas9- mediated homologous recombination were constructed using Q5 site-directed mutagenesis ( Nexv England Biolabs) using pTargetF plasmid ( Addgene #62226) as a template. All genomic modifications w ere verified via sequencing

[0314] Table 2. Strain list

[0315] Table 3. Plasmid list

[0316] Culture media. Overnight cultures were grown at 37 °C in 3 mL of Luria-Bertani (LB) media with appropriate antibiotics. Antibiotic concentrations were as follows: spectinomycin (50 pg mL'1), ampicillin (200 pg mL'1), kanamycin (50 pg mL'1), gentamycin (15 pg mL'1). M9P media for L-sorbose production consists of M9 minimal media supplemented with 5 g L'1of yeast extract and appropriate antibiotics. M9 minimal media consists of 33.7 mM Na2HPO4, 22 mM KH2PO4, 8.6 mM NaCl, 9.4 mM NH4C1, 2 mM MgSO4, 0.1 mM CaCh, A5 trace metals mix (2.86mg L'1H3BO3, 1.81 mg L'1MnCl2-4H2O, 0.079 mg L1CuSO4-5H2O, 49.4 g L'1Co(NO3)2-6H2O), varying concentrations of glucose, and appropriate antibiotics. Inducer concentrations are as follows: isopropyl-|3-D-l -thiogalactopyranoside (IPTG) (1 mM). anhydrotetracycline (aTc) (100 ng ml;1). ODeoo was measured with a Synergy Hl hybrid plate reader (BioTek Instruments, Inc.).

[0317] L-sorbose production conditions. Overnight cultures were inoculated at 1% into 3 mL of M9P media. Cells were grown at 37 °C until ODeoo -0.4 unless otherwise described, then induced with IPTG if necessary, and grown at 30 °C for 24 or 48 h.

[0318] High-Performance Anion-Exchange chromatography (HPAE) Analysis. Analysis of L- sorbose, D-sorbitol, D-fructose, D-sorbitol, and D-glucose concentrations was also performed using HPAE with Pulsed Amperometric Detection (PAD) on an ICS-5000 system with a CarboPac PA10 4x250mm (Thermo Fisher). Samples were run with an injection volume of 10 iiL. The column oven was maintained at 30 °C. Each run consisted of an equilibration phase, an analysis phase, and a wash phase. Equilibration used a mobile phase comprising 50 mM NaOH in degassed MilliQ water at a flow rate of 1.5 mL min'1for 15 min. Analysis used a mobile phase consisting of 50 mM NaOH in degassed MilliQ water at a flow rate of 1.5 mL min'1for 15 min. Wash used a mobile phase consisting of 200 mM NaOH in degassed MilliQ water at a flow rate of 1.5 mL min'1for 10 min. To prepare samples for HPAE-PAD analysis, 300 pL of lOx diluted supernatant in filter-sterilized milliQ water was applied to a 0.2 m PVDF hydrophilic membrane 96 well filter plate and centrifuged at 17,000 g for 2 min into a polystyrene 96 well.Production plasmid nucleic acid sequences

[0319] pAL2521:Ċ

[0326] pAL2541: Ptiacoi: codon optimized srlD P05707 - hxpBTGGCAGCACTGCATAATTCTCTGTACTCAACCAAGTCATTCTGAReferences:1. Mijailovic, N., Nesler, A., Perazzolli, M., Ait Barka, E. & Aziz, A. Rare Sugars: Recent Advances and Their Potential Role in Sustainable Crop Protection. Molecules 26, (2021).2. Van Laar, A. D. E., Grootaert, C. & Van Camp, J. Rare mono- and disaccharides as healthy alternative for traditional sugars and sweeteners? Crit. Rev. Food Set. Nutr. 61, 713— 741 (2021).3. Smith, A. et al. Rare sugars: metabolic impacts and mechanisms of action: a scoping review. Br. J. Nutr. 128, 389-406.4. Mu, W., Zhang, W., Feng, Y., Jiang, B. & Zhou, L. Recent advances on applications and biotechnological production of D-psicose. Appl. Microbiol. Biotechnol. 94, 1461-1467 (2012).5. Naha, N. et al. Rare sugar d-allose induces programmed cell death in hormone refractory prostate cancer cells. Apoptosis 13, 1121-1134 (2008).6. Xu, H.-L. et al. 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[0042] L-sorbose -1-phosphate reductase, in Methods in Enzymology' vol. 89 248-251 (Academic Press, 1982).31. Kuznetsova, E. et al. Genome- wide analysis of substrate specificities of the Escherichia coli haloacid dehalogenase-like phosphatase family. J. Biol. Chem. 281, 36149- 36161 (2006).32. Lutz, R. & Bujard, H. Independent and tight regulation of transcriptional units in escherichia coli via the LacR / O, the TetR / O and AraC / Il-I2 regulatory elements. Nucleic Acids Res. 25, 1203-1210 (1997).33. Sevin, D. C., Fuhrer, T., Zamboni, N. & Sauer, U. Nontargeted in vitro metabolomics for high-throughput identification of novel enzymes in Escherichia coli. Nat. Methods 14, 187-194 (2017).34. Yang, J. et al. Biosynthesis of L-sorbose and L-psicose based on COC bond formation catalyzed by aldolases in an engineered Corynebacterium glutamicum strain. Appl. Environ. Microbiol. 81, 4284-4294 (2015).Example 2 — Further Detail forProduction of L-sorbose in Escherichia coli

[0334] This Example includes some data that is duplicative of data presented in Example 1 as well as including new detail not provided in Example 1.Designing an L-sorbose production pathway

[0335] We theorized the thermodynamically favorable process of phosphorylating and dephosphorylating sugars could be used to produce L-sorbose from D-glucose (Fig. 24A) (6). Within E. coli, D-glucose is simultaneously phosphorylated and transferred across the cell membrane by the phosphotransferase system (PTS) (19). Alternatively, D-glucose can be assimilated by the galactose proton symporter GalP (20), after which it is phosphorylated to G6P by glucokinase Glk (21). D-glucose 6-phosphate (G6P) is then isomerized to F6P by G6P isomerase Pgi (19). F6P is typically directed into glycolysis but could be interconverted between Sbtl6P by a Sbtl6P dehydrogenase, known as SorD or SrlD (18, 22, 23). From there, Sbtl6P can be converted to SIP by an SIP reductase SorE (23-25). SorD and SorE are oxidoreductases in the Sor operon, a catabolic pathway for L-sorbose (17, 18, 23). Although this pathway usually runs from SIP to Sbtl6P to F6P as part of the sorbose and sorbitol degradation pathways, an accumulation of intracellular F6P could cause the reaction to run in reverse to produce S IP (22). Finally, dephosphorylation of SIP by a phosphatase provides thermodynamic incentive for flux through the production pathway and generates free L-sorbose to be excreted from the cell. Overall, the additional expression of genes encoding for SorD, SorE, and a phosphatase should lead to the biosynthesis of L-sorbose (Fig. 24AB).

[0336] Directing carbon flux towards L-sorbose production requires eliminating competing pathways. We performed the following experiments using a previously established strain, AL4386, containing genetic knockouts for enzymes at key branch points where these pathways diverge (Table 4). These genetic knockouts include Azu / (encoding G6P dehydrogenase, which diverts G6P into the Pentose Phosphate Pathway (PPP)), ^pfkA (encoding phosphofructokinaseA, which diverts F6P into glycolysis), AmanA (encoding D-mannose 6-phsophate isomerase, which diverts F6P into the D-mannose biosynthesis), kalsE (encoding D-allulose 6-phosphate 3- epimerase, which diverts F6P into the D-psicose biosynthesis), bpgm (encoding phosphoglucomutase, which diverts F6P into glycogen biosynthesis), and EgatZ (encoding a putative tagatose 1,6-bisphosphate aldolase 2 chaperone, which diverts F6P into D-tagatose biosynthesis) (6, 19, 26-32).

[0337] In vitro analysis of enzyme candidates for L-sorbose production

[0338] We selected three microbial representatives of each enzyme based on annotations in the UniProt database. SorD candidates included SorD C3SXZ2 (£. coli), SrlD P05707 (£. coli), SorD P37079 (Klebsiella pneumoniae), which are henceforth referred to as SorDl, SorD2, and SorD3 respectively (Fig. 24C). SorE candidates included SorE A0A2K9PGB6 (Citrobacter freimdii complex sp. CFNIH2), SorE P37084 (K. pneumoniae), and SorE A0A066SY94 (£. coli), which are henceforth referred to as SorEl, SorE2, and SorE3 respectively (Fig. 24C). All nucleic acid sequences were codon optimized for expression within £. coli. At least one representative of each enzyme had been previously characterized in vitro for L-sorbose catabolism but not L-sorbose production (17, 18, 33).

[0339] To assess activity, SorD enzymes were incubated with excess F6P and NADH, and SorE enzymes with excess SIP and NAD+for 24 h. Reaction products were analyzed for a change in mass using HPLC-MS and compared to product standards, demonstrating that all three tested SorD enzymes (SorDl, SorD2, and SorD3) and two out of three SorE enzymes (SorEl and SorE3) completed the desired reaction (Fig. 24C, 27 & 28). These promising candidates from the in vitro screen were brought forward to test L-sorbose production in £. coli.

[0340] Establishing microbial D-sorbitol production to identify SorD

[0341] Within the production pathway, SorD converts F6P to Sbtl6P. In addition to being converted to SIP by SorE, Sbtl6P may also be dephosphorylated by a phosphatase to produce free D-sorbitol (Fig. 24A). As such, measuring D-sorbitol production provides a “checkpoint” at which the efficacy of different SorD enzymes can be evaluated. Initial in vivo screening showed no production from codon-optimized SorD2, so the native sequence was substituted, henceforth called SorD2* (Fig. 24C). The genes sorDl, sorD2*, and sorD3 were expressed from the IPTG- inducible promoter PijacOi, generating pAL2526, pAL2553, and pAL2528 respectively (Table 4). AL4386 with pAL2526, pAL2553, or pAL2528 were evaluated for D-sorbitol production (Strain 4, 7, and 5, Table 4) (34). Cultures were grown in M9P media with 5 g L'1glucose at 30°C for 24 h, but only minimal D-sorbitol production was detected (Fig. 25A).

[0342] Previous work has shown that the additional expression of a phosphatase may be necessary to produce D-sorbitol (6). Hexitol phosphatase B (HxpB) was identified as a potential phosphatase for converting Sbtl6P to D-sorbitol (35, 36). The hxpB gene was cloned downstream sorDl, sorD2*. and sorD3, generating pAL2540, pAL2532, and pAL2542 (Table 4). AL4386 with pAL2540, pAL2532, or pAL2542 were evaluated for D-sorbitol production (Strain 8-10, Table 4). Cells were cultured on M9P media with 5 g L'1glucose at 30°C for 24 h. Increased D-sorbitol was detected in production Strain 9 and 10 (Fig. 25 A). Strain 9 (sorD2*-hxpB) produced 0.73 g L'1D-sorbitol, while production Strain 10 (sorD3-hxpB) produced 0.50 g L-i. The following L- sorbose production experiments were therefore carried out using SorD2* and SorD3.

[0343] Establishing microbial L-sorbose production to identify SorE

[0344] After confirming the functionality of SorD2* and SorD3, our next step was to additionally express a SorE enzyme to convert Sbtl6P to SIP and a phosphatase to dephosphorylate SIP to free L-sorbose. The two SorE enzymes yielding positive results from initial in vitro testing (Fig. 24C & 28), SorEl and SorE3, were tested for L-sorbose production in E. coll.

[0345] The sorEl and sorE3 genes were cloned downstream of either sorD2* or sorD3, along with gene yqaB, which encodes E. coll fructose 1 -phosphate phosphatase YqaB. YqaB is a promiscuous haloacid dehalogenase (HAD)-like hydrolase with phosphatase activity towards L- sorbose 1-phsophate (37). The resulting four plasmids, pAL2538 (Priacoi: sorD2*-sorEl-yqaB), pAL2539 (Pnacoi: sorD2*-sorE3-yqaB), pAL2554 (Puacoi: sorD3-sorEl-yqaB), and pAL2555 (Pnacoi: sorD3-sorE3-yqaB') were introduced into AL4386, generating production Strain 11-14, and tested for L-sorbose production (Table 4). Cultures were grown on M9P media with 15 g L'1glucose at 30°C for 24 h. The presence of D-fructose as a side product made explicit quantification of L-sorbose difficult, but cultures expressing sori)2* produced more L-sorbose relative to those expressing sorD3 (Fig. 29). Therefore, the combinations of sorD2*-sorEl-yqaB and sorD2*- sorE3-yqaB were used for further testing.

[0346] In addition to D-fructose as a side product, chromatographic analysis also indicated the presence of D-sorbitol and D-sedoheptulose in culture supernatant. The formation of these side products suggests an overaccumulation of F6P and inefficient utilization of carbon flux in the L- sorbose production pathway. First, if SorE activity allows for a buildup of Sbtl6P, the additional expression of promiscuous phosphatase YqaB and endogenous expression of HxpB may lead to the dephosphorylation of Sbtl6P to free D-sorbitol (Fig. 24A). In the case of F6P, it can be dephosphorylated by a number of phosphatases, leading to free D-fructose (Fig. 24A) (38). While E. coli can utilize D-fructose as a carbon source, the cells prioritize D-glucose consumption due to carbon catabolism and do not utilize D-fructose until glucose is depleted (39). F6P is also a keymetabolite in the non-oxidative branch of the PPP, and along with D-erythrose 4-phosphate is reversibly interconverted to D-sedoheptulose 7-phosphate and glyceraldehyde 3-phosphate by transaldolases TalA and TalB (Fig. 24A) (40). An excess of intracellular F6P may lead to flux favoring the production of sedoheptulose 7-phosphate, which in the presence of a promiscuous enzymes such as YqaB could be dephosphorylated to free D-sedoheptulose.

[0347] Improving L-sorbose production using the stationary phase promoter PgadB

[0348] Until this point, we used the IPTG-inducible promoter PuacOi to express genes for L- sorbose production. While it allows for controlled timing of gene expression, chemical induction may lead to competition for carbon flux between endogenous cellular pathways and production (41). To maximize cellular health and growth, carbon flux should be directed towards endogenous pathways during growth (42). Once the cells enter stationary phase, production pathway genes can be expressed to redirect carbon flux towards production. Previously, we described the use of E. coli promoter PgadB to express genes during the stationary' phase of growth without the need for chemical induction (6). Success with using PgadB to produce D-psicose prompted us to try this strategy in producing L-sorbose. pAL1989 (PgadB.sorD2*-sorE l-yqaB) and pAL1993 (Pgads: sorD2*-sorE3-yqaB ) were introduced into AL4386 (Strain 15 & 16, Table 4) and tested against their PuacOi counterparts (Strain 11 and 12, Table 4) for L-sorbose production. Cultures were grown on M9P media with 15 g L'1glucose at 30°C for 48 h (Fig. 25B). Strain 15 (PgadB'. sorD2*-sorEl-yqaB) produced the most L-sorbose, at 1.60 g L'1(Fig. 25B). Strain 16 (PgadB:sorD2*-sorE3-yqaB) produced slightly less L-sorbose, at 1.05 g L’1, further supporting sorD2* and sorEl as the superior L-sorbose enzyme combination. In comparison, Strain 11 and 12 generated comparable levels of L-sorbose, at 1.08 g L'1and 1.23 g L’1respectively (Fig. 25B). These results confirm PgadB as an inducer-free option for effective gene expression.

[0349] In addition to L-sorbose, D-fructose was detected in uninduced cultures of Strain 11 and 12. D-sorbitol and D-sedoheptulose were present in all cultures, irrespective of induction or promoter (Fig. 25B). As described above, the presence of these side products may indicate an intracellular accumulation of F6P, and “leaking” of carbon flux into competing pathways. As such, we decided to screen additional phosphatases in an effort to increase L-sorbose and minimize side product dephosphorylation.

[0350] Screening phosphatases for increased L-sorbose production

[0351] E. coll possesses a number of haloacid dehalogenase (HAD)-like hydrolases with promiscuous phosphatase activity towards hexoses (38). Although we originally used YqaB during the screen for SorE enzymes, other phosphatases may more specifically dephosphorylate S 1 P. Increased specificity may help alleviate carbon flux bottlenecks and prevent the prematuredephosphorylation of Sbtl6P or the extraneous dephosphorylation of D-sedoheptulose 7- phosphate. Six additional phosphatases were inserted downstream of PgadB'.sorD2*-sorEl . The phosphatases tested were hexitol phosphatase A (HxpA) (pAL2656), hexitol phosphatase B (HxpB) (pAL2657), sugar phosphatase YbiV (pAL2571), sugar phosphatase YidA (pAL2659), phosphosugar phosphatase YigL (pAL2660), and a-D-glucose 1 -phosphate phosphatase YihX (pAL2661) (Table 4) (38).

[0352] AL4386 transformed with plasmids carrying each phosphatase gene (pAL2656, pAL2657, pAL2571, pAL2659, and pAL2660) was tested for L-sorbose production (Strain 18- 23, Table 4). Out of the seven tested phosphatases, Strain 20 (ybiV) performed best at 3.51 g L1L-sorbose, and the least amount of D-sedoheptulose, at 0.39 g L'1(Fig. 25C). Interestingly, while earlier studies identified sorbitol 6-phosphate as a favored substrate for YbiV, subsequent research demonstrated that HxpB exhibits greater activity (36, 43). Despite this propensity, negligible D- sorbitol was detected in these cultures.

[0353] Minimizing diversion of F6P to the Pentose Phosphate Pathway

[0354] To enhance carbon flux through the L-sorbose production pathway by increasing upstream F6P pools, the transaldolase genes talA and talB were knocked out in AL4386 (Table 4, Fig. 24A) (40). These strains harboring pAL2571 (PgadB:sorD2*-sorEl-ybiV, Strain 24 and 25, Table 4) were tested for L-sorbose production. Cells were grown on M9P media with 15 g L1glucose at 30°C for 48 h (Fig. 30). The deletion of talA slightly increased L-sorbose from 3.51 g L'!to 3.76 g L'1, but also increased D-sorbitol formation from below limits of quantification to 0.05 g L'1(Fig. 30). The deletion of talB decreased L-sorbose production to 2.07 g L'1and increased D- sorbitol and D-sedoheptulose formation to 0.37 g L1and 0.83 g L’1respectively (Fig. 30). Without more efficient carbon utilization by the L-sorbose production pathway, increasing upstream F6P by reducing flux into the PPP did little to improve titers, and AL4386 was used for subsequent experiments.

[0355] Enhancing glucose uptake through galP anftglk additional expression

[0356] To maximize production by minimizing carbon flux towards cell growth, we sought to mimic the high culture density conditions found in industrial production. Before attempting high-density production, a supplemental glucose import pathway was introduced to help increase carbon flux through the L-sorbose pathway. Similarly to our previous study on the microbial production of D-psicose (6), the genes encoding galactose proton symporter GalP and glucokinase Glk were cloned under the IPTG-inducible Puacoi promoter (pAL2263, Table 4). AL4386 harboring pAL2263 and pAL2571 (PgadB: sorD2*-sorEl-ybiV) was tested for L-sorbose production (Strain 26, Table 4) (6). When induced with IPTG, Strain 26 produced more L-sorboseat 5.26 g L'!, compared to uninduced cultures which only made 3.69 g L'1(Fig. 31). Induced cultures also produced more D-sorbitol, at 1.20 g L'1, and D-sedoheptulose, at 0.62 g L1. The increase in L-sorbose and side products demonstrate that additionally expressing an alternative mode of glucose import results in increased flux through the L-sorbose pathway, although more work is needed to prevent side product formation.

[0357] L-sorbose production under high density conditions

[0358] Strain 26 (PgadB'. sorD2*-sorEl-ybiV, Pu^ov. galP-glk, Table 4) was cultured under high density conditions for a total of 96 h. Samples were taken and additional glucose was added to each flask every 24 h. Over the course of 96 h, cultures produced an average of 14.5 g L’1of L-sorbose, with an average productivity of 0.15 g L'!h'!, specific titer of 1.00 g L'!ODeoo’1, and yield of 24.2% (Fig. 26). D-sorbitol and D-sedoheptulose were detected at final titers of 7.62 g L’1and 4.01 g L’1respectively.

[0359] Cultures were able to consume all 15 g L'1of media glucose every 24 h. While full consumption of substrate is a desirable property of microbial production, further experiments should be performed to elucidate the upper threshold of glucose this strain is able to import and utilize. The presence of D-sorbitol and D-sedoheptulose can be addressed by allowing a final “starvation period” during fermentation, where no D-glucose is added to the culture, and cells are allowed to reassimilate side products back into central carbon metabolism.

[0360] Conclusion

[0361] In this study, we successfully implemented a novel biosynthetic pathway for L- sorbose production in E. coli. By integrating phosphorylation and dephosphorylation strategies, we demonstrated the direct conversion of D-glucose to L-sorbose with minimal side product contamination. Our optimized production strain, combining pathway enzymes SorD, SorE, and YbiV with carbon flux-enhancing strategies, achieved titers of 3.51 g L ' in regular density conditions after 48 h and 14.5 g L1in high-density fermentation after 96 h. These results highlight the potential of this system for efficient and cost-effective L-sorbose production. This work establishes a strong foundation for the broader application of L-sorbose as a sugar alternative, therapeutic agent, and precursor for Vitamin C.Materials and MethodsEnzyme Identification

[0362] The UniProt database was used to identify enzymes potentially capable of the following transformations: F6P to Sbtl6P and Sbtl6P to SIP. SorD and SorE enzymes were identified as candidates for each chemical transformation, respectively. Three enzymes of each class wereselected for characterization (Fig. 24C) and were chemically synthesized by Twist Biosciences, encoded in a pet29b+ plasmid.Enzyme Expression and Purification

[0363] BL21(DE3)pLysS cells were transformed with a pET29b- plasmid (encoding polypeptides of interest with a C-terminal His-tag). Cells suspended in a 10 mL volume of Terrific Broth with 50 pg mL’1kanamycin, 1 mM MgSO4 were grown at 37 °C for 24 h. Cultures were spun down at 5,000 g for 10 min and resuspended in 10 mL of auto-induction media (Terrific Broth, 50 pg mL’1kanamycin, 1 mM MgSO4, I xNPS and 1 ' 5052) for induction at 18 °C for 27 h. After the induction period, cells were spun down, washed with 500 pL wash buffer (100 mM HEPES, 50 mM NaCl, 5 mM imidazole) and resuspended in 500 pL lysis buffer (wash buffer, 1.6 mg mL’1lysozyme, 0.2 mg mL’1PMSF, 0.2 mg mL’1DNase). The lysis mixture was incubated with gentle agitation for 30 min, followed by centrifugation at 5,000 g for 30 min. The supernatant was loaded onto a gravity flow column with 300 pL Cobalt bead slurry and was washed with 500 pL of wash buffer five times. Proteins were eluted with 300 pL of elution buffer (80 mM HEPES, 40 mM NaCl, 200 mM imidazole). Protein concentrations were determined using a Synergy Hl spectrophotometer (Biotek) by measuring absorbance at 280 nm using calculated extinction coefficients. Enzymes containing a concentration of at least 0.1 mg mL’1were then assayed.Enzvme Assavs

[0364] For each SorD and SorE enzyme, 50 pL of substrate mix was combined with 50 pL of purified enzyme, incubated at room temperature (21 °C) for 24 h, and then quenched in 300 pL of methanol. For SorD, substrate mix was an aqueous solution of 8 mM F6P , 20 mM NADH. For SorE, substrate mix was an aqueous solution of 8 mM Sbtl6P, 20 mM NAD . Controls were included wherein enzyme solution was replaced with elution buffer, or the substrate mix was replaced with water.HPLC-MS Analysis of Enzyme Assay Results

[0365] After the 24 h incubation at 21 °C, the quenched samples were prepared for High Performance Liquid Chromatography and Mass Spectrometry (HPLC-MS) analysis by centrifuging for 10 min at 5,000 g. Supernatant was collected to be used in HPLC-MS analysis. The column, Shodex HILICpak VT-50 2D (2.0 mm I.D. x 150 mm), was used to separate the analytes in the reaction. The analysis was run at 0.3 mL min1flow rate with Mobile Phase A (25 mM Ammonium Formate, aqueous) and Mobile Phase B (100% ACN). The analysis was started at 80% MPA / 20% MPB until minute 13. At minute 13, a gradient process altered the concentrations to 5%MPA / 95% MPB by minute 15. The concentration was held constant untilminute 16.5, when a gradient process altered the concentrations to 80% MPA / 20% MPB by minute 17. The concentration was then held constant until minute 50, when the run ended. To detect the conversion between sugar-phosphates (F6P, SIP) and sugar alcohol-phosphates (Sbtl6P), Single Ion Mode was used to scan for the unique mass of the sugars (260 g mol’1) or sugar alcohols (262 g mol'1) (Fig. 27 & 28).Cloning and standard reagents

[0366] All enzymes involved in molecular cloning were purchased from New England Biolabs, except for repliQa HiFi ToughMix, which was purchased from Quantabio. All synthetic oligonucleotides were synthesized by Integrated DNA Technologies. Sanger Sequencing was provided by Azenta / Genewiz. L-sorbose was purchased from TCI America. D-sorbitol and D- sedoheptulose were purchased from Sigma Aldrich. D-fructose was purchased from Fisher Scientific. D-glucose was purchased from Thermo Scientific.Strains and plasmids

[0367] The relevant strains and plasmids used for L-sorbose production in this study are listed in Table 4. All oligonucleotides are listed in Table 5. A guide to the construction of plasmids used in this study is detailed in Table 6. Genome modifications such as gene deletion and gene insertion were constructed using CRISPR-Cas9-mediated homologous recombination (44). Linear DNA repair fragments for gene deletions and insertions were constructed by amplifying genomic or plasmid DNA via PCR assembly (45). Plasmids encoding sgRNA for CRISPR-Cas9-mediated homologous recombination were constructed using Q5 site-directed mutagenesis (New England Biolabs) using pTargetF plasmid (Addgene #62226) as a template. All genomic modifications were verified via sequencing.Culture media

[0368] Overnight cultures were grown at 37 °C in 3 nil of Luria-Bertani (LB) media with appropriate antibiotics. Antibiotic concentrations were as follows: spectinomycin (50 pg mL'1), ampicillin (200 pg mL'1), kanamycin (50 pg mL'1), gentamycin (3.75 pg mL'1). M9P media for L-sorbose production consists of M9 minimal media supplemented with 5 g L'1of yeast extract and appropriate antibiotics. M9 minimal media consists of 33.7 mM Na2HPO4, 22 mM KH2PO4, 8.6 mM NaCl, 9.4 mM NH4Q, 2 mM MgSO4, 0.1 mM CaCh, A5 trace metals mix (2.86 mg L'1H3BO3, 1.81 mg L'1MnCl2-4H2O, 0.079 mg L'1CuSO4-5H2O, 49.4 g L'1Co(NO3)2-6H2O), varying concentrations of glucose, and appropriate antibiotics. Inducer concentrations are as follows: isopropyl-|3-D-l -thiogalactopyranoside (IPTG) (1 mM), anhydrotetracycline (aTc) (100 ng mL'1). ODsoo was measured with a Synergy Hl hybrid plate reader (BioTek Instruments, Inc.).L-sorbose production

[0369] For regular cell density production experiments, overnight cultures were inoculated at 1% into 3 mL of M9P media supplemented with antibiotics. Cells were grown at 37 °C until ODeoo -0.4 to 1 unless otherwise described, then induced with IPTG if necessary, and grown at 30 °C for 24 or 48 h.

[0370] For high-density production experiments, overnight cultures were inoculated at 1% into 50 mL of M9P media supplemented with antibiotics. Cells were grown at 37 °C until ODeoo -0.4 to 1, then induced with ImM IPTG and grown for an additional 30 m. Cells were then pelleted at 5,000 g for 15 m, before being resuspended to an ODeoo of -9.5 in 5 mL of M9P containing 15 g L'1glucose and 1 mM IPTG. Cultures were grown for 96 h, with samples taken every 24 h. An additional 15 g L'1of glucose was added every 24 h by replacing 10% of culture volume with media.High-Performance Anion-Exchange chromatography (HPAE) Analysis

[0371] Analysis of L-sorbose, D-sorbitol, D-fructose, D-sorbitol, and D-glucose concentrations was also performed using HPAE with Pulsed Amperometric Detection (PAD) on an ICS-5000 system with a CarboPac PA10 4x250mm (Thermo Fisher). Samples were run with an injection volume of 10 pL. The column oven was maintained at 30 °C. Each run consisted of an equilibration phase, an analysis phase, and a wash phase. Equilibration used a mobile phase comprising 50 mM NaOH in degassed MilliQ water at a flow rate of 1.5 mL min'1for 15 min. Analysis used a mobile phase consisting of 50 mM NaOH in degassed MilliQ water at a flow rate of 1.5 mL min'1for 15 min. Wash used a mobile phase consisting of 200 mM NaOH in degassed MilliQ water at a flow rate of 1.5 mL min'1for 10 min. To prepare samples for HPAE-PAD analysis, 300 pL of lOx diluted supernatant in filter-sterilized milliQ water was applied to a 0.2 m PVDF hydrophilic membrane 96 well filter plate and centrifuged at 17,000 g for 2 min into a polystyrene 96 well plate.Table 4. Strains and plasmids used in this study.Table 5Table 6. Plasmids construction guide

[0372] 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 the 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 matter, 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 matter, 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 matter. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

[0373] 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 that produces an increased amount of an L-sorbose, D- fructose, D-sorbitol and / or D-sedoheptulose as compared to a naturally occurring microorganism, wherein the recombinant microorganism comprises at least one exogenous oxidoreductase and at least one exogenous phosphatase.

2. The recombinant microorganism of claim 1, wherein the at least one exogenous oxidoreductase is a sorbitol-6-phosphate dehydrogenase (SorD / SrlD), a L-sorbose 1- phosphate reductase (SorE), or a combination thereof.

3. The recombinant microorganism of claim 1, wherein the SorD / SrlD comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 4.

4. The recombinant microorganism of claim 2 or 3, wherein the SorD / SrlD comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO : 2, or SEQ ID NO: 4.

5. The recombinant microorganism of claim 4, wherein the SorD / SrlD comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2.

6. The recombinant microorganism of any one of claims 2-5, wherein the SorE comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

7. The recombinant microorganism of any one of claims 2-6, wherein the SorE comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

8. The recombinant microorganism of claim 7, wherein the SorE comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9.

9. The recombinant microorganism of any one of claims 1-8, wherein the phosphatase is a hexitol phosphatase B (HxpB), a fructose- 1 -phosphate phosphatase (YqaB), a sugar phosphatase YbiV (YbiV), Hexitol phosphatase A (HxpA), sugar phosphatase YidA, phosphosugar phosphatase YigL, sugar phosphatase YihXor a combination thereof.

10. The recombinant microorganism of claim 9, wherein the HxpB comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 15.

11. The recombinant microorganism of claim 9 or 10, wherein the HxpB comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15.

12. The recombinant microorganism of any one of claims 9-11, wherein the YqaB comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 17.

13. The recombinant microorganism of any one of claims 9-12, wherein the YqaB comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17.

14. The recombinant microorganism of any one of claims 9-13, wherein the YbiV comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 3.

15. The recombinant microorganism of any one of claims 9-14, wherein the YbiV comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3.

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

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

18. The recombinant microorganism of any one of claims 1-17, further comprising a mutation of a gene encoding an enzyme of glycogen biosynthesis.

19. The recombinant microorganism of claim 18, wherein the enzyme of glycogen biosynthesis is phosphoglucomutase (Pgm).

20. The recombinant microorganism of any one of claims 1-19, further comprising a mutation of a gene encoding an enzyme of the mannose biosynthesis pathway.

21. The recombinant microorganism of claim 20, wherein the enzyme of the mannose biosynthesis pathway is mannose-6-phosphate isomerase (ManA).

22. The recombinant microorganism of any one of claims 1-21, further comprising a mutation of a gene encoding an enzyme of glycolysis.

23. The recombinant microorganism of claim 22, wherein the enzyme of glycolysis is selected from phosphofructokinase A (PfkA), phosphofructokinase B (PfkB), fructose-biphosphate aldolase, triosephosphate isomerase, glyceraldehyde-3 -phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, pyruvate kinase, or a combination thereof.

24. The recombinant microorganism of claim 22 or 23, wherein the enzyme of glycolysis is phosphofructokinase A (PfkA), phosphofructokinase B (PfkB), or a combination thereof.

25. The recombinant microorganism of any one of claims 1-24, further comprising a mutation of a gene encoding an enzyme of D-tagatose biosynthesis.

26. The recombinant microorganism of claim 25, wherein the enzyme of D-tagatose biosynthesis is D-tagatose- 1,6-bisphosphate aldolase subunit GatZ (GatZ).

27. The recombinant microorganism of any one of claims 1 -26, further comprising a mutation of a gene encoding an enzyme of allulose biosynthesis.

28. The recombinant microorganism of claim 27, wherein the enzyme of allulose biosynthesis is D-allulose-6-phosphate 3-epimerase (AlsE).

29. The recombinant microorganism of any one of claims 1-28, wherein the recombinant microorganism further comprises exogenous galactose:H+symporter (GalP) and glucokinase (Glk).

30. The recombinant microorganism of any one of claims 1-29, wherein at least one exogenous oxidoreductase and / or at least one exogenous phosphatase are expressed by a stationary phase promoter or an inducible promoter.

31. The recombinant microorganism of any one of claims 1-30, wherein at least one exogenous oxidoreductase and / or at least one exogenous phosphatase are expressed by a stationary phase promoter.

32. The recombinant microorganism of 30 or 31, wherein the stationary phase promoter is PgadB.

33. A recombinant microorganism that produces an increased amount of an L-sorbose, D- fructose, D-sorbitol and / or D-sedoheptulose as compared to a naturally occurring microorganism, wherein the recombinant microorganism comprises a recombinant polynucleotide encoding at least one exogenous oxidoreductase and at least one exogenous phosphatase.

34. The recombinant microorganism of claim 33, wherein the at least one exogenous oxidoreductase is a sorbitol-6-phosphate dehydrogenase (SorD / SrlD), a L-sorbose 1- phosphate reductase (SorE), or a combination thereof.

35. The recombinant microorganism of claim 33 or 34, wherein the phosphatase is a hexitol phosphatase B (HxpB), a fructose- 1 -phosphate phosphatase (YqaB), a sugar phosphatase YbiV (YbiV), ), Hexitol phosphatase A (HxpA), sugar phosphatase YidA, phosphosugar phosphatase YigL, sugar phosphatase YihXor a combination thereof.

36. The recombinant microorganism of any one of claims 33-35, further comprising a mutation of a gene encoding an enzyme of the pentose phosphate pathway, a mutation of a gene encoding an enzyme of glycogen biosynthesis, a mutation of a gene encoding an enzyme of the mannose biosynthesis pathway, a mutation of a gene encoding an enzymeof glycolysis, a mutation of a gene encoding an enzyme of D-tagatose biosynthesis, a mutation of a gene encoding an enzyme of allulose biosynthesis, or a combination thereof.

37. The recombinant microorganism of claim 36, wherein the enzyme of the pentose phosphate pathway is glucose-6-phosphate dehydrogenase (Zwf).

38. The recombinant microorganism of claim 36, wherein the enzyme of glycogen biosynthesis is phosphoglucomutase (Pgm).

39. The recombinant microorganism of claim 36, wherein the enzyme of the mannose biosynthesis pathway is mannose-6-phosphate isomerase (ManA).

40. The recombinant microorganism of claim 36, wherein the enzyme of glycolysis is phosphofructokinase A (PfkA), phosphofructokinase B (PfkB), or a combination thereof.

41. The recombinant microorganism of claim 36, wherein the enzyme of D-tagatose biosynthesis is D-tagatose- 1,6-bisphosphate aldolase subunit GatZ (GatZ).

42. The recombinant microorganism of claim 36, wherein the enzyme of allulose biosynthesis is D-allulose-6-phosphate 3-epimerase (AlsE).

43. The recombinant microorganism of any one of claims 33-42, wherein the recombinant microorganism further comprises exogenous galactose:H+symporter (GalP) and glucokinase (Glk).

44. The recombinant microorganism of any one of claims 33-43, wherein recombinant polynucleotide comprises a stationary phase promoter or an inducible promoter.

45. The recombinant microorganism of 44, wherein the stationary phase promoter is PgadB.

46. The recombinant microorganism of any one of claims 16-32 and 36-45, wherein the mutation is a deletion.

47. The recombinant microorganisms of claim 46, wherein the mutation reduces or eliminates expression or activity of the enzyme.

48. The recombinant microorganism of any one of claims 1-47, wherein the microorganism is E. colt, Bacillus suhiilis. or Lactococcus lactis.

49. A method for producing L-sorbose, D-fructose, D-sorbitol and / or D-sedoheptulose comprising culturing the microorganism of any one of claims 1-47 under conditions suitable for converting a substrate to L-sorbose, D-fructose, D-sorbitol and / or D- sedoheptulose.

50. The method of claim 49, wherein the substrate comprises D-glucose.

51. A method of making a food product comprising an L-sorbose, D-fructose, D-sorbitol and / or D-sedoheptulose comprising:a) culturing the microorganism of any one of claims 1-47 under conditions suitable for converting a substrate to L-sorbose, D-fructose, D-sorbitol and / or D-sedoheptulose; and b) admixing the L-sorbose with one or more foods to form a food comprising the L- sorbose, D-fructose, D-sorbitol and / or D-sedoheptulose.

52. The method of claim 51 , wherein the substrate comprises D-glucose.

53. The method of claim 51 or 52, wherein the food product is a beverage, yogurt, ice cream, a baked good, or a nutritional bar.

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