Microorganisms used to produce allosugar

Abstract

This disclosure relates to microorganisms that can be used for the biosynthesis of D-allose. Methods for producing the disclosed microorganisms and for producing D-allose are also provided.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 537,136, filed September 7, 2023, the contents of which are incorporated herein by reference in their entirety, and this application claims priority to that U.S. Provisional Patent Application Serial No. 63 / 537,136.

[0003] sequence list

[0004] This application contains a sequence list, which has been electronically submitted in XML format and is incorporated herein by reference in its entirety. The XML copy was created on September 4, 2024, named 081906-1460211_253410PC_SL, and is 248,312 bytes in size. Technical Field

[0005] The subject matter of this disclosure relates to compositions and methods for producing low-calorie sugars in microorganisms. The subject matter of this disclosure also relates to compositions and methods for producing allose in microorganisms.

[0006] background

[0007] Current industrial production of D-allose involves challenging, multi-step in vitro enzymatic synthesis. These methods include isomerization of allulose to allose via L-rhamnose isomerase (RI) (EC 5.3.1.14), ribose-5-phosphate isomerase (RpI) (EC 5.3.1.6), or D-glucose isomerase (EC 5.3.1.5), or isomerization of allulose-6-phosphate to allose-6-phosphate via RpI. The main drawbacks of these methods include the need for multiple separation and purification processes, the use of thermodynamically unfavorable reactions, and the addition of expensive cofactors and other enzymes. Therefore, new methods and compositions for the production of D-allose are needed.

[0008] Overview

[0009] On the one hand, this disclosure relates to recombinant microorganisms containing exogenous epimerases, exogenous isomerases and exogenous phosphatases, wherein the recombinant microorganisms produce an increased amount of allose compared to naturally occurring microorganisms.

[0010] In some embodiments, the epimerase is allulose-6-phosphate 3-epomerase (AlsE). In some embodiments, the epimerase is *E. coli* AlsE. In some embodiments, the epimerase comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the epimerase comprises the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the epimerase consists of the amino acid sequence shown in SEQ ID NO: 1.

[0011] In some embodiments, the phosphatase is hexitol phosphatase A (HxpA). In some embodiments, the phosphatase is *E. coli* HxpA. In some embodiments, the phosphatase comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO: 62. In some embodiments, the phosphatase comprises the amino acid sequence shown in SEQ ID NO: 62. In some embodiments, the phosphatase consists of the amino acid sequence shown in SEQ ID NO: 62. In some embodiments, the phosphatase comprises an adenine-thymine-guanine (ATG) start codon.

[0012] In some embodiments, the exogenous phosphatase is fructose-1-phosphate phosphatase (YqaB). In some embodiments, the recombinant microorganism is a bacterium. In some embodiments, the bacterium is *Escherichia coli*. In some embodiments, the phosphatase comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, the phosphatase comprises the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, the phosphatase consists of the amino acid sequence shown in SEQ ID NO: 60.

[0013] In some embodiments, the recombinant microorganism further comprises an exogenous isomerase. In some embodiments, the isomerase is *Escherichia coli* RpiB. In some embodiments, the isomerase comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO: 24. In some embodiments, the isomerase comprises the amino acid sequence shown in SEQ ID NO: 24. In some embodiments, the isomerase consists of the amino acid sequence shown in SEQ ID NO: 24.

[0014] In some implementations, the recombinant microorganisms also contain exogenous galactose:H +The recombinant microorganism contains a symporter (GalP) and a glucokinase (Glk). In some embodiments, GalP is *E. coli* GalP and Glk is *E. coli* Glk. In some embodiments, GalP contains at least about 80% of the amino acid sequence identical to the amino acid sequence shown in SEQ ID NO: 38 and Glk contains at least about 80% of the amino acid sequence identical to the amino acid sequence shown in SEQ ID NO: 40. In some embodiments, GalP contains the amino acid sequence shown in SEQ ID NO: 38 and Glk contains the amino acid sequence shown in SEQ ID NO: 40. In some embodiments, the recombinant microorganism also contains an exogenous glucose-6-phosphate isomerase (Gpi).

[0015] In some embodiments, the recombinant microorganisms also contain mutations in the genes encoding enzymes of the pentose phosphate pathway compared to naturally occurring microorganisms. In some embodiments, the enzyme of the pentose phosphate pathway is glucose-6-phosphate 1-dehydrogenase (Zwf).

[0016] In some embodiments, the recombinant microorganisms also contain mutations in genes encoding glycolytic enzymes compared to naturally occurring microorganisms. In some embodiments, the glycolytic enzymes are phosphofructokinase-1 (PfkA), phosphofructokinase-2 (PfkB), or pyruvate kinase (PykF). In some embodiments, the glycolytic enzyme is phosphofructokinase-1 (PfkA).

[0017] In some embodiments, the recombinant microorganism also contains a mutation in the gene encoding an aldolase. In some embodiments, the aldolase is deoxyribose-phosphoaldolase (DeoC).

[0018] In some embodiments, the recombinant microorganism further comprises a mutation in at least one gene encoding a phosphatase. In some embodiments, the at least one gene encoding a phosphatase is selected from the genes of fructose-1-phosphate phosphatase (YqaB), sugar phosphatase YbiV, hexitol phosphatase B (HxpB), sugar phosphatase YidA, or combinations thereof.

[0019] In some embodiments, the recombinant microorganism further comprises a mutation in a gene encoding at least one enzyme of allosugar metabolism. In some embodiments, the gene encoding at least one enzyme of allosugar metabolism is selected from genes of D-allosugar input ATP-binding protein (AlsA), D-allosugar-binding periplasmic protein (AlsB), D-allosugar transport system permease protein (AlsC), D-allosugar kinase (AlsK), HTH-type transcriptional regulator (AlsR), or combinations thereof.

[0020] In some embodiments, the recombinant microorganism also contains a mutation in the gene encoding an enzyme in the mannose biosynthesis pathway. In some embodiments, the enzyme in the mannose biosynthesis pathway is mannose-6-phosphate isomerase (ManA).

[0021] In some implementations, the recombinant microorganism also contains mutations in the genes encoding enzymes of the phosphotransferase system (PTS).

[0022] In some embodiments, the microorganism also contains a mutation in the gene encoding an enzyme for glycogen biosynthesis, said enzyme being selected from phosphoglucomutase (Pgm), UDP-glucose pyrophosphorylase, glycogen synthase, glycogen branching enzyme, and glycogen protein. In some embodiments, the enzyme for glycogen biosynthesis is phosphoglucomutase (Pgm).

[0023] In some embodiments, the exogenous epimerase, exogenous isomerase, and exogenous phosphatase are expressed by a stationary promoter. In some embodiments, the exogenous epimerase, exogenous isomerase, and exogenous phosphatase are expressed by an inducible promoter.

[0024] On the other hand, this disclosure relates to microorganisms containing recombinant polynucleotides encoding epimerases, isomerases, and phosphatases, wherein the expression of epimerases, isomerases, and phosphatases leads to increased allose production compared to microorganisms lacking recombinant polynucleotides.

[0025] In some embodiments, the epimerase is allulose-6-phosphate 3-epomerase (AlsE). In some embodiments, the epimerase is *E. coli* AlsE. In some embodiments, the epimerase comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the epimerase comprises the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the epimerase consists of the amino acid sequence shown in SEQ ID NO: 1.

[0026] In some embodiments, the phosphatase is hexitol phosphatase A (HxpA). In some embodiments, the phosphatase is *E. coli* HxpA. In some embodiments, the phosphatase comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO: 62. In some embodiments, the phosphatase comprises the amino acid sequence shown in SEQ ID NO: 62. In some embodiments, the phosphatase consists of the amino acid sequence shown in SEQ ID NO: 62. In some embodiments, the phosphatase comprises an adenine-thymine-guanine (ATG) start codon.

[0027] In some embodiments, the phosphatase is fructose-1-phosphate phosphatase (YqaB). In some embodiments, the phosphatase is *Escherichia coli* YqaB. In some embodiments, the phosphatase comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, the phosphatase comprises the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, the phosphatase consists of the amino acid sequence shown in SEQ ID NO: 60.

[0028] In some embodiments, the recombinant microorganism further comprises an exogenous isomerase. In some embodiments, the isomerase is *Escherichia coli* RpiB. In some embodiments, the isomerase comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO: 24. In some embodiments, the isomerase comprises the amino acid sequence shown in SEQ ID NO: 24. In some embodiments, the isomerase consists of the amino acid sequence shown in SEQ ID NO: 24.

[0029] In some implementations, the recombinant microorganisms also contain exogenous galactose:H + The recombinant microorganism contains a cotransporter (GalP) and a glucokinase (Glk). In some embodiments, GalP is *E. coli* GalP and Glk is *E. coli* Glk. In some embodiments, GalP contains at least about 80% of the amino acid sequence identical to the amino acid sequence shown in SEQ ID NO: 38 and Glk contains at least about 80% of the amino acid sequence identical to the amino acid sequence shown in SEQ ID NO: 40. In some embodiments, GalP contains the amino acid sequence shown in SEQ ID NO: 38 and Glk contains the amino acid sequence shown in SEQ ID NO: 40. In some embodiments, the recombinant microorganism also contains an exogenous glucose-6-phosphate isomerase (Gpi).

[0030] In some embodiments, the microorganism also contains a mutation in the gene encoding an enzyme of the pentose phosphate pathway. In some embodiments, the enzyme of the pentose phosphate pathway is glucose-6-phosphate 1-dehydrogenase (Zwf). In some embodiments, the microorganism also contains a mutation in the gene encoding an enzyme of glycolysis. In some embodiments, the enzyme of glycolysis is phosphofructokinase-1 (PfkA), phosphofructokinase-2 (PfkB), or pyruvate kinase (PykF). In some embodiments, the enzyme of glycolysis is phosphofructokinase-1 (PfkA).

[0031] In some embodiments, the recombinant microorganism also contains a mutation in the gene encoding an aldolase. In some embodiments, the aldolase is deoxyribose-phosphoaldolase (DeoC).

[0032] In some embodiments, the recombinant microorganism further comprises a mutation in at least one gene encoding a phosphatase. In some embodiments, the at least one gene encoding a phosphatase is selected from the genes of fructose-1-phosphate phosphatase (YqaB), sugar phosphatase YbiV, hexitol phosphatase B (HxpB), sugar phosphatase (YidA), or combinations thereof.

[0033] In some embodiments, the recombinant microorganism further comprises a mutation in a gene encoding at least one enzyme of allosugar metabolism. In some embodiments, the gene encoding at least one enzyme of allosugar metabolism is selected from genes of D-allosugar input ATP-binding protein (AlsA), D-allosugar-binding periplasmic protein (AlsB), D-allosugar transport system permease protein (AlsC), D-allosugar kinase (AlsK), HTH-type transcriptional regulator (AlsR), or combinations thereof.

[0034] In some embodiments, the microorganism also contains mutations in the genes encoding enzymes of the mannose biosynthesis pathway. In some embodiments, the enzymes of the mannose biosynthesis pathway are mannose-6-phosphate isomerases (ManA).

[0035] In some implementations, the recombinant microorganism also contains mutations in the genes encoding enzymes of the phosphotransferase system (PTS).

[0036] In some embodiments, the microorganism also contains a mutation in the gene encoding an enzyme for glycogen biosynthesis, said enzyme being selected from phosphoglucomutase (Pgm), UDP-glucose pyrophosphorylase, glycogen synthase, glycogen branching enzyme, and glycogen protein. In some embodiments, the enzyme for glycogen biosynthesis is phosphoglucomutase (Pgm).

[0037] In some embodiments, the exogenous epimerase, exogenous isomerase, and exogenous phosphatase are expressed by a stationary promoter. In some embodiments, the exogenous epimerase, exogenous isomerase, and exogenous phosphatase are expressed by an inducible promoter.

[0038] On one hand, this disclosure relates to microorganisms comprising recombinant polynucleotides encoding epimerases, isomerases, and phosphatases; mutations in genes encoding enzymes of the pentose phosphate pathway; mutations in genes encoding enzymes of glycolysis; mutations in genes encoding aldolases; mutations in genes encoding enzymes of the mannose biosynthesis pathway; recombinant polynucleotides optionally encoding GalP, Glk, or both; recombinant polynucleotides optionally encoding Gpi; and mutations in genes optionally encoding phosphotransferase systems (PTS). In some embodiments, the microorganisms further comprise mutations in at least one enzyme encoding a phosphatase. In some embodiments, the microorganisms further comprise mutations in at least one allose-metabolizing enzyme.

[0039] On the other hand, this disclosure relates to microorganisms comprising recombinant polynucleotides encoding allose-6-phosphate isomerase (RpiB) and hexitol phosphatase A (HxpA); mutations in glucose-6-phosphate 1-dehydrogenase (Zwf); mutations in phosphofructokinase-1 (PfkA); mutations in deoxyribose-phosphoaldolase (DeoC); mutations in mannose-6-phosphate isomerase (ManA); and optionally recombinant polynucleotides encoding GalP, Glk, or both. In some embodiments, the microorganism further comprises at least one mutation selected from the genes of fructose-1-phosphate phosphatase (YqaB), sugar phosphatase YbiV, hexitol phosphatase B (HxpB), sugar phosphatase (YidA), ATP-binding protein (AlsA), D-allose-binding periplasmic protein (AlsB), D-allose transport system permease protein (AlsC), D-allose kinase (AlsK), HTH-type transcription regulator (AlsR), or combinations thereof.

[0040] On the other hand, this disclosure relates to microorganisms comprising recombinant polynucleotides encoding allulose-6-phosphate 3-epimerase (AlsE), allulose-6-phosphate isomerase (RpiB), and fructose-1-phosphate phosphatase (YqaB); mutations in glucose-6-phosphate 1-dehydrogenase (Zwf); mutations in phosphofructokinase-1 (PfkA); mutations in deoxyribose-phosphoaldolase (DeoC); mutations in mannose-6-phosphate isomerase (ManA); recombinant polynucleotides optionally encoding GalP, Glk, or both; recombinant polynucleotides optionally encoding Gpi; and mutations in genes optionally encoding enzymes of a phosphotransferase system (PTS). In some embodiments, the microorganism further comprises a mutation of at least one gene selected from the following: sugar phosphatase YbiV, hexitol phosphatase B (HxpB), sugar phosphatase (YidA), ATP-binding protein (AlsA), D-allose-binding periplasmic protein (AlsB), D-allose transport system permease protein (AlsC), D-allose kinase (AlsK), HTH-type transcription regulator (AlsR), or a combination thereof.

[0041] Furthermore, this disclosure relates to microorganisms comprising recombinant polynucleotides encoding allulose-6-phosphate 3-epimerase (AlsE); recombinant polynucleotides encoding allulose-6-phosphate isomerase (RpiB); recombinant polynucleotides encoding hexitol phosphatase A (HxpA); mutations in glucose-6-phosphate 1-dehydrogenase (Zwf); mutations in phosphofructokinase-1 (PfkA); mutations in deoxyribose-phosphoaldolase (DeoC); mutations in mannose-6-phosphate isomerase (ManA); recombinant polynucleotides optionally encoding GalP, Glk, or both; recombinant polynucleotides optionally encoding Gpi; and mutations in genes optionally encoding enzymes of a phosphotransferase system (PTS). In some embodiments, the microorganism further comprises at least one mutation selected from the genes of fructose-1-phosphate phosphatase (YqaB), sugar phosphatase YbiV, hexitol phosphatase B (HxpB), sugar phosphatase (YidA), ATP-binding protein (AlsA), D-allose-binding periplasmic protein (AlsB), D-allose transport system permease protein (AlsC), D-allose kinase (AlsK), HTH-type transcription regulator (AlsR), or combinations thereof.

[0042] Furthermore, this disclosure relates to microorganisms comprising recombinant polynucleotides encoding allulose-6-phosphate 3-epimerase (AlsE); recombinant polynucleotides encoding allulose-6-phosphate isomerase (RpiB); recombinant polynucleotides encoding fructose-1-phosphate phosphatase (YqaB); mutations in glucose-6-phosphate 1-dehydrogenase (Zwf); mutations in phosphofructokinase-1 (PfkA); mutations in deoxyribose-phosphoaldolase (DeoC); mutations in mannose-6-phosphate isomerase (ManA); recombinant polynucleotides optionally encoding GalP, Glk, or both; recombinant polynucleotides optionally encoding Gpi; and mutations in genes optionally encoding enzymes of a phosphotransferase system (PTS). In some embodiments, the microorganism further comprises a mutation of at least one gene selected from the following: sugar phosphatase YbiV, hexitol phosphatase B (HxpB), sugar phosphatase (YidA), ATP-binding protein (AlsA), D-allose-binding periplasmic protein (AlsB), D-allose transport system permease protein (AlsC), D-allose kinase (AlsK), HTH-type transcription regulator (AlsR), or a combination thereof.

[0043] In some implementations, the mutation is a deletion. In some implementations, the mutation reduces or eliminates the expression or activity of the enzyme. In some implementations, the microorganism is *Escherichia coli*, *Bacillus subtilis*, etc. Bacillus subtilis ) or Lactococcus lactis ( Lactococcus lactis ).

[0044] On the one hand, this disclosure also relates to a method for producing allose, which includes culturing the microorganisms disclosed herein under conditions suitable for converting a substrate into allose. In some embodiments, the substrate includes glucose.

[0045] Furthermore, this disclosure relates to a method for preparing a food product containing allosugar, the method comprising culturing the microorganisms disclosed herein under conditions suitable for converting a substrate into allosugar; and mixing allosugar with one or more food products to form a food product containing allosugar. In some embodiments, the food product is a beverage, yogurt, ice cream, baked goods, or nutrition bars. Brief description of the attached diagram

[0047] Figure 1A and Figure 1B The pathway for the biosynthesis of allosugar is illustrated. Glucose is introduced and phosphorylated to glucose-6-phosphate (G6P) via a phosphotransferase system (PTS) or GalP / Glk. G6P is then isomerized to fructose-6-phosphate (F6P) by glucose-6-phosphate isomerase. F6P is then epimerized to allulose-6-phosphate (P6P) by D-allulose-6-phosphate 3-epimerase (AlsE). P6P is then isomerized to allosugar-6-phosphate (A6P) by allosugar-6-phosphate isomerase (RpiB), and then dephosphorylated to free allosugar by a native phosphatase such as hexitol phosphatase A (HxpA). Finally, the free allosugar can diffuse across the cell membrane into the supernatant. Competitive pathways include the pentose phosphate pathway catalyzed by glucose-6-phosphate dehydrogenase (Zwf), glycolysis catalyzed by phosphofructokinases A and B (PfkA and B), and mannose biosynthesis catalyzed by mannose-6-phosphate isomerase (ManA). Other competitive pathways include glycogen biosynthesis catalyzed by phosphoglucose mutase (Pgm), alloose degradation catalyzed by DeoC, and alloose input via alloose transporter / phosphorylation mechanisms (AlsA, AlsB, AlsC, and AlsK).

[0048] Figure 2A-2C The biosynthetic strategy of D-allose is shown. Figure 2A The current method for D-allose production is shown, which results in a limited yield (approximately 50%) due to the overall positive ΔG'°. Figure 2B The proposed D-allose biosynthesis pathway was demonstrated, in which the dephosphorylation step, at a 1 mM cellular reactant concentration, is affected by a large negative ΔG'. m And it is thermodynamically driven to produce. Figure 2CThe pathway for the biosynthesis of D-allose in *E. coli* is shown. Deleted steps are shown in blue. Overexpressed steps are shown in red. PTS, phosphotransferase system; AlsE, D-allose-6-phosphate-3-epimerase; RpiB, D-ribose-6-phosphate-isomerase B; HxpA, hexitol phosphatase A.

[0049] Figure 3 Screening for D-allose-6-phosphate phosphatase in *E. coli* was demonstrated. Cells were incubated at 37°C with 10 g L... -1 Glucose was grown to OD in M9P medium 600 ~0.4, and then grown at 30°C for 24 h. At OD 600 When the concentration is ~0.4, add 1 mM IPTG. Test P LlacO1 D-allose production of various sugar phosphatase genes containing alsE and rpiB (Table 5). (-) indicates no additional expression of the phosphatase gene. Error bars indicate sd. (n = 3 biological replicates).

[0050] Figure 4 The effect of gene deletion on D-allose production was shown. Cells were incubated at 37°C in a solution containing 10 g / L... -1 Glucose was grown to OD in M9P medium 600 ~0.4, and then grown at 30°C for 24 h. At OD 600 When the concentration is ~0.4, add 1 mM MIPTG. alsE , rpiB The operator of hxpA in P LlacO1 The expression was performed using pAL2310 (Table 4). Yield (%) was calculated based on the probability that 1 mol of D-allose could be produced for every 1 mol of D-glucose consumed, leading to 100% of the theoretical maximum yield. Error bars indicate SD. (n = 3 biological replicates).

[0051] Figure 5 The fusion of AlsE and RpiB was shown to produce D-allose. Cells were incubated at 37°C with a solution containing 10 g / L... -1 Glucose was grown to OD in M9P medium 600 ~0.4, and then grown at 30°C for 24 h. At OD 600 When the concentration is ~0.4, add 1 mM IPTG. Peptide linkage. alsE - rpiB and hxpA The operator in P LlacO1 (pAL2562-pAL2565, Table 5) were expressed in AL4387 (Table 1). The peptide linker was (Gly-Ser-Gly).n (such as “(Gly-Ser-Gly)3” disclosed in SEQ ID NO: 79) or (Gly-Gly-Gly-Gly-Ser) n (n=1, 3) (consisting of “(Gly-Gly-Gly-Gly-Ser)1” and “(Gly-Gly-Gly-Gly-Ser)3” as disclosed in SEQ ID NO: 80 and 81, respectively). Error bars indicate sd. (n = 3 biological replicates).

[0052] Figure 6 This demonstrates the regulation of gene expression in the D-allose pathway. Cells were incubated at 37°C with a solution containing 10 g / L... -1 Glucose was grown to OD in M9P medium 600 ~0.4, and then grown at 30°C for 24 h. At OD 600 When the concentration is ~0.4, add 1 mM IPTG for P LlacO1 Components. Genes alsE , rpiB and hxpA In P LlacO1 or P gadB The expression of genes differs depending on their order. hxpA instruct hxpA The gene start codon has been changed from GTG to ATG. These plasmids (pAL2580, pAL2581, pAL2609, pAL2623, and pAL2624 (Table 5)) were transformed into AL4387 (Table 4). Error bars indicate sd. (n = 3 biological replicates).

[0053] Figures 7A-7B The study showed how supplementing glucose input with GalP and Glk and regulating the allulose:allose production ratio were achieved. Figure 7A Shown in P LlacO1 The following expression galP and glk Gene (pAL2264, Table 5). pAL2310, pAL2609, pAL2623, and pAL2624 (Table 2) were transformed into AL4387 along with pAL2264. Cells were then subjected to a treatment with 40 g / L... -1 Glucose was grown to OD in M9P medium 600 ~0.4, then grown at 30℃ for 24 h. At OD 600 When the concentration is ~0.4, add 1 mM IPTG to induce P. LlacO1Plasmid. Based on the probability that 1 mol of D-allulose can be produced for every 1 mol of D-glucose consumed, the yield (%) was calculated, leading to the theoretical maximum yield of 100%. Specific potency (g) -1 L -1 OD 600 -1 Indicator valence / final OD 600 Error bars indicate sd. (n = 3 biological replicates). Figure 7B This shows the P in AL4387. LlacO1 Genes expressed under pAL2310 and pAL2313 (Table 5, respectively) alsE, rpiB and hxpA or yqaB Cells were grown at 37°C in M9P medium containing 10 g L⁻¹ glucose until OD₀. 600 ~0.4, and then grown at 30°C for 24 h. At OD 600 When the value is ~0.4, add 1 mM IPTG. The error bar indicates sd. (n = 3 biological replicates).

[0054] Figures 8A-8C High cell density D-allose production was observed. Cultures of this strain were incubated at 37°C in a 40 g / L container. -1 Glucose was grown in M9P medium until OD 600 ~1, then induced with 1 mM IPTG and allowed to regrow for 30 minutes. The culture was then centrifuged and resuspended in a solution containing 40 g L. -1 glucose and 1 mM IPTG in M9P medium to OD 600 ~10, and grown at 30℃. Every 24 h, 10% of the culture volume was removed and replaced with a solution containing 400 g L. -1 Replace the medium with M9P medium containing glucose and 1 mM IPTG. Figure 8A It showed that it contained P within 120 hours gadB : rpiB-alsE-hxpA and P LlacO1 : galP- glk Production of D-allose and D-allulose in strain 9 (AL4387, Table 4) (pAL2609 and pAL2264, respectively, Table 5). Figure 8B The data shows the consumption of D-glucose over 120 hours. Figure 8C Display OD within 120 hours 600 Measurements. Error bars indicate sd. (n = 3 biological replicates).

[0055] Detailed Explanation

[0056] The market for rare sugars as food, dietary supplements, and health adjuvants is expanding. Among rare sugars, D-allose has attracted particular attention. However, current methods for producing D-allose are costly, inefficient, and thermodynamically unfavorable, limiting their potential for widespread application. Notably, this disclosure addresses several obstacles in D-allose production, including thermodynamic barriers, limited yields, the need for purification enzymes, and the addition of cofactors. Another important finding disclosed herein is that *E. coli* naturally possesses a thermodynamically favorable pathway for D-allose production, which allows for increased D-allose production by increasing the expression of the native gene and eliminating competing pathways without introducing heterologous genes. The subject matter of this disclosure facilitates the industrial-scale production of D-allose without requiring expensive enzyme purification or difficult separation of feedstocks and products.

[0057] This disclosure is based in part on the discovery that microorganisms containing specific genetic modifications (e.g., gene deletions) can be prepared to produce low-calorie sugars. In some embodiments, the low-calorie sugar is allosugar. For clarity and not as a limitation, the detailed description of the subject matter of this disclosure is divided into the following subsections: 1. Definition; 2. Microorganisms that produce allosugar; 3. Methods for producing and generating microorganisms; and 4. Methods for producing allosugar.

[0058] 1. Definition

[0059] The terms used in this specification generally have their ordinary meaning in the art, both in the context of the invention and in the specific context in which 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 they are prepared and used.

[0060] As used herein, the words “a” or “an” may mean “one” when used in conjunction with the term “comprising” in the claims and / or specification, but they are also consistent with the meanings of “one or more”, “at least one” and “one or more”.

[0061] The term "about" or "approximately" means within an acceptable range of error for a particular value as determined by a person skilled 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, according to practice in the art, "about" may mean within three or more standard deviations. Alternatively, "about" may mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly for biological systems or processes, the term may mean within orders of magnitude of the value, preferably within 5 times, and more preferably within 2 times.

[0062] As used herein, the terms “comprise(s)”, “include(s)”, “having”, “has”, “may”, “contain”, and variations thereof are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional actions or structures. This disclosure also contemplates other embodiments that “comprising”, “compose” of, and “substantially” consist of the embodiments or elements presented herein, whether or not explicitly stated.

[0063] As used herein, the term "microorganism" refers to any organism existing as a microscopic cell, contained within the domains Archaea, Bacteria, or Eukaryotes, the latter including yeasts and filamentous fungi, protozoa, algae, or higher protozoa. In some embodiments, the term includes prokaryotic or eukaryotic cells or organisms having a microscopic size, including but not limited to bacteria, archaea, and eubacteria of all species, as well as eukaryotic microorganisms such as yeasts and fungi. In some embodiments, the term microorganism includes cells that can be cultured for the production of chemicals (e.g., sugars). In some embodiments, the microorganism is a prokaryotic microorganism. In some embodiments, the prokaryotic microorganism is a bacterium.

[0064] As used herein, the terms “bacterium,” “bacteria,” or “eubacteria” refer to the domain of prokaryotes. In some embodiments, bacteria include Gram-negative bacteria, Gram-positive bacteria, Proteobacteria, cyanobacteria, spirochetes and related species, planctomyces, bacteroides, chlamydia, green sulfur bacteria, green non-sulfur bacteria, radioresistant micrococci, and thermococci, as well as Thermosiphothermophiles.

[0065] As used herein, the term "Gram-negative bacteria" includes cocci, non-enterobacteria, and enterobacteria. Genus Gram-negative bacteria include, for example, but not limited to, *Neisseria* spp. (…). Neisseria Spirulina ( ) Spirillum Pasteurella ( ) Pasteurella Brucella ( ) Brucella Yersinia spp. Yersinia ), Francisella genus ( Francisella Haemophilus spp. Haemophilus ), Bordetella spp. Bordetella ), Escherichia coli ( Escherichia Salmonella ( Salmonella ), Shigella genus ( Shigella ), Klebsiella spp. Klebsiella ), Proteus spp. Proteus ), Vibrio ( Vibrio ), Pseudomonas spp. Pseudomonas ), Bacteroides ( Bacteroides Acetobacter spp. Acetobacter Aerobacterium spp. Aerobacter ), Agrobacterium ( Agrobacterium ), nitrogen-fixing bacteria ( Azotobacter ), Spirulina ( Spirilla ), Serratia ( Serratia ), Vibrio ( Vibrio Rhizobium ( ) Rhizobium Chlamydia ( ) Chlamydia ), Rickettsia species ( Rickettsia ), genus *Treponema* Treponema ) and Fusobacterium genus ( Fusobacterium ).

[0066] As used herein, the term "Gram-positive bacteria" includes cocci, nonsporulating rods, and sporulating rods. Genus Gram-positive bacteria includes, for example, but not limited to, *Actinomyces*. Actinomyces ), Bacillus spp. Bacillus Clostridium ( Clostridium Corynebacterium spp. Corynebacterium ), Erysipelothrix ( Erysipelothrix Lactobacillus () Lactobacillus Listeria ( ) Listeria ), Mycobacterium ( Mycobacterium ), Myxococcus ( Myxococcus Nocardia ( ) Nocardia Staphylococcus spp. Staphylococcus Streptococcus spp. Streptococcus ) and Streptomyces ( Streptomyces ).

[0067] As used in this article, the term "recombinant microorganism" refers to a microorganism containing one or more recombinant polynucleotides.

[0068] As used herein, the term "exogenous" refers to molecules that are not naturally occurring in yeast, bacteria, organisms, microorganisms, or cells given in nature, and / or molecules that are not naturally produced by yeast, bacteria, organisms, microorganisms, or cells given in nature. The term "endogenous" as used herein refers to molecules that are naturally occurring in yeast, bacteria, organisms, microorganisms, or cells given in nature, and / or molecules that are naturally produced by yeast, bacteria, organisms, microorganisms, or cells given in nature.

[0069] As used herein, the terms “nucleic acid molecule,” “nucleotide sequence,” or “polynucleotide” refer to a sequence of single-stranded or double-stranded covalently linked nucleotides, wherein the 3' and 5' ends of each nucleotide are linked by a phosphodiester bond. Nucleic acid molecules may comprise deoxyribonucleotide or ribonucleotide bases and may be synthesized in vitro or isolated from natural sources.

[0070] As used herein, "recombinant polynucleotide" refers to a polynucleotide in which the exact nucleotide sequence is foreign for a given host (i.e., not naturally present in the given host). In some embodiments, the recombinant polynucleotide sequence is naturally present in a given host, but in a non-natural (e.g., greater or less than expected) amount, or additionally, if the polynucleotide sequence contains two or more subsequences that do not exist in nature in the same relationship to each other. For example, but without limitation, a recombinant polynucleotide may have two or more sequences derived from unrelated polynucleotides or from endogenous nucleotides, which are arranged to form a new polynucleotide. In some embodiments, this disclosure provides for introducing recombinant polynucleotides into microorganisms, wherein the polynucleotide encodes a polypeptide that is not normally present in the microorganism. For the genome of the microorganism, the polynucleotide sequence encoding the polypeptide is recombinant or heterologous.

[0071] As used herein, "gene" refers to a DNA region (including exons and introns) that encodes a gene product, as well as all DNA regions that regulate the production of gene products, regardless of whether these regulatory sequences are adjacent to coding and / or transcriptional sequences. In some non-limiting embodiments, a gene includes promoter sequences, terminators, translation regulatory sequences (e.g., ribosome binding sites and internal ribosome entry sites), enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions.

[0072] The terms “polypeptide,” “peptide,” “amino acid sequence,” and “protein,” used interchangeably herein, refer to a molecule formed by the linkage of at least two amino acids. The linkage between one amino acid residue and the next is an amide bond, and is sometimes called a peptide bond. Polypeptides can be obtained by suitable methods known in the art, including isolation from natural sources, expression in recombinant expression systems, chemical synthesis, or enzymatic synthesis. The term may be applied to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, as well as to both naturally occurring and non-naturally occurring amino acid polymers.

[0073] As used herein, the term "amino acid" can refer to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, and those subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs and derivatives can refer to compounds having the same basic chemical structure as naturally occurring amino acids, i.e., carbon atoms bound to hydrogen, carboxyl groups, amino groups, and R groups, such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids. Non-limiting examples of amino acids include tryptophan, phenylalanine, histidine, glycine, cysteine, alanine, tyrosine, serine, methionine, asparagine, leucine, asparagine, threonine, isoleucine, proline, glutamic acid, aspartic acid, hydroxyproline, arginine, cystine, glutamine, lysine, valine, ornithine, taurine, and combinations thereof.

[0074] As used herein, the term “separation” refers to material removed from at least one component that is naturally associated with it (e.g., removed from its original environment).

[0075] As used herein, the terms “reduce” and “reduction” mean that an endpoint (e.g., enzyme activity, compound production, protein expression) is measurably reduced by at least about 10%, at least about 50%, at least about 75%, or at least about 90%. In some embodiments, the reduction can be from about 10% to about 100%.

[0076] As used herein, the terms “increase,” “elevate,” and “elevation” refer to a measurable increase of at least about 10%, at least about 50%, at least about 75%, or at least about 90% in an endpoint (e.g., enzyme activity, compound production, protein expression). In some embodiments, the increase can be from about 10% to about 100%. In some embodiments, the increase can be at least about 10-fold, about 100-fold, or about 1000-fold or more. In some embodiments, the increase can be about 100-fold or more, about 1000-fold or more, or about 10,000-fold or more.

[0077] As used herein, the term "isomerase" refers to any EC 5 enzyme that catalyzes a change in geometry or structure within a single molecule. Depending on the type of isomerization they catalyze, they may be classified as racemic or epimerases (EC 5.1 subclass); cis-trans isomerases (EC 5.2 subclass); intramolecular oxidoreductases (EC 5.3 subclass); intramolecular transferases (mutases) (EC 5.4 subclass); or intramolecular lyases (EC 5.5 subclass); other isomerases are classified in EC 5.99 subclass.

[0078] As used in this article, the term "epomerase" refers to a class of enzymes that catalyze the inversion of asymmetric groups in a substrate with several asymmetric centers.

[0079] As used herein, the term "dehydrogenase" refers to any enzyme that catalyzes the removal of hydrogen atoms (e.g., dehydrogenation) in biological reactions. Dehydrogenases are present in many biochemical pathways and participate in the electron transport chain reactions that drive cellular respiration. In some embodiments, dehydrogenases act in conjunction with hydrogen-accepting coenzymes NAD and FAD.

[0080] As used herein, the term "phosphatase" refers to a class of enzymes that catalyze the removal of phosphate groups from organic compounds. In some embodiments, phosphatases catalyze the removal of phosphate groups from sugars. In some embodiments, the sugar is a hexose.

[0081] As used in this article, the term "aldolase" refers to an EC4.1.2 subclass of enzymes that catalyze aldol condensation and its reversal (i.e., aldehyde lyases).

[0082] Techniques for determining the identity of nucleic acid and amino acid sequences are known in the art. Typically, such techniques involve determining the nucleotide sequence of a gene's mRNA and / or the amino acid sequence it encodes, and comparing these sequences to second nucleotide or amino acid sequences. Genomic sequences can also be determined and compared in this manner. Generally, identity refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence, respectively, between two polynucleotide or polypeptide sequences. Two or more sequences (polynucleotides or amino acids) can be compared by determining their percentage of identity. For both nucleic acid and amino acid sequences, the percentage of identity between two sequences is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence and multiplied by 100. Unless otherwise indicated, the percentage of identity between two sequences is determined when comparing and aligning the largest correspondences over a comparison window or specified region, as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters. See, for example, the NCBI website at ncbi.nlm.nih.gov / BLAST. For example, BLASTN and BLASTP can be used with the following default parameters: Genetic code = standard; Filter = none; Chain = both; Cutoff = 60; Expected value = 10; Matrix = BLOSUM62; Description = 50 sequences; Sort = high score; Database = non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these procedures can be found on the GenBank website.

[0083] A “mutation” in a gene can include, for example, nucleotide alterations, deletions of one or more nucleotides (which may include the entire coding sequence and / or promoters or other regulatory sequences), and insertions of one or more nucleotides, which can occur in the coding sequence of a gene or its regulatory components (e.g., the gene’s promoter). Mutations can also include, for example, mutations that reduce or eliminate function (e.g., nonsense mutations) and genomic alterations that reduce or knock out the expression of gene products.

[0084] As used herein, the terms "linker," "peptide linker," or "flexible peptide linker" refer to short polypeptide sequences, typically from 1 to 20 amino acid residues, that appear between polypeptides. Linkers are typically composed of flexible residues such as glycine and serine, allowing adjacent polypeptides to move freely relative to each other. Longer linkers are generally used when it is necessary to ensure that two adjacent domains do not spatially interfere with each other. Non-limiting examples of linkers include (Gly–Ser–Gly)n (such as "(Gly-Ser-Gly)3" disclosed in SEQ ID NO: 79) and (Gly-Gly-Gly-Gly-Ser)n, where n = 1 or 3 (such as "(Gly-Gly-Gly-Gly-Ser)1" and "(Gly-Gly-Gly-Gly-Ser)3" disclosed in SEQ ID NO: 80 and 81, respectively).

[0085] 2. Microorganisms that produce allosugar

[0086] This disclosure provides genetically engineered microorganisms. In some embodiments, the microorganisms of this disclosure can produce an increased amount of allosugar, for example, an increased amount compared to naturally occurring control microorganisms.

[0087] D-Allose is a natural but rare monosaccharide. It is an aldose with the same empirical formula as common monosaccharides such as glucose and fructose. It is epimeric with glucose at the 3-position. Its enantiomer, L-Allose, is unknown in nature but has been synthesized. D-Allose has the following formula: .

[0088] D-Allose has 80% the sweetness of sucrose (e.g., castor sugar), but it has a lower nutritional energy (e.g., calorie) value (Mooradian et al., Clinical nutrition eSPen 18(2017): 1-8.). Therefore, D-allose can be used as a substitute for sucrose and artificial sweeteners.

[0089] This disclosure provides genetically engineered microorganisms with increased allosugar production compared to naturally occurring microorganisms. Figure 1 illustrates the regulated biochemical pathways in an exemplary microorganism of this disclosure. In this exemplary microorganism, glucose is introduced and phosphorylated to glucose-6-phosphate (G6P) via a phosphotransferase system (PTS) or GalP / Glk. G6P is then isomerized to fructose-6-phosphate (F6P) by glucose-6-phosphate isomerase. F6P is then epimerized to allulose-6-phosphate (P6P) by D-allulose-6-phosphate 3-epimerase (AlsE). P6P is then isomerized to allosugar-6-phosphate (A6P) by allosugar-6-phosphate isomerase (RpiB), and then dephosphorylated to free allosugar by a natural phosphatase such as hexitol phosphatase A (HxpA). Finally, the free allosugar can diffuse across the cell membrane into the supernatant. Competitive pathways include the pentose phosphate pathway catalyzed by glucose-6-phosphate dehydrogenase (Zwf), glycolysis catalyzed by phosphofructokinases A and B (pfkA and B), and the mannose biosynthesis pathway catalyzed by mannose-6-phosphate isomerase (ManA).

[0090] Glucose can be introduced into cells and phosphorylated using a phosphotransferase system (PTS). In some embodiments, the microorganism contains mutations that reduce PTS activity. In some embodiments, the microorganism contains mutations in the genes encoding enzymes of the phosphotransferase system (PTS).

[0091] 2.1. Allosugar-producing enzyme

[0092] In some embodiments, the microorganisms of this disclosure comprise overexpression of at least one gene encoding an enzyme catalyzing a reaction for the production of allose. In some embodiments, the microorganisms of this disclosure comprise a recombinant polynucleotide encoding at least one enzyme catalyzing a reaction for the production of allose. In some embodiments, said enzyme is an epimerase, such as an epimerase that converts fructose-6-phosphate (F6P) to allulose-6-phosphate. Non-limiting examples of epimerases include D-allulose-6-phosphate 3-epomerase, methylmalonyl-CoA epimerase, UDP-galactose 4-epomerase, UDP-glucose 4-epomerase, UDP-glucuronide 4-epomerase, UDP-glucuronide 5'-epomerase, ribose-5-phosphate epimerase, GDP-mannose 3,5-epomerase, L-ribulose phosphate 4-epomerase, UDP-N -Acetylglucosamine 2-epomerase, UDP-N-acetylglucosamine 4-epomerase, UDP-galactose 4-epomerase, UDP-glucose 4-epomerase, UDP-glucuronide 4-epomerase, UDP-glucuronide 5'-epomerase, GDP-mannose 3,5-epomerase, methylmalonyl-CoA epimerase, ribose-5-phosphate epimerase, and UDP-N-acetylglucosamine 2-epomerase.

[0093] In some embodiments, the epimerase is D-allulose-6-phosphate 3-epomerase (AlsE) (UniProt No. P32719). AlsE catalyzes the reversible epimerization of D-allulose-6-phosphate to D-fructose-6-phosphate. In some embodiments, AlsE is *E. coli* AlsE. In some embodiments, AlsE comprises 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% of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, AlsE comprises the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, AlsE consists of the amino acid sequence shown in SEQ ID NO: 1. SEQ ID NO: 1 is provided below.

[0094] MKISPSLMCMDLLKFKEQIEFIDSHADYFHIDIMDGHFVPNLTLSPFFVSQVKKLATKPLDCHLMVTRPQDYIAQLARAGADFITLHPETINGQAFRLIDEIRRHDMKVGLILNP ETPVEAMKYYIHKADKITVMTVDPGFAGQPFIPEMLDKLAELKAWREREGLEYEIEVDGSCNQATYEKLMAAGADVFIVGTSGLFNHAENIDEAWRIMTAQILAAKSEVQPHAKTA [SEQ ID NO: 1]

[0095] In some implementations, genes alsE 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 shown in SEQ ID NO: 2. In some embodiments, alsE It contains the nucleotide sequence shown in SEQ ID NO: 2. In some embodiments, alsE It consists of the nucleotide sequence shown in SEQ ID NO: 2. SEQ ID NO: 2 is provided below.

[0096] ATGAAAATCTCCCTCGTTAATGTGTATGGATCTGCTGAAATTTAAAGAACAGATCGAATTTATCGACAGCCATGCCGATTACTTCCACATCGATATCATGGACGGTCACTTTGTCCCCAATCTGACACTCTCACCGTTCTTCGTAAGTCAGGTTAAAAAACTGGCAACTAAA CCGCTCGACTGTCATCTGATGGTGACGCGGCCGCAGGATTACATTGTCAACTGGCGCGTGCGGGAGCAGATTTCATCACTCTGCATCCGGAAACCATCAACGGCCAGGCGTTCCGCCTGATTGATGAAATCCGCCGTCATGACATGAAAGTGGGGCTGATCCTTAACCCGGAG ACGCCAGTTGAGGCCATGAAATACTATATCCATAAGGCCGATAAAATTACGTCATGACTGTCGATCCCGGCTTTGCCGGACAACCGTTCATTCCTGAAATGCTGGATAAACTTGCCGAACTGAAGGCATGGCGTGAACGAGAAGGTCTGGAGTACGAAATTGAGGTGGACGGT TCCTGCAACCAGGCAACTTACGAAAAACTGATGGCGGCAGGGGCGGATGTCTTTATCGTCGGCACTTCCGGCCTGTTTAATCATGCGGAAAATATCGACGAAGCATGGAGAATTATGACCGCGCAGATTCTGGCTGCAAAAAGCGAGGTACAGCCTCATGCAAAAACAGCATAA [SEQ ID NO: 2]

[0097] In some embodiments, the enzyme is an isomerase, such as an isomerase that catalyzes the conversion of allulose-6-phosphate to allose-6-phosphate. In some embodiments, the isomerase is allose-6-phosphate isomerase (RpiB) (Entrez gene ID: 948602). RpiB catalyzes the interconversion of ribulose-5-P and ribose-5-P, as well as the interconversion of D-allose-6-phosphate (All6P) and D-allulose-6-phosphate. In some embodiments, RpiB is *E. coli* RpiB. (Gene) rpiBThe representative nucleotide sequence is shown in SEQ ID NO: 8, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 8. SEQ ID NO: 8 is provided below.

[0098] ATGAAAAAGATTGCATTTGGCGTTTCATTTTAAAACATGAAATAGTGGCACATTTAGTTGAGCGTGGCGTTGAAGTGATTGATAAAGGAACCTGGTCGTCAGAGCGTACTGATTATCCACATTACGCCAGTCAAGTCGCACTGGCTGTTGCTGGCGGAGAGGTTGATGGCGGGATTTTGATTTGTGGTACTGGCGTCGGTATTTCGATAGCG GCGAACAAGTTTGCCGGAATTCGCGCGGTCGTCTGTAGCGAACCTTATTCCGCGCAACTTTCGCGGCAGCATAACGACACCAACGTGCTGGCTTTTGGTTCACGAGTGGTTGGCCTCGAACTGGCAAAAATGATTGTGGATGCGTGGCTGGGCGCACAGTACGAAGGCGGTCGTCATCAACAACGCGTGGAGGCGATTACGGCAATAGAGCAGCGGAGAAATTGA [SEQ ID NO: 8]

[0099] In some embodiments, *E. coli* RpiB comprises 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% of the amino acid sequence shown in SEQ ID NO: 24. In some embodiments, *E. coli* RpiB comprises the amino acid sequence shown in SEQ ID NO: 24. SEQ ID NO: 24 is provided below.

[0100] MKKIAFGCDHVGFILKHEIVAHLVERGVEVIDKGTWSSERTDYPHYASQVALAVAGGEVDGGILICGTGVGISIAANKFAGIRAVVCSEPYSAQLSRQHNDTNVLAFGSRVVGLELAKMIVDAWLGAQYEGGRHQQRVEAITAIEQRRN[SEQ ID NO: 24]

[0101] In some implementations, RpiB is a Bacillus subtilis gene. rpiB The representative amino acid sequence of RpiB is found in A0A6M4JQ63(Uniprot) / BSU36920(KEGG), or is shown in SEQ ID NO: 14, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 14. SEQ ID NO: 14 is provided below.

[0102] MKVAIASDHGGVHIRNEIKELMDELQIEYIDMGCDCGSGSVDYPDYAFPVAEKVVSGEVDRGILICGTGIGMSISANKVKGIRCALAHDTFSAKATREHNDTNILAMGERVIGPGLAREIAKIWLTTEFTGGRHQTRIGKISDYEEKNL(SEQ ID NO: 14)

[0103] Bacillus subtilis gene rpiB The representative nucleotide sequence is shown in SEQ ID NO: 28, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 28. SEQ ID NO: 28 is provided below.

[0104] atgaaagtagccattgcatcggatcatggcggcgttcacattcgaaatgaaatcaaagagttaatggacgaattgcaaattgaatatattgatatgggctgtgactgcggcag cggctctgtcgattatccggattatgcttttccggtggccgaaaaagtggttagcggcgaagttgacagaggcattttaatttgcgggacaggcatcggcatgagcatttccgc taataaagtaaaagggattcgctgcgcgctggcgcacgataccttcagcgcgaaggcgacgagggagcataatgacacaaacatccttgcgatgggtgaacgggtgatcggac ctggtttggctcgggaaatcgcaaaaatctggctgactactgagtttaccgggggaagacaccaaacgcgtattggaaaaatctccgattatgaagagaaaaacctgtag(SEQ ID NO: 28)

[0105] In some implementations, RpiB is *Lactococcus lactis* RpiB. (Gene) rpiB The representative amino acid sequence is found in LLA12_RS12460: ribose-5-phosphate isomerase, EC5.3.1.6, or is shown in SEQ ID NO: 15, or is at least 90%, 95%, 95% or 99% identical to SEQ ID NO: 15, which is provided below.

[0106] MDNLKKQVGIKAAEFVKSGMVVGLGTGSTAAYFVEELGRRIAEEQLEITGVTTSNVTSSQARALGIPLASIDEVDYVDLTVDGADEIDSSLNGIKGGGAALLMEKIVATYSKDY IWIVDESKLSENLGSFKIPVEVIPYGSQQVFKKFEAAGYAPTWRLNEENERLITDMHHFIIDLHISQIKEPEKLAEELDLMVGVVEHGLFNNMVKKVIVAGNEGVRIINK(SEQ ID NO: 15)

[0107] Lactococcus lactis gene rpiB The representative nucleotide sequence is shown in SEQ ID NO: 34, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 34. SEQ ID NO: 34 is provided below.

[0108] (SEQ ID NO:34)

[0109] In some embodiments, the enzyme is a phosphatase, such as a phosphatase that dephosphorylates allosugar-6-phosphate to free allosugar. In some embodiments, the phosphatase catalyzes the removal of a phosphate group from the sugar. In some embodiments, the sugar is a hexose.

[0110] In some embodiments, the phosphatase is hexitol phosphatase A (HxpA) (UniProt No. P77625). HxpA catalyzes the dephosphorylation of D-allose-6-phosphate. In some embodiments, HxpA is *Escherichia coli* HxpA. In some embodiments, HxpA comprises 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% of the amino acid sequence shown in SEQ ID NO: 62. In some embodiments, HxpA comprises the amino acid sequence shown in SEQ ID NO: 62. SEQ ID NO: 62 is provided below.

[0111] MRCKGFLFLDGTLVDSLPAVERAWSNWARRHHGLAPEEVLAFIHGKQAITSLRHFMAGKSEADIAAEFTRLEHIEATETEGITALPGAIALLSHLNKAGIPWAIVTSGSMP VARARHKIAGLPAPEVFVTAERVKRGKPEPDAYLLGAQLLGLAPQECVVVEDAPAGVLSGLAAGCHVIAVNAPADTPRLNEVDLVLHSLEQITVTKQPNGDVIIQ[SEQID NO: 62]

[0112] In some implementations, genes hxpA 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 shown in SEQ ID NO: 63. In some embodiments, hxpA It contains the nucleotide sequence shown in SEQ ID NO: 63. In some embodiments, hxpA It consists of the nucleotide sequence shown in SEQ ID NO: 63. SEQ ID NO: 63 is provided below.

[0113] GTGCGGTGCAAAGGTTTTCTGTTTGATCTTGATGGAACGCTGGTGGATTCCCTGCCTGCGGTAGAACGGGCGTGGAGCAACTGGGCCAGACGTCATGGGTTAGCGCCGGAAGAGGTGCTGGCTTTCATTCACGGTAAACAGGCGATCACCTCTCTGCGCCAT TTTATGGCGGGCAAATCCGAGGCTGATATTGCCGCCGAGTTTACGCGTCTGGAGCACATCGAGGCCACGGAAACCGAAGGTATTACCGCGCTTTCCGGGGGCAATCGCCTTACTCAGTCATTTGAATAAAGCAGGTATTCCGTGGGCCATTGTGACTTCTGGCT CCATGCCGGTAGCGCGAGCGCGCCATAAAATAGCTGGGCTTCCCGCACCAGAGGTGTTTGTAACCGCTGAGCGAGTGAAGCGCGGAAAACCAGAACCTGATGCGTATCTGTTAGGCGCGCAGCTGCTGGGGCTTGCGCCGCAGGAGTGTGTGGTGGTGGAAGA TGCTCCCGCTGGCGTGCTTTCTGGCCTGGCGGCGGGTTGTCATGTCATTGCGGTTAACGCTCCGGCAGATACCCCGCGCCTGAATGAGGTCGATTTGGTCCTCCACAGTCTGGAGCAAATTACTGTGACCAAACAGCCAAATGGCGATGTTATTATTCAGTGA [SEQ ID NO: 63]

[0114] In some implementations, genes hxpA It contains the guanine-thymine-guanine (GTG) start codon. In some implementations, the gene... hxpA It contains the adenine-thymine-guanine (ATG) start codon. In some implementations, the gene... hxpA Encoded by 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 nucleotide sequences to the nucleotide sequence shown in SEQ ID NO: 76. In some embodiments, hxpA It contains the nucleotide sequence shown in SEQ ID NO: 76. In some embodiments, hxpAComposed of the nucleotide sequence shown in SEQ ID NO: 76. SEQ ID NO: 76 is provided below.

[0115] ATGCGGTGCAAAGGTTTTCTGTTTGATCTTGATGGAACGCTGGTGGATTCCCTGCCTGCGGTAGAACGGGCGTGGAGCAACTGGGCCAGACGTCATGGGTTAGCGCCGGAAGAGGTGCTGGCTTTCATTCACGGTAAACAGGCGATCACCTCTCTGCGCCATTTTATGGCGGGCAAATCCGAGGCTGATATTGCCGCCGAGTTTACGCGTCTGGAGCACATCGAGGCCACGGAAACCGAAGGTATTACCGCGCTTCCGGGGGCAATCGCCTTACTCAGTCATTTGAATAAAGCAGGTATTCCGTGGGCCATTGTGACTTCTGGCTCCATGCCGGTAGCGCGAGCGCGCCATAAAATAGCTGGGCTTCCCGCACCAGAGGTGTTTGTAACCGCTGAGCGAGTGAAGCGCGGAAAACCAGAACCTGATGCGTATCTGTTAGGCGCGCAGCTGCTGGGGCTTGCGCCGCAGGAGTGTGTGGTGGTGGAAGATGCTCCCGCTGGCGTGCTTTCTGGCCTGGCGGCGGGTTGTCATGTCATTGCGGTTAACGCTCCGGCAGATACCCCGCGCCTGAATGAGGTCGATTTGGTCCTCCACAGTCTGGAGCAAATTACTGTGACCAAACAGCCAAATGGCGATGTTATTATTCAGTGA [SEQ ID NO: 76]

[0116] In some embodiments, the phosphatase is a sugar phosphatase, such as phosphatose-phosphatase (YigL), (UniProtP27848). In some embodiments, YigL is *E. coli* YigL. In some embodiments, YigL comprises 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% of the amino acid sequence shown in SEQ ID NO: 74. In some embodiments, YigL consists of the amino acid sequence shown in SEQ ID NO: 74. SEQ ID NO: 74 is provided below.

[0117] MYQVVASDLDGTLLSPDHTLSPYAKETLKLLTARGINFVFATGRHHVDVGQIRDNLEIKSYMITSNGARVHDLDGNLIFAHNLDRDIASDLFGVVNDNPDIITNVYRDDEWFMNRHRPEEMRFFKEAVFQYALYE PGLLEPEGVSKVFFTCDSHEQLLPLEQAINARWGDRVNVSFSTLTCLEVMAGGVSKGHALEAVAKKLGYSLKDCIAFGDGMNDAEMLSMAGKGCIMGSAHQRLKDLHPELEVIGTNADDAVPHYLRKLYLS[SEQ ID NO: 74]

[0118] In some implementations, genes yigL 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 shown in SEQ ID NO: 75. In some embodiments, yigL It contains the nucleotide sequence shown in SEQ ID NO: 75. In some embodiments, yigL It consists of the nucleotide sequence shown in SEQ ID NO: 75. SEQ ID NO: 75 is provided below.

[0119] ATGTACCAGGTTGTTGCGTCTGATTTAGATGGCACGTTACTTTCTCCCGACCATACGTTATCCCCTTACGCCAAAGAAACTCTGAAGCTGCTCACCGCGCGCGGCATCAACTTTGTGTTTGCGACCGGTCGTCACCACGTTGATGTGGGGCAAATTCGCGATAATCTGGAGATTAAGTCTTACATGATTACCTCCAATGG TGCGCGCGTTCACGATCTGGATGGTAATCTGATTTTTGCTCATAACCTGGATCGCGACATTGCCAGCGATCTGTTTGGCGTAGTCAACGACAATCCGGACATCATTACTAACGTTTATCGCGACGACGAATGGTTTATGAATCGCCATCGCCCGGAAGAGATGCGCTTTTTTAAAGAAGCGGTGTTCCAATATGCGCTGT ATGAGCCTGGATTACTGGAGCCGGAAGGCGTCAGCAAAGTGTTCTTCACCTGCGATTCCCATGAACAACTGCTGCCGCTGGAGCAGGCGATTAACGCTCGTTGGGGCGATCGCGTCAACGTCAGTTTCTCTACCTTAACCTGTCTGGAAGTGATGGCGGGCGGCGTTTCAAAAGGCCATGCGCTGGAAGCGGTGGCGAAG AAACTGGGCTACAGCCTGAAGGATTGTATTGCGTTTGGTGACGGGATGAACGACGCCGAAATGCTGTCGATGGCGGGGAAAGGCTGCATTATGGGCAGTGCGCACCAGCGTCTGAAAGACCTTCATCCCGAGCTGGAAGTGATTGGTACTAATGCCGACGACGCGGTGCCGCATTATCTGCGTAAACTCTATTTATCGTAA [SEQ ID NO: 75]

[0120] In some embodiments, the phosphatase is fructose-1-phosphate phosphatase (YqaB) (Entrez gene ID: 945776). YqaB catalyzes the dephosphorylation of allulose-6-phosphate. In some embodiments, YqaB is *E. coli* YqaB. (Gene) yqaBThe representative nucleotide sequence is shown in SEQ ID NO: 61 or SEQ ID NO: 80, or is at least 90%, 95%, 95% or 99% identical to SEQ ID NO: 61 or SEQ ID NO: 80, which is provided below.

[0121] In some embodiments, *E. coli* YqaB comprises 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% of the amino acid sequence shown in SEQ ID NO: 60. In some embodiments, *E. coli* YqaB comprises the amino acid sequence shown in SEQ ID NO: 60.

[0122] In some embodiments, one or more transport proteins that transport glucose into cells may be expressed (i.e., overexpressed) in the cells, thereby increasing glucose levels in the cells. In some embodiments, galactose:H is expressed in microorganisms. + One or both of the cotransporter (GalP) and glucokinase (Glk). For example, in some embodiments, GalP transports glucose into the cell, where it is phosphorylated to glucose-6-phosphate by Glack and assimilated into central carbon metabolism.

[0123] In some implementations, galactose:H + The cotransporter protein (GalP) is as described in EG12148 (EcoCyc) or POAEP1 (UniProt). In some embodiments, GalP is *E. coli* GalP. In some embodiments, GalP comprises 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% of the amino acid sequence shown in SEQ ID NO: 38. In some embodiments, GalP consists of the amino acid sequence shown in SEQ ID NO: 38. SEQ ID NO: 38 is provided below.

[0124] MPDAKKQGRSNKAMTFFVCFLAALAGLLFGLLDIGVIAGALPFIADEFQITSHTQEWVVSSMMFGAAVGAVGSGWLSFKLGRKKSLMIGAILFVAGSLFSAAAPNVEVLILSRVLLGL AVGVASYTAPLYLSEIAPEKIRGSMISMYQLMITIGILGAYLSDTAFSYTGAWRWMLGVIIIPAILLLIGVFFLPDSPRWFAAKRRFVDAERVLLRLRDTSAEAKRELDEIRESLQV KQSGWALFKENSNFRRAVFLGVLLQVMQQFTGMNVIMYYAPKIFELAGYTNTTEQMWGTVIVGLTNVLATFIAIGLVDRWGRKPTLTLGFLVMAAGMGVLGTMMHIGIHSPSAQYFA IAMLLMFIVGFAMSAGPLIWVLCSEIQPLKGRDFGITCSTATNWIANMIVGATFLTMLNTLGNANTFWVYAALNVLFILLTLWLVPETKHVSLEHIERNLMKGRKLREIGAHD(SEQ ID NO:38)

[0125] In some implementations, genes galP 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 shown in SEQ ID NO: 39. In some embodiments, galP It contains the nucleotide sequence shown in SEQ ID NO: 39. In some embodiments, galP It consists of the nucleotide sequence shown in SEQ ID NO: 39. SEQ ID NO: 39 is provided below.

[0126]

[0127] In some embodiments, glucokinase (Glk) is as described in EG12957 (EcoCyc) or POA6V8 (UniProt). In some embodiments, Glk is *E. coli* Glk. In some embodiments, Glk comprises 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% of the amino acid sequence shown in SEQ ID NO: 40. In some embodiments, Glk consists of the amino acid sequence shown in SEQ ID NO: 40. SEQ ID NO: 40 is provided below.

[0128] MTKYALVGDVGGTNARLALCDIASGEISQAKTYSGLDYPSLEAVIRVYLEEHKVEVKDGCIAIACPITGDWVAMTNHTWAFSIAEMKKNLGFSHLEIINDFTAVSMAIPMLKKEHLIQFGGAEPVEGKPIAVYGAGTLGGVAHLVHVDKRWVSLPGEGGHVD FAPNSEEEAIILEILRAEIGHVSAERVLSGPGLVNLYRAIVKADNRLPENLKPKDITERALADSCTDCRRALSLFCVIMGRFGGNLALNLGTFGGVFIAGGIVPRFLEFFKASGFRAAFEDKGRFKEYVHDIPVYLIVHDNPGLLGSGAHLRQTLGHIL(SEQ ID NO:40)

[0129] In some implementations, genes glk 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 shown in SEQ ID NO:41. In some embodiments, glk It contains the nucleotide sequence shown in SEQ ID NO: 41. In some embodiments, glk It consists of the nucleotide sequence shown in SEQ ID NO: 41. SEQ ID NO: 41 is provided below.

[0130] atgACAAAGTATGCATTAGTCGGTGATGTGGGCGGCACCAACGCACGTCTTGCTCTGTGTGATATTGCCAGTGGTGAAATCTCGCAGGCTAAGACCTATTCAGGGCTTGATTACCCCAGCCTCGAAGCGGTCATTCGCGTTTATCTTGAAGAACATAAGGTCGAGGTGAAAGACGGCTGTATTGCCATCGCTTGCCCAATTACCGGTGACTGGGTGGCGATGACCAACCATACCTGGGCGTTCTCAATTGCCGAAATGAAAAAGAATCTCGGTTTTAGCCATCTGGAAATTATTAACGATTTTACCGCTGTATCGATGGCGATCCCGATGCTGAAAAAAGAGCATCTGATTCAGTTTGGTGGCGCAGAACCGGTCGAAGGTAAGCCTATTGCGGTTTACGGTGCCGGAACGGGGCTTGGGGTTGCGCATCTGGTCCATGTCGATAAGCGTTGGGTAAGCTTGCCAGGCGAAGGCGGTCACGTTGATTTTGCGCCGAATAGTGAAGAAGAGGCCATTATCCTCGAAATATTGCGTGCGGAAATTGGTCATGTTTCGGCGGAGCGCGTGCTTTCTGGCCCTGGGCTGGTGAATTTGTATCGCGCAATTGTGAAAGCTGACAACCGCCTGCCAGAAAATCTCAAGCCAAAAGATATTACCGAACGCGCGCTGGCTGACAGCTGCACCGATTGCCGCCGCGCATTGTCGCTGTTTTGCGTCATTATGGGCCGTTTTGGCGGCAATCTGGCGCTCAATCTCGGGACATTTGGCGGCGTGTTTATTGCGGGCGGTATCGTGCCGCGCTTCCTTGAGTTCTTCAAAGCCTCCGGTTTCCGTGCCGCATTTGAAGATAAAGGGCGCTTTAAAGAATATGTCCATGATATTCCGGTGTATCTCATCGTCCATGACAATCCGGGCCTTCTCGGTTCCGGTGCACATTTACGCCAGACCTTAGGTCACATTCTGtaa(SEQ ID NO:41)

[0131] Unbound by any theory, the inventors of this disclosure believe that any enzyme performing a similar function to the enzymes described above can be used in the microorganisms of this disclosure. For example, but not limited to, the microorganisms of this disclosure may contain any enzyme that catalyzes the reversible epimerization of D-fructose-6-phosphate to D-allulose-6-phosphate. In another non-limiting example, the microorganisms of this disclosure may contain any enzyme that catalyzes the reversible isomerization of D-allulose-6-phosphate to D-allose-6-phosphate. In another non-limiting example, the microorganisms of this disclosure may contain any enzyme that dephosphates allose-6-phosphate to free allose.

[0132] 2.2. Competitive Approaches

[0133] In some embodiments, the microorganisms of this disclosure contain mutations in one or more genes encoding one or more enzymes that regulate biochemical pathways that reduce allose production. In some embodiments, the microorganisms of this disclosure contain reduced expression of genes encoding enzymes that regulate biochemical pathways that reduce allose production. Physiologically, cells catalyze the production of energy (e.g., ATP) from sugars via the pentose phosphate pathway and glycolysis. The inventors of this disclosure have found that the deletion or reduced expression of genes encoding enzymes in certain metabolic pathways leads to increased allose production.

[0134] In some embodiments, the microorganisms of this disclosure contain mutations in the genes encoding enzymes of the pentose phosphate pathway. In some embodiments, the microorganisms of this disclosure contain reduced expression of genes encoding enzymes of the pentose phosphate pathway. In some embodiments, the enzymes of the pentose phosphate pathway are selected from glucose-6-phosphate dehydrogenase, 6-phosphogluconolactonease, phosphoglucuronide dehydrogenase, pentose phosphate isomerase, pentose phosphate epimerase, transketolase, and transaldolase. In some embodiments, the enzyme of the pentose phosphate pathway is glucose-6-phosphate dehydrogenase (Zwf) (Entrez gene ID: 946370). Zwf catalyzes the oxidation of glucose-6-phosphate to 6-phosphogluconolactone. In some embodiments, Zwf is *Escherichia coli* Zwf. zwf The representative nucleotide sequence is shown in SEQ ID NO: 6, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 6. SEQ ID NO: 6 is provided below.

[0135]

[0136] In some embodiments, *E. coli* Zwf contains 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% of the amino acid sequence shown in SEQ ID NO: 20. In some embodiments, *E. coli* Zwf contains the amino acid sequence shown in SEQ ID NO: 20. SEQ ID NO: 20 is provided below.

[0137] MAVTQTAQACDLVIFGAKGDLARRKLLPSLYQLEKAGQLNPDTRIIGVGRADWDKAAYTKVVREALETFMKETIDEGLWDTLSARLDFCNLDVNDTAAFSRLGAMLDQKNRITINYFAMPPST FGAICKGLGEAKLNAKPARVVMEKPLGTSLATSQEINDQVGEYFEECQVYRIDHYLGKETVLNLLALRFANSLFVNNWDNRTIDHVEITVAEEVGIEGRWGYFDKAGQMRDMIQNHLLQILCMI AMSPPSDLSADSIRDEKVKVLKSLRRIDRSNVREKTVRGQYTAGFAQGKKVPGYLEEEGANKSSNTETFVAIRVDIDNWRWAGVPFYLRTGKRLPTKCSEVVVYFKTPELNLFKESWQDLPQNK LTIRLQPDEGVDIQVLNKVPGLDHKHNLQITKLDLSYSETFNQTHLADAYERLLLETMRGIQALFVRRDEVEEAWKWVDSITEAWAMDNDAPKPYQAGTWGPVASVAMITRDGRSWNEFE[SEQ ID NO: 20]

[0138] In some embodiments, Zwf is Bacillus subtilis Zwf. A representative amino acid sequence of Bacillus subtilis Zwf can be found in P54547(Uniprot) / BSU23850(KEGG) or as shown in SEQ ID NO: 10, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 10. SEQ ID NO: 10 is provided below.

[0139] MKTNQQPKAVIVIFGATGDLAKRKLYPSIHRLYQNGQIGEEFAVVGVGRRPWSNEDLRQTVKTSISSSADKHIDDFTSHFYYHPFDVTNPGSYQELNVLLNQLEDTYQIPNNRMFYLAMAPEF FGTIAKTLKSEGVTATTGWSRLVIEKPFGHDLPSAQALNKEIREAFTEDQIYRIDHYLGKQMVQNIEVIRFANAIFEPLWTNRYISNIQITSSESLGVEDRARYYEKSGALRDMVQNHIMQMV ALLAMEPPIKLNTEEIRSEKVKVLRALRPIAKDEVDEYFVRGQYHAGEIDGVPVPAYTDEDNVAPDSNTETFVAGKLLIDNFRWAGVPFYIRTGKRMKEKSTKIVVQFKDIPMNLYYGNENNM NPNLLVIHIQPDEGITLYLNAKKLGGAAHAQPIKLDYCSNCNDELNTPEAYEKLIHDCLLGDATNFAHWDEVALSWSFVDSISETWAANKTLSPNYESGSMGPKESDDLLVKDGLHWWNI(SEQ ID NO:10)

[0140] Bacillus subtilis zwf The representative nucleotide sequence of the gene is shown in SEQ ID NO: 26, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 26. SEQ ID NO: 26 is provided below.

[0141]

[0142] In some embodiments, Zwf is *Lactococcus lactis* Zwf. A representative amino acid sequence of *Lactococcus lactis* Zwf is found in LA12_RS12225: glucose-6-phosphate dehydrogenase, EC1.1.1.49, or as shown in SEQ ID NO: 11, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 11. SEQ ID NO: 11 is provided below.

[0143] MTEQKQALFTIFGATGDLAKRKLYPSLFFRLFKKGELADNFAVIGTARRPWTNEYYREVVLESIKDLMNSKTEAENFASHFYYQSHDVSDSSHYVNLKDLGEKLRKQYKTAGNQVFFLAMAPQFFGT IAEHLKSENILTGEGFERIVIEKPFGTSYDTAKSLNDSLAKVFSEEQIFRIDHYLGKEMIQAVSAVRFANPIFESLWNNQHIDNVQITFAEFIGVEDRGGYYETSGALKDMIQNHVLQVLSLIAMEK PEKFDESYIVKEKVKALNAIRQYSSEEALENFVRGQYIAGRFDGEDYLGYREEDSVATDSRTETFAAGKFVIDNERWSGVPFYVRSGKRMTEKGTRINIVFKKDKDNLFAENCDDQSVQNVLTIYIQ PTEGFSLSVNGKAAGQGFHLEPLRLNFRHDSEFLGNSPEAYEKLFLDVLNGDGTNFSHWEEAARAWELIDVIREAWDKETSELPTYAARTMGPKAAFDLLEKNGHEWAWQPDLWYQERGYYNK(SEQ ID NO:11)

[0144] Lactococcus lactis zwf The representative nucleotide sequence of the gene is shown in SEQ ID NO: 33, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 33. SEQ ID NO: 33 is provided below.

[0145]

[0146] In some embodiments, the microorganisms of this disclosure contain mutations in genes encoding enzymes for glycogen biosynthesis. In some embodiments, the microorganisms of this disclosure contain reduced expression of genes encoding enzymes for glycogen biosynthesis. In some embodiments, the enzymes for glycogen biosynthesis are selected from phosphoglucomutase (Pgm), UDP-glucose pyrophosphorylase, glycogen synthase, glycogen branching enzyme, and glycogen protein. In some embodiments, the enzyme for glycogen biosynthesis is phosphoglucomutase (Pgm) (Entrez gene ID: 946370). Pgm (EC5.4.2.2) is an enzyme that transfers a phosphate group on an α-D-glucose monomer from a forward 1 position to a forward 6 position or from a reverse 6 position to a reverse 1 position. In some embodiments, Pgm is *Escherichia coli* Pgm. [Gene] pgm The representative nucleotide sequence is shown in SEQ ID NO: 42, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 47. SEQ ID NO: 47 is provided below.

[0147]

[0148] In some embodiments, *E. coli* Pgm comprises 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% of the amino acid sequence shown in SEQ ID NO: 48. In some embodiments, *E. coli* Pgm comprises the amino acid sequence shown in SEQ ID NO: 48. SEQ ID NO: 48 is provided below.

[0149] MAIHNRAGQPAQQSDLINVAQLTAQYYVLKPEAGNAEHAVKFGTSGHRGSAARHSFNEPHILAIAQAIAEERAKNGITGPCYVGKDTHALSEPAFISVLEVLAANGVDVIVQENNGFTPTPAVSNAILVHNKKGGP LADGIVITPSHNPPEDGGIKYNPPNGGPADTNVTKVVEDRANALLADGLKGVKRISLDEAMASGHVKEQDLVQPFVEGLADIVDMAAIQKAGLTLGVDPLGGSGIEYWKRIGEYYNLNLTIVNDQVDQTFRFMHLDK DGAIRMDCSSECAMAGLLALRDKFDLAFANDPDYDRHGIVTPAGLMNPNHYLAVAINYLFQHRPQWGKDVAVGKTLVSSAMIDRVVNDLGRKLVEVPVGFKWFVDGLFDGSFGFGGEESAGASFLRFDGTPWSTDK DGIIMCLLAAEITAVTGKNPQEHYNELAKRFGAPSYNRLQAAATSAQKAALSKLSPEMVSASTLAGDPITARLTAAPGNGASIGGLKVMTDNGWFAARPSGTEDAYKIYCESFLGEEHRKQIEKEAVEIVSEVLKNA [SEQ ID NO: 48]

[0150] In some implementations, Pgm is Bacillus subtilis Pgm. (Gene) pgm The representative nucleotide sequence is shown in SEQ ID NO: 49, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 49. SEQ ID NO: 49 is provided below.

[0151]

[0152] In some embodiments, *Bacillus subtilis* Pgm comprises 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% of the amino acid sequence shown in SEQ ID NO: 50. SEQ ID NO: 50 is provided below.

[0153] MSKKPAALIILDGFGLRNETVGNAVALAKKPNFDRYWNQYPHQTLTASGEAVGLPEGQMGNSEVGHLNIGAGRIVYQSLTRVNVAIREGEFERNQTFLDAISNAKENNKALHLFGLLSDGGVHSHINH LFALLKLAKKEGLTKVYIHGFLDGRDVGPQTAKTYINQLNDQIKEIGVGEIASISGRYYSMDRDKRWDRVEKAYRAMAYGEGPSYRSALDVVDDSYANGIYDEFVIPSVITKENGEPVAKIQDGDSVIF YNFRPDRAIQISNTFTNKDFRDFDRGENYPKNLYFVCLTHFSETVDGYVAFKPINLDNTVGEVLSQHGLKQLRIAETEKYPHVTFFMSGGREAEFPGEERILINSPKVATYDLKPEMSAYEVKDALVKE IEADKHDAIILNFANPDMVGHSGMVEPTIKAIEAVDECLGEVVDAILAKGGHAIITADHGNADILITESGEPHTAHTTNPVPVIVTKEGITLREGGILGDLAPTLLDLLGVEKPKEMTGTSLIQK(SEQ ID NO:50)

[0154] In some embodiments, Pgm is Bacillus subtilis Pgm. A representative nucleotide sequence of the gene pgm is shown in SEQ ID NO: 81, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 81. SEQ ID NO: 81 is provided below.

[0155]

[0156] In some embodiments, *Bacillus subtilis* Pgm comprises 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% of the amino acid sequence shown in SEQ ID NO: 82. SEQ ID NO: 82 is provided below.

[0157] (SEQ ID NO: 82)

[0158] In some implementations, Pgm is *Lactococcus lactis* Pgm. (Gene) pgm The representative nucleotide sequence is shown in SEQ ID NO: 51, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 51. SEQ ID NO: 51 is provided below.

[0159] ATGTTTAAAGCAGTATTGTTTGATTTAGATGGTGTAATTACAGATACCGCAGAGTATCATTTTAGAGCTTGGAAAGCTTTGGCTGAAGAAATTGGCATTAATGGTGTTGACCGCCAATTTAATGAGCAATTAAAAGGGGTCTCACGAGAAGACTCGCTTCAGAAAA TTCTAGATTTAGCTGATAAAAAAGTATCAGCTGAGGAATTTAAAGAACTTGCTAAGAGAAAAAATGATAACTATGTGAAAATGATTCAGGATGTGTCGCCAGCCGATGTCTATCCTGGAATTTTACAATTACTCAAAGATTTACGTTCAAATAAAATCAAAATTGCT TTAGCATCGGCTTCTAAGAATGGTCCATTTTTATTAGAGAGAATGAATTTAACTGGATTTTTGATGCAATTGCTGATCCGGCTGAAGTTGCAGCATCAAAACCAGCACCAGATATTTTTTGCAGCAGCACATGCAGTGGGTGTTGCCCCTCTGAATCAATTG GGTTAGAGGATTCTCAAGCTGGAATTCAAGCCATCAAAGATTCAGGGGCTTTACCAATTGGTGTAGGGCGCCCAGAAGATTTGGGAGATGATATCGTCATTGTGCCTGATACTTCACACTATACATTAGAATTTTTGAAAGAAGTTTTGGCTTCAAAAGCAAAAATGA [SEQ IDNO: 51]

[0160] In some embodiments, *Lactococcus lactis* Pgm contains 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% of the amino acid sequence shown in SEQ ID NO: 52. SEQ ID NO: 52 is provided below.

[0161] MFKAVLFDLDGVITDTAEYHFRAWKALAEEIGINGVDRQFNEQLKGVSREDSLQKILDLADKKVSAEEFKELAKRKNDNYVKMIQDVSPADVYPGILQLLKDLRSNKIKIAL ASASKNGPFLLEKMNLTGYFDAIADPAEVAASKPAPDIFIAAAHAVGVAPSESIGLEDSQAGIQAIKDSGALPIGVGRPEDLGDDIVIVPDTSYYTLEFLKEVWLQKQK(SEQ ID NO: 52)

[0162] In some embodiments, the microorganisms of this disclosure contain mutations in genes encoding enzymes of glycolysis. In some embodiments, the microorganisms of this disclosure contain reduced expression of genes encoding enzymes of glycolysis. In some embodiments, the enzymes of glycolysis are selected from phosphofructokinase A, phosphofructokinase B, fructose-bisphosphate aldolase, triose phosphoisomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, and pyruvate kinase. In some embodiments, the enzyme of glycolysis is phosphofructokinase B (PfkB). In some embodiments, the enzyme of glycolysis is pyruvate kinase. In some embodiments, the enzyme of glycolysis is phosphofructokinase A (PfkA) (Entrez gene ID: 948412). PfkA catalyzes the phosphorylation of D-fructose-6-phosphate to fructose-1,6-bisphosphate via ATP catalysis, which is the first key step in glycolysis. In some embodiments, PfkA is *Escherichia coli* PfkA. pfkA The representative nucleotide sequence is shown in SEQ ID NO: 7. SEQ ID NO: 7 is provided below.

[0163] ATGATTAAGAAAATCGGTGTGTTGACAAGCGGCGGTGATGCGCCAGGCATGAACGCCGCAATTCGCGGGGTTGTTCGTTCTGCGCTGACAGAAGGTCTGGAAGTAATGGGTATTTATGACGGCTATCTGGGTCTGTATGAAGACCGTATGGTACAGCTAGACCGTTACAGCGTGTCTGACATGATCAACCGTGGCGGTACGTTCCTCGGTTCTGCGCGTTTCCCGGAATTCCGCGACGAGAACATCCGCGCCGTGGCTATCGAAAACCTGAAAAAACGTGGTATCGACGCGCTGGTGGTTATCGGCGGTGACGGTTCCTACATGGGTGCAATGCGTCTGACCGAAATGGGCTTCCCGTGCATCGGTCTGCCGGGCACTATCGACAACGACATCAAAGGCACTGACTACACTATCGGTTTCTTCACTGCGCTGAGCACCGTTGTAGAAGCGATCGACCGTCTGCGTGACACCTCTTCTTCTCACCAGCGTATTTCCGTGGTGGAAGTGATGGGCCGTTATTGTGGAGATCTGACGTTGGCTGCGGCCATTGCCGGTGGCTGTGAATTCGTTGTGGTTCCGGAAGTTGAATTCAGCCGTGAAGACCTGGTAAACGAAATCAAAGCGGGTATCGCGAAAGGTAAAAAACACGCGATCGTGGCGATTACCGAACATATGTGTGATGTTGACGAACTGGCGCATTTCATCGAGAAAGAAACCGGTCGTGAAACCCGCGCAACTGTGCTGGGCCACATCCAGCGCGGTGGTTCTCCGGTGCCTTACGACCGTATTCTGGCTTCCCGTATGGGCGCTTACGCTATCGATCTGCTGCTGGCAGGTTACGGCGGTCGTTGTGTAGGTATCCAGAACGAACAGCTGGTTCACCACGACATCATCGACGCTATCGAAAACATGAAGCGTCCGTTCAAAGGTGACTGGCTGGACTGCGCGAAAAAACTGTATTAA [SEQ ID NO: 7]

[0164] In some embodiments, *E. coli* PfkA comprises 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% of the amino acid sequence shown in SEQ ID NO: 21. In some embodiments, *E. coli* PfkA comprises the amino acid sequence shown in SEQ ID NO: 21. SEQ ID NO: 21 is provided below.

[0165] MIKKIGVLTSGGDAPGMNAAIRGVVRSALTEGLEVMGIYDGYLGLYEDRMVQLDRYSVSDMINRGGTFLGSARFPEFRDENIRAVAIENLKKRGIDALVVIGGDGSYMGAMRLTEMGFPCIGLPGTIDNDIKGTDYTIGFFTALSTVVEAIDRLRDTSSSHQ RISVVEVMGRYCGDLTLAAAIAAGGCEFVVVPEVEFSREDLVNEIKAGIAKGKKHAIVAITEHMCDVDELAHFIEKETGRETRATVLGHIQRGGSPVPYDRILASRMGAYAIDLLLAGYGGRCVGIQNEQLVHHDIIDAIENMKRPFKGDWLDCAKKLY[SEQ ID NO: 21]

[0166] In some embodiments, PfkA is Bacillus subtilis PfkA. A representative amino acid sequence of Bacillus subtilis PfkA is found in O34529(Uniprot) / BSU29190(KEGG) or as shown in SEQ ID NO: 12, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 12. SEQ ID NO: 12 is provided below.

[0167] MKRIGVLTSGGDSPGMNAAVRAVVRKAIYHDVEVYGIYNGYAGLISGKIEKLELGSVGDIIHRGGTKLYTARCPEFKTVEGREKGIANLKKLGIEGLVVIGGDGSYMGAKKLTEHGFPCVGVPGTIDNDIPGTDFTIGFDTALNTVIDAIDKIRDTATSHE RTYVIEVMGRHAGDIALWAGLAGGAESILIPEADYDMHEIIARLKRGHERGKKHSIIIVAEGVGSGVEFGKRIEEETNLETRVSVLGHIQRGGSPSAADRVLASRLGAYAVELLLEGKGGRCVGIQNNKLVDHDIIEILETKHTVEQNMYQLSKELSI(SEQ ID NO: 12)

[0168] Bacillus subtilis gene pfkA The representative nucleotide sequence is shown in SEQ ID NO: 27, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 22. SEQ ID NO: 27 is provided below.

[0169] (SEQ ID NO: 27)

[0170] In some embodiments, PfkA is *Lactococcus lactis* PfkA. A representative amino acid sequence of *Lactococcus lactis* PfkA can be found in LLA12_RS07020: ATP-dependent 6-phosphofructokinase, EC2.7.1.11, or as shown in SEQ ID NO: 13, or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 13, which is provided below.

[0171] MKRIAVLTSGGDAPGMNAAIRAVVRKAISEGIEVYGINHGYAGMVAGDIFPLTSASVGDKIRGGGTFLYSARYPEFAQVEGQLAGIEQLKKFGIEGVVVIGGDGSYHGAMRLTEHGFPAVGLPGTIDNDIVGTDFTIGFDTAVSTVVDALDKIRDTSSSHNRTFVVEVMGRN AGDIALNAGIAAGADDICIPEKEFKFENVVNNINKGYEKGKNHHIIVLAEGVMTGEEFATKLKEAGYKGDLRVSVLGHIQRGGSPTARDRVLASRMGARAVELLRDGIGGVAVGIRNEELVESPILGTAEEGALFSLTTEGGIKVNNPHKAGLELYRLNSALNNLNLN(SEQ ID NO:13)

[0172] Lactococcus lactis gene pfkA The representative nucleotide sequence is shown in SEQ ID NO: 32, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 32. SEQ ID NO: 32 is provided below.

[0173]

[0174] In some implementations, PfkB is the *E. coli* PfkB gene. pfkB The representative nucleotide sequence is shown in SEQ ID NO: 22. SEQ ID NO: 22 is provided below.

[0175] atgGTACGTATCTATACGTTGACACTTGCGCCCTCTCTCGATAGCGCAACAATTACCCCGCAAATTTATCCCGAAGGAAAACTGCGCTGTACCGCACCGGTGTTCGAACCCGGGGGCGGCGGCATCAACGTCGCCCGCGCCATTGCCCATCTTGGAGGCAGTGCCACAGCGATCTTCCCGGCGGGTGGCGCGACCGGCGAACACCTGGTTTCACTGTTGGCGGATGAAAATGTCCCCGTCGCTACTGTAGAAGCCAAAGACTGGACCCGGCAGAATTTACACGTACATGTGGAAGCAAGCGGTGAGCAGTATCGTTTTGTTATGCCAGGCGCGGCATTAAATGAAGATGAGTTTCGCCAGCTTGAAGAGCAAGTTCTGGAAATTGAATCCGGGGCCATCCTGGTCATAAGCGGAAGCCTGCCGCCAGGTGTGAAGCTGGAAAAATTAACCCAACTGATTTCCGCTGCGCAAAAACAAGGGATCCGCTGCATCGTCGACAGTTCTGGCGAAGCGTTAAGTGCAGCACTGGCAATTGGTAACATCGAGTTGGTTAAGCCTAACCAAAAAGAACTCAGTGCGCTGGTGAATCGCGAACTCACCCAGCCGGACGATGTCCGCAAAGCCGCGCAGGAAATCGTTAATAGCGGCAAGGCCAAACGGGTTGTCGTTTCCCTGGGTCCACAAGGAGCGCTGGGTGTTGATAGTGAAAACTGTATTCAGGTGGTGCCACCACCGGTGAAAAGCCAGAGTACCGTTGGCGCTGGTGACAGCATGGTCGGCGCGATGACACTGAAACTGGCAGAAAATGCCTCTCTTGAAGAGATGGTTCGTTTTGGCGTAGCTGCGGGGAGTGCAGCCACACTCAATCAGGGAACACGTCTGTGCTCCCATGACGATACGCAAAAAATTTACGCTTACCTTTCCCGCtaa[SEQ ID NO: 22]

[0176] In some embodiments, *E. coli* PfkB comprises 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% of the amino acid sequence shown in SEQ ID NO: 23. In some embodiments, *E. coli* PfkB comprises the amino acid sequence shown in SEQ ID NO: 23. SEQ ID NO: 23 is provided below.

[0177] MVRIYTLTLAPSLDSATITPQIYPEGKLRCTAPVFEPGGGGINVARAIAHLGGSATAIFPAGGATGEHLVSLLADENVPVATVEAKDWTRQNLHVHVEASGEQYRFVMPGAALNEDEFRQLEEQVLEIESGAILVISGSLPPGVKLEKLTQLISAA QKQGIRCIVDSSGEALSAALAIGNIELVKPNQKELSALVNRELTQPDDVRKAAQEIVNSGKAKRVVVSLGPQGALGVDSENCIQVVPPPVKSQSTVGAGDSMVGAMTLKLAENASLEEMVRFGVAAGSAATLNQGTRLCSHDDTQKIYAYLSR[SEQ ID NO: 23]

[0178] In some embodiments, the microorganisms disclosed herein do not include the absence or reduced expression of hexokinase. In some embodiments, the microorganisms disclosed herein do not include the absence, disruption, or reduced expression of glucokinase. In some embodiments, the microorganisms disclosed herein do not include the absence or reduced expression of glucose-6-phosphate isomerase.

[0179] In some embodiments, the microorganisms of this disclosure contain mutations in genes encoding enzymes of the mannose biosynthesis pathway. In some embodiments, the microorganisms of this disclosure contain reduced expression of genes encoding enzymes of the mannose biosynthesis pathway. In some 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 GDP-mannose and polyterpene-phosphate-mannose required for many key mannose transfer reactions. ManA also catalyzes the interconversion of fructose-6-phosphate and mannose-6-phosphate. In some embodiments, ManA is *Escherichia coli* ManA. (Gene) manAThe representative nucleotide sequence is shown in SEQ ID NO: 9, or is at least 90%, 95%, 95% or 99% identical to SEQ ID NO: 9, which is provided below.

[0180]

[0181] In some embodiments, *E. coli* ManA comprises 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% of the amino acid sequence shown in SEQ ID NO: 25. SEQ ID NO: 25 is provided below.

[0182] MQKLINSVQNYAWGSKTALTELYGMENPSSQPMAELWMGAHPKSSSRVQNAAGDIVSLRDVIESDKSTLLGEAVAKRFGELPFLFKVLCAAQPLSIQVHPNKHNSEIGFAKENAAGIPMDAAERNYKDPNHKPELVFALTPFLAMNAFREFSEIVSLLQPVAGAHPAIAHFLQQPDAERLSELFASLLNMQGEEKSR ALAILKSALDSQQGEPWQTIRLISEFYPEDSGLFSPLLLNVVKLNPGEAMFLFAETPHAYLQGVALEVMANSDNVLRAGLTPKYIDIPELVANVKFEAK PANQLLTQPVKQGAELDFPIPVDDFAFSLHDLSDKETTISQQSAAILFCVEGDATLWKGSQQLQLKPGESAFIAANESPVTVKGHGRLARVYNKL[SEQ ID NO: 25]

[0183] In some embodiments, ManA is Bacillus subtilis ManA. The representative amino acid sequence of ManA is found in O31646(Uniprot) / BSU12020(KEGG), or is shown in SEQ ID NO: 16, or is at least 90%, 95%, 95% or 99% identical to SEQ ID NO: 16.

[0184] MTTEPLFFKPVFKERIWGGTALADFGYTIPSQRTGECWAFAAHQNGQSVVQNGMYKGFTLSELWEHHRHLFGQLEGDRFPLLTKILDADQDLSVQVHPNDEYANIHENGELGKTECWYIIDCQKDAEIIYGHNATTKEELTTMIERGEWDELLRRVKVK PGDFFYVPSGTVHAIGKGILALETQQNSDTTYRLYDYDRKDAEGKLRELHLKKSIEVIEVPSIPERHTVHHEQIEDLLTTTLIECAYFSVGKWNLSGSASLKQQKPFLLISVIEGEGRMISGEYVYPFKKGDHMLLPYGLGEFKLEGYAECIVSHL(SEQ ID NO:16)

[0185] Bacillus subtilis gene manA The representative nucleotide sequence is shown in SEQ ID NO: 29, or is at least 90%, 95%, 95% or 99% identical to SEQ ID NO: 29.

[0186] atgacgactgaaccgttatttttcaagcctgttttcaaagaaagaatttggggcgggaccgctttagctgattttggctataccattccgtcacaacgaacaggggagtgctgggcttttgccgcgcatcaaaatggtcaaagcgttgttcaaaacggaatgtataaggggttcacgctcagcgaattatgggaacatcacagacatttattcggacagcttgaaggggaccgtttccctctgcttacaaaaatattagatgctgaccaggacttatctgttcaggtgcatccgaatgatgaatatgccaacatacatgaaaacggtgagcttggaaaaacagaatgctggtacattattgattgccaaaaagatgccgagattatttatggccacaatgcaacaacaaaggaagaactaactaccatgatagagcgtggagaatgggatgagctcttgcgccgtgtaaaggtaaagccgggggattttttctatgtgccaagcggtactgttcatgcgattggaaaaggaattcttgctttggagacgcagcagaactcagacacaacctacagattatatgattatgaccgaaaagatgcagaaggcaagctgcgcgagcttcatctgaaaaagagcattgaagtgatagaggtcccgtctattccagaacggcatacagttcaccatgaacaaattgaggatttgcttacaacgacattgattgaatgcgcttacttttcggtggggaaatggaacttatcaggatcagcaagcttaaagcagcaaaaaccattccttcttatcagtgtgattgaaggggagggccgtatgatctctggtgagtatgtctatcctttcaaaaaaggagatcatatgttgctgccttacggtcttggagaatttaaactcgaaggatatgcagaatgtatcgtctcccatctgtaa(SEQ ID NO:29)

[0187] In some implementations, Bacillus subtilis, optionally in combination with the deletion of Bacillus subtilis ManA, also has one or both of the following missing.

[0188] (i) YvyI of Bacillus subtilis. A representative amino acid sequence of Yvy1 is found in P39841(Uniprot) / BSU35790(KEGG), or is shown in SEQ ID NO: 17, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 17. SEQ ID NO: 17 is provided below.

[0189] MTQSPIFLTPVFKEKIWGGTALRDRFGYSIPSESTGECWAISAHPKGPSTVANGPYKGKTLIELWEEHREVFGGVEGDRFPLLTKLLDVKEDTSIKVHPDDYYAGENEEGELGKTECWYIIDCKENAEIIYGHTARSKTELVTMINSGDWEGLLRRIKIK PGDFYYVPSGTLHALCKGALVLETQQNSDATYRVYDYDRLDSNGSPRELHFAKAVNAATVPHVDGYIDESTESRKGITIKTFVQGEYFSVYKWDINGEAEMAQDESFLICSVIEGSGLLKYEDKTCPLKKGDHFILPAQMPDFTIKGTCTLIVSHI(SEQ ID NO:17)

[0190] Bacillus subtilis gene yvyI The representative nucleotide sequence is shown in SEQ ID NO: 30, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 30. SEQ ID NO: 30 is provided below.

[0191] atgacgcaatcaccgatttttctaacgcctgtgtttaaagaaaaaatctggggcggaaccgctttacgagatagatttggatacagtattccttcagaatcaacgggggaatgctgggccatttccgctcatccaaaaggaccgagcactgttgcaaatggcccgtataaaggaaagacattgatcgagctttgggaagagcaccgtgaagtattcggcggcgtagagggggatcggtttccgcttctgacaaagctgctggatgtgaaggaagatacgtcaattaaagttcaccctgatgattactatgccggagaaaacgaagagggagaactcggcaagacggaatgctggtacattatcgactgtaaggaaaacgcagaaatcatttacgggcatacggcccgctcaaaaaccgaacttgtcacaatgatcaacagcggtgactgggagggcctgctgcgaagaatcaaaattaaaccgggtgatttctattatgtgccgagcggaacgctgcacgcattgtgcaagggggcccttgttttagagactcagcaaaattcagatgccacataccgggtgtacgattatgaccgtcttgatagcaacggaagtccgagagagcttcattttgccaaagcggtcaatgccgccacggttccccatgtggacgggtatatagatgaatcgacagaatcaagaaaaggaataaccattaaaacatttgtccaaggggaatatttttcggtttataaatgggacatcaatggcgaagctgaaatggctcaggatgaatcctttctgatttgcagcgtgatagaaggaagcggtttgctcaagtatgaggacaaaacatgtccgctcaaaaaaggtgatcactttattttgccggctcaaatgcccgattttacgataaaaggaacttgtacccttatcgtgtctcatatttaa(SEQ ID NO: 30)

[0192] (ii) GmuF of Bacillus subtilis. A representative amino acid sequence of GmuF is found in O05511(Uniprot) / BSU05870(KEGG), or is shown in SEQ ID NO: 18, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 18. SEQ ID NO: 18 is provided below.

[0193] MTHPLFLEPVFKERLWGGTKLRDAFGYAIPSQKTGECWAVSAHAHGSSSVKNGPLAGKTLDQVWKDHPEIFGFPDGKVFPLLVKLLDANMDLSVQVHPDDDYAKLHENGDLGKTECWYIIDCKDDAELILGHHASTKEEFKQRIESGDWNGLLRRIKIK PGDFFYVPSGTLHALCKGTLVLEIQQNSDTTYRVYDYDRCNDQGQKRTLHIEKAMEVITIPHIDKVHTPEVKEVGNAEIIVYVQSDYFSVYKWKISGRAAFPSYQTYLLGSVLSGSGRIINNGIQYECNAGSHFILPAHFGEFTIEGTCEFMISHP(SEQ ID NO: 18)

[0194] Bacillus subtilis gene gmuF The representative nucleotide sequence is shown in SEQ ID NO: 31, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 31. SEQ ID NO: 31 is provided below.

[0195] (SEQ ID NO: 31)

[0196] In some embodiments, ManA is *Lactococcus lactis* ManA. A representative amino acid sequence of *Lactococcus lactis* ManA is found in LLA12_RS03920: mannose-6-phosphate isomerase, EC5.3.1.8, or is shown in SEQ ID NO: 19, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 19. SEQ ID NO: 19 is provided below.

[0197] MKEPLFLNSVLQEKIWGGDHLKEFGYDLPSDKVGEYWAISAHPHGVSTIANGEFKGQKLDQLYASHRELFGDSKKEVFPLLTKILDANDWLSVQVHPDDEYGQKHEGELGKTECWYIISAEPGAEIIYGHNAKSREELAEMIKSGDWDHLLRKVKVKTG DFFHVPSGTMHAIGAGIVILETQQSSDTTYRVYDFDRKDDQGNLRELHIQQSIDVLNIPGDKVPENQVKTEKFADAEITTLVKSDFFDVYKWQIHGDHEFTKVADYTLVSVLDGQGKLTVDGNEYPVEKGAHFILPSNIEKWNLSGQLEIIASNPA(SEQ ID NO: 19)

[0198] Lactococcus lactis gene manA The representative nucleotide sequence is shown in SEQ ID NO: 35, or is at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 35. SEQ ID NO: 35 is provided below.

[0199] (SEQ ID NO: 35)

[0200] In some embodiments, the phosphatase is hexitol phosphatase B (HxpB) (UniProt No. P77247 or UniProt No. Q7ADF8). HxpB catalyzes the dephosphorylation of D-allulose 6-phosphate. In some embodiments, HxpB is *Escherichia coli* HxpB. In some embodiments, HxpB comprises 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% of the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, HxpB comprises the amino acid sequence shown in SEQ ID NO: 3. SEQ ID NO: 3 is provided below.

[0201] MSTPRQILAAIFDMDGLLIDSEPLWDRAELDVMASLGVDISRRNELPDTLGLRIDMVVDLWYARQPWNGPSRQEVVERVIARAISLVEETRPLLPGVREAVALCKEQGLLVGL ASASPLHMLEKVLTMFDLRDSFDALASAEKLPYSKPHPQVYLDCAAKLGVDPLTCVALEDSVNGMIASKAARMRSIVVPAPEAQNDPRFVLADVKLSSLTELTAKDLLG[SEQ ID NO: 3]

[0202] In some embodiments, HxpB comprises the amino acid sequence shown in SEQ ID NO: 4. In some embodiments, HxpB consists of the amino acid sequence shown in SEQ ID NO: 4. SEQ ID NO: 4 is provided below.

[0203] MSTPRQILAAIFDMDGLLIDSEPLWDRAELDVMASLGVDISRRNELPDTLGLRIDMVVDLWYARQPWNGPSRQEVVERVIARAISLVEETRPLLPGVREAVALCKEQGLLVGL ASASPLHMLEKVLTMFDLRDSFDALASAEKLPYSKPHPQVYLDCAAKLGVDPLTCVALEDSVNGMIASKAARMRSIVVPAPEAQNDPRFVLANVKLSSLTELTAKDLLG[SEQ ID NO: 4]

[0204] In some implementations, genes hxpBEncoded 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 shown in SEQ ID NO: 5. In some embodiments, hxpB It contains the nucleotide sequence shown in SEQ ID NO: 5. In some embodiments, hxpB It consists of the nucleotide sequence shown in SEQ ID NO: 5. SEQ ID NO: 5 is provided below.

[0205] ATGTCAACCCCGCGTCAGATTCTTGCTGCAATTTTTGATATGGATGGATTACTTATCGACTCAGAACCTTTATGGGATCGAGCCGAACTGGATGTGATGGCAAGCCTGGGGGTGGATATCTCCCGTCGTAACGAGCTGCCGGACACCTTAGGTTTACGCATCGATAT GGTGGTCGATCTTTTGGTACGCCCGGCAACCGTGGAATGGGCCAAGCCGTCAGGAAGTAGTAGAACGGGTTATTGCCCGTGCCATTTCACTGGTTGAAGAGACACGTCCATTATTACCAGGCGTGCGCGAAGCCGTTGCGTTATGCAAAGAACAAGGTTTATTGGTGG GACTGGCCTCCGCGTCACCACTACATATGCTGGAAAAAGTGTTGACCATGTTTGACTTACGCGACAGTTTCGATGCCCTCGCCTCGGCCGAAAAACTGCCTTACAGCAAGCCGCATCCGCAAGTATATCTCGACTGCGCAGCAAAACTGGGCGTTGACCCTCTGACC TGCGTAGCGCTGGAAGATTCGGTAAATGGCATGATCGCCTCTAAAGCAGCCCGCATGCGTTCCATCGTCGTTCCTGCGCCAGAAGCGCAAAATGATCCACGTTTTGTATTAGCAGACGTCAAACTTTCATCGCTGACAGAACTCACCGCAAAAGACCTTCTCGGTTGA [SEQ IDNO: 5]

[0206] In some embodiments, the phosphatase is a sugar phosphatase, such as YbiV, G6425(EcoCyc) P75792(UniProt), and catalyzes the dephosphorylation of D-allulose-6-phosphate. In some embodiments, YbiV is *E. coli* YbiV. In some embodiments, YbiV comprises 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% of the amino acid sequence shown in SEQ ID NO: 36. In some embodiments, YbiV consists of the amino acid sequence shown in SEQ ID NO: 36. SEQ ID NO: 36 is provided below.

[0207] MSVKVIVTDMGTFLNDAKTYNQPRFMAQYQELKKRGIKFVVASGNQYYQLISFFPELKDEISFVAENGALVYEHGKQLFHGELTRHESRIVIGELLKDKQLNFVACGLQSAYVSENAPEAFVALMAKHYHRLKPVK DYQEIDDVLFKFSLNLPDEQIPLVIDKLHVALDGIMKPVTSGFGFIDLIIPGLHKANGISRLLKRWDLSPQNVVAIGDSGNDAEMLKMARYSFAMGNAAENIKQIARYATDDNNHEGALNVIQAVLDNTSPFNS[SEQ ID NO: 36]

[0208] In some implementations, genes ybiV 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 shown in SEQ ID NO: 37. In some embodiments, ybiV It contains the nucleotide sequence shown in SEQ ID NO: 37. In some embodiments, ybiV It consists of the nucleotide sequence shown in SEQ ID NO: 37. SEQ ID NO: 37 is provided below.

[0209] atgAGCGTAAAAGTTATCGTCACAGACATGGACGGTACTTTTCTTAACGACGCCAAAACGTACAACCAACCACGTTTTATGGCGCAATATCAGGAACTGAAAAAGCGCGGCATTAAGTTCGTTGTTGCCAGCGGTAATCAGTATTACCAGCTTATTTCATTCTTTCCTGAGCTAAAGGATGAGATCTCTTTTGTCGCGGAAAACGGCGCACTGGTTTACGAACATGGCAAGCAGTTGTTCCACGGCGAACTGACCCGACATGAATCGCGGATTGTTATTGGCGAGTTGCTAAAAGATAAGCAACTCAATTTTGTCGCCTGCGGTCTGCAAAGTGCATATGTCAGCGAAAATGCCCCCGAAGCATTTGTCGCACTGATGGCAAAACACTACCATCGCCTGAAACCTGTAAAAGATTATCAGGAGATTGACGACGTACTGTTCAAGTTTTCGCTCAACCTGCCGGATGAACAAATCCCGTTAGTGATCGACAAACTGCACGTAGCGCTCGATGGCATTATGAAACCCGTTACCAGTGGTTTTGGCTTTATCGACCTGATTATTCCCGGTCTACATAAAGCAAACGGTATTTCGCGGTTACTGAAACGCTGGGATCTGTCACCGCAAAATGTGGTAGCGATTGGCGACAGCGGTAACGATGCGGAGATGCTGAAAATGGCGCGTTATTCCTTTGCGATGGGCAATGCTGCGGAAAACATTAAACAAATCGCCCGTTACGCTACCGATGATAATAATCATGAAGGCGCGCTGAATGTGATTCAGGCGGTGCTGGATAACACATCCCCTTTTAACAGCtga[SEQ ID NO:37]

[0210] In some embodiments, the microorganisms of this disclosure contain mutations in the gene encoding an aldolase. In some embodiments, the microorganisms of this disclosure contain reduced expression of the gene encoding an aldolase. In some embodiments, the aldolase is deoxyribophosphate aldolase (DeoC) (Entrez gene ID: 948902). DeoC catalyzes the reversible conversion of allose-6-phosphate to erythrose-4-phosphate and glycolaldehyde. In some embodiments, DeoC is *Escherichia coli* DeoC. [Gene] deoC The representative nucleotide sequence is shown in SEQ ID NO: 59, or is at least 90%, 95%, 95% or 99% identical to SEQ ID NO: 59, which is provided below.

[0211] ATGACTGATCTGAAAGCAAGCAGCCTGCGTGCACTGAAATTGATGGACCTGACCACCCTGAATGACGACGACACCGACGAGAAAGTGATCGCCCTGTGTCATCAGGCCAAAACTCCGGTCGGCAATACCGCCGCTATCTGTATCTATCCTCGCTTTATCCCGATTGCTCGCAAAACTCTGAAAGAGCAGGGCACC CCGGAAATCCGTATCGCTACGGTAACCAACTTCCCACACGGTAACGACGACATCGACATCGCGCTGGCAGAAACCCGTGCGGCAATCGCCTACGGTGCTGATGAAGTTGACGTTGTGTTCCCGTACCGCGCGCTGATGGCGGGTAACGAGCAGGTTGGTTTTGACCTGGTGAAAGCCTGTAAAGAGGCTTGCGCG GCAGCGAATGTACTGCTGAAAGTGATCATCGAAACCGGCGAACTGAAAGACGAAGCGCTGATCCGTAAAGCGTCTGAAATCCATCAAAGCGGGTGCGGACTTCATCAAAACCTCTACCGGTAAAGTGGCTGTGAACGCGACGCCGGAAAGCGCGCGCATCATGATGGAAGTGATCCGTGATATGGGCGTAGAA AAAACCGTTGGTTTCAAACCGGCGGGCGGCGTGCGTACTGCGGAAGATGCGCAGAAATATCTCGCCATTGCAGATGAACTGTTCGGTGCTGACTGGGCAGATGCGCGTCACTACCGCTTTGGCGCTTCCAGCCTGCTGGCAAGCCTGCTGAAAGCGCTGGGTCACGGCGACGGTAAGAGCGCCAGCAGCTACTAA [SEQ ID NO: 59]

[0212] In some embodiments, *E. coli* DeoC comprises 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% of the amino acid sequence shown in SEQ ID NO: 58. In some embodiments, *E. coli* DeoC comprises the amino acid sequence shown in SEQ ID NO: 58. SEQ ID NO: 58 is provided below.

[0213] MTDLKASSLRALKLMDLTTLNDDDTDEKVIALCHQAKTPVGNTAAICIYPRFIPIARKTLKEQGTPEIRIATVTNFPHGNDDIDIALAETRAAIAYGADEVDVVFPYRALMAGNEQVGFDLVKACKEACAA ANVLLKVIIETGELKDEALIRKASEISIKAGADFIKTSTGKVAVNATPESARIMMEVIRDMGVEKTVGFKPAGGVRTAEDAQKYLAIADELFGADWADARHYRFGASSLLASLLKALGHGDGKSASSY[SEQ ID NO: 58]

[0214] In some embodiments, the microorganisms of this disclosure contain mutations in the gene encoding the phosphatase. In some embodiments, the microorganisms of this disclosure contain reduced expression of the gene encoding the phosphatase. In some embodiments, the phosphatase is fructose-1-phosphate phosphatase (YqaB) (Entrez gene ID: 945776; EcoCyc ID: G7408; UniProt ID: P77475). YqaB catalyzes the dephosphorylation of allulose-6-phosphate. In some embodiments, YqaB is *Escherichia coli* YqaB. [Gene] yqaB The representative nucleotide sequence is shown in SEQ ID NO: 61 or SEQ ID NO: 80, or is at least 90%, 95%, 95% or 99% identical to SEQ ID NO: 61 or SEQ ID NO: 80, which is provided below.

[0215] ATGACTGATCTGAAAGCAAGCAGCCTGCGTGCACTGAAATTGATGGACCTGACCACCCTGAATGACGACGACACCGACGAGAAAGTGATCGCCCTGTGTCATCAGGCCAAAACTCCGGTCGGCAATACCGCCGCTATCTGTATCTATCCTCGCTTTATCCCGATTGCTCGCAAAACTCTGAAAGAGCAGGGCACCCCGGAAATCCGTATCGCTACGGTAACCAACTTCCCACACGGTAACGACGACATCGACATCGCGCTGGCAGAAACCCGTGCGGCAATCGCCTACGGTGCTGATGAAGTTGACGTTGTGTTCCCGTACCGCGCGCTGATGGCGGGTAACGAGCAGGTTGGTTTTGACCTGGTGAAAGCCTGTAAAGAGGCTTGCGCGGCAGCGAATGTACTGCTGAAAGTGATCATCGAAACCGGCGAACTGAAAGACGAAGCGCTGATCCGTAAAGCGTCTGAAATCTCCATCAAAGCGGGTGCGGACTTCATCAAAACCTCTACCGGTAAAGTGGCTGTGAACGCGACGCCGGAAAGCGCGCGCATCATGATGGAAGTGATCCGTGATATGGGCGTAGAAAAAACCGTTGGTTTCAAACCGGCGGGCGGCGTGCGTACTGCGGAAGATGCGCAGAAATATCTCGCCATTGCAGATGAACTGTTCGGTGCTGACTGGGCAGATGCGCGTCACTACCGCTTTGGCGCTTCCAGCCTGCTGGCAAGCCTGCTGAAAGCGCTGGGTCACGGCGACGGTAAGAGCGCCAGCAGCTACTAA [SEQ ID NO: 61]

[0216] [SEQ ID NO: 80]

[0217] In some embodiments, *E. coli* YqaB comprises 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% of the amino acid sequence shown in SEQ ID NO: 60. SEQ ID NO: 60 is provided below.

[0218] MYERYAGLIFDMDGTILDTEPTHRKAWREVLGHYGLQYDIQAMIALNGSPTWRIAQAIIELNQADLDPHALAREKTEAVRSMLLDSVEPLPLVDVVKSWHGRRPMAVGTGSESAIAEALLAHLGLRHYFDAVVAADHVKHHKPAPDTFLLCAQRMGVQPTQCVVFEDADFGIQAARAAGMDAVDVRLL[SEQ ID NO: 60]

[0219] In some embodiments, the phosphatase is a sugar phosphatase, such as YidA, P0A8Y5 (UniProt), or EG11195 (EcoCyc), and catalyzes the dephosphorylation of D-allulose 6-phosphate. In some embodiments, YidA is *E. coli* YidA. In some embodiments, YidA comprises 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% of the amino acid sequence shown in SEQ ID NO: 77. In some embodiments, YidA comprises the amino acid sequence shown in SEQ ID NO: 77. SEQ ID NO: 77 is provided below.

[0220] MAIKLIAIDMDGTLLLPDHTISPAVKNAIAAARARGVNVVLTTGRPYAGVHNYLKELHMEQPGDYCITYNGALVQKAADGSTVAQTALSYDDYRFLEKLSREVGSHFHALDRTTLYTANRDISYYTVHESFVATIPL VFCEAEKMDPNTQFLKVMMIDEPAILDQAIARIPQEVKEKYTVLKSAPYFLEILDKRVNKGTGVKSLADVLGIKPEEIMAIGDQENDIAMIEYAGVGVAMDNAIPSVKEVANFVTKSNLEDGVAFAIEKYVLN[SEQ ID NO: 77]

[0221] In some implementations, genes yidA Encoded by 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 nucleotide sequences to the nucleotide sequence shown in SEQ ID NO: 78. In some embodiments, yidA comprises the nucleotide sequence shown in SEQ ID NO: 78. In certain embodiments, yidA consists of the nucleotide sequence shown in SEQ ID NO: 78. SEQ ID NO: 78 is provided below.

[0222] atgAGCGTAAAAGTTATCGTCACAGACATGGACGGTACTTTTCTTAACGACGCCAAAACGTACAACCAACCACGTTTTATGGCGCAATATCAGGAACTGAAAAAGCGCGGCATTAAGTTCGTTGTTGCCAGCGGTAATCAGTATTACCAGCTTATTTCATTCTTTCCTGAGCTAAAGGATGAGATCTCTTTTGTCGCGGAAAACGGCGCACTGGTTTACGAACATGGCAAGCAGTTGTTCCACGGCGAACTGACCCGACATGAATCGCGGATTGTTATTGGCGAGTTGCTAAAAGATAAGCAACTCAATTTTGTCGCCTGCGGTCTGCAAAGTGCATATGTCAGCGAAAATGCCCCCGAAGCATTTGTCGCACTGATGGCAAAACACTACCATCGCCTGAAACCTGTAAAAGATTATCAGGAGATTGACGACGTACTGTTCAAGTTTTCGCTCAACCTGCCGGATGAACAAATCCCGTTAGTGATCGACAAACTGCACGTAGCGCTCGATGGCATTATGAAACCCGTTACCAGTGGTTTTGGCTTTATCGACCTGATTATTCCCGGTCTACATAAAGCAAACGGTATTTCGCGGTTACTGAAACGCTGGGATCTGTCACCGCAAAATGTGGTAGCGATTGGCGACAGCGGTAACGATGCGGAGATGCTGAAAATGGCGCGTTATTCCTTTGCGATGGGCAATGCTGCGGAAAACATTAAACAAATCGCCCGTTACGCTACCGATGATAATAATCATGAAGGCGCGCTGAATGTGATTCAGGCGGTGCTGGATAACACATCCCCTTTTAACAGCtga[SEQ ID NO:78]

[0223] In some embodiments, the gene yidA is encoded by 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 nucleotide sequences to the nucleotide sequence shown in SEQ ID NO: 83. In some embodiments, yidA comprises the nucleotide sequence shown in SEQ ID NO: 83. SEQ ID NO: 83 is provided below.

[0224] [SEQ ID NO: 83]

[0225] In some embodiments, the microorganisms of this disclosure contain mutations in genes encoding enzymes of allose metabolism. In some embodiments, the microorganisms of this disclosure contain reduced expression of genes encoding enzymes of allose metabolism.

[0226] In some embodiments, the enzyme involved in allose metabolism is D-allose input ATP-binding protein (AlsA) (Entrez gene ID: 948593; EcoCyc ID: EG11959; UniProt ID: P32721). In some embodiments, AlsA is *E. coli* AlsA. alsA The representative nucleotide sequence is shown in SEQ ID NO: 65, or is at least 90%, 95%, 95% or 99% identical to SEQ ID NO: 65, which is provided below.

[0227]

[0228] In some embodiments, *E. coli* AlsA comprises 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% of the amino acid sequence shown in SEQ ID NO: 64. In some embodiments, *E. coli* AlsA comprises the amino acid sequence shown in SEQ ID NO: 64. SEQ ID NO: 64 is provided below.

[0229] MATPYISMAGIGKSFGPVHALKSVNLTVYPGEIHALLGENGAGKSTLMKVLSGIHEPTKGTITINNISYNKLDHKLAAQLGIGIIYQELSVIDELTVLENLYIGRHLTKKICGVNIIDWREMRVRAAM MLLRVGLKVDLDEKVANLSISHKQMLEIAKTLMLDAKVIIMDEPTSSLTNKEVDYLFLIMNQLRKEGTAIVYISHKLAEIRRICDRYTVMKDGSSVCSGIVSDVSNDDIVRLMVGRELQNRFNAMKENV SNLAHETVFEVRNVTSRDRKKVRDISFSVCRGEILGFAGLVGSGRTELMNCLFGVDKRAGGEIRLNGKDISPRSPLDAVKKGMAYITESRRDNGFFPNFSIAQNMAISRSLKDGGYKGAMGLFHEVDE QRTAENQRELLALKCHSVNQNITELSGGNQQKVLISKWLCCCPEVIIFDEPTRGIDVGAKAEIYKVMRQLADDGKVILMVSSELPEIITVCDRIAVFCEGRLTQILTNRDDMSEEEIMAWALPQE[SEQ ID NO: 64]

[0230] In some embodiments, the enzyme metabolizing allosugar is D-allosugar-binding periplasmic protein (AlsB) (Entrez gene ID: 948604; EcoCyc ID: EG12458; UniProt ID: P39265). In some embodiments, AlsB is *E. coli* AlsB. (Gene) alsB The representative nucleotide sequence is shown in SEQ ID NO: 67, or is at least 90%, 95%, 95% or 99% identical to SEQ ID NO: 67, which is provided below.

[0231] ATGAATAAATATCTGAAATATTTCAGCGGCACACTCGTGGGCTTAATGTTGTCAACCAGCGCTTTTGCTGCCGCCGAATATGCTGTCGTATTGAAAACCCTCTCCAACCCATTTTGGGTAGATATGAAAAAAGGCATTGAAGATGAAGCAAAAACACTGGGCGTCAGCGTTGATATTTTTGCCTCTCCTTCAGAAGGCGATTTTCAATCTCAATTGCAGTTATTTGAAGATCTCAGTAATAAAAATTACAAAGGTATCGCCTTCGCTCCATTATCCTCAGTGAATCTGGTCATGCCTGTCGCCCGCGCATGGAAAAAAGGCATTTATCTGGTTAATCTCGATGAAAAAATCGACATGGATAATCTGAAAAAAGCTGGCGGCAATGTGGAAGCTTTTGTCACCACCGATAACGTTGCTGTCGGGGCGAAAGGCGCGTCGTTCATTATTGACAAATTGGGCGCTGAAGGTGGTGAAGTCGCAATCATTGAGGGTAAAGCCGGTAACGCCTCCGGTGAAGCGCGTCGTAATGGTGCCACCGAAGCCTTCAAAAAAGCAAGCCAGATCAAGCTTGTCGCCAGCCAGCCTGCCGACTGGGACCGCATTAAAGCACTGGATGTCGCCACTAACGTGTTGCAACGTAATCCGAATATTAAAGCGATCTATTGCGCGAATGACACGATGGCAATGGGTGTTGCTCAGGCAGTCGCAAACGCCGGAAAAACGGGAAAAGTGCTGGTCGTCGGTACAGATGGCATTCCGGAAGCCCGCAAAATGGTGGAAGCCGGACAAATGACCGCGACGGTTGCCCAGAACCCGGCGGATATCGGCGCAACGGGTCTGAAGCTGATGGTTGACGCTGAGAAATCCGGCAAGGTTATCCCGCTGGATAAAGCACCGGAATTTAAACTGGTCGATTCAATCCTGGTCACTCAATAA [SEQ ID NO: 67]

[0232] In some embodiments, *E. coli* AlsB comprises 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% of the amino acid sequence shown in SEQ ID NO: 66. In some embodiments, *E. coli* AlsB comprises the amino acid sequence shown in SEQ ID NO: 66. SEQ ID NO: 66 is provided below.

[0233] MNKYLKYFSGTLVGLMLLSTSAFAAAEYAVVLKTLSNPFWVDMKKGIEDEAKTLGVSVDIFASPSEGDFQSQLQLFEDLSNKNYKGIAFAPLSSVNLVMPVARAWKKGIYLVNLDEKIDMDNLKKAGGNVEAFVTTDNVAVGAKGASFIIDKLGAEGG EVAIIEGKAGNASGEARRNGATEAFKKASQIKLVASQPADWDRIKALDVATNVLQRNPNIKAIYCANDTMAMGVAQAVANAGKTGKVLVVGTDGIPEARKMVEAGQMTATVAQNPADIGATGLKLMVDAEKSGKVIPLDKAPEFKLVDSILVTQ[SEQ ID NO: 66]

[0234] In some embodiments, the enzyme metabolizing allosugar is the D-allosugar transporter permease protein (AlsC) (Entrez gene ID: 948594; EcoCyc ID: EG11958; UniProt ID: P32720). In some embodiments, AlsC is *E. coli* AlsC. gene. alsC The representative nucleotide sequence is shown in SEQ ID NO: 69, or is at least 90%, 95%, 95% or 99% identical to SEQ ID NO: 69, which is provided below.

[0235] ATGGGCTTTACCACAAGAGTAAAAAGCGAAGCGAGCGAGAAGAAACCGTTCAACTTTGCGCTGTTCTGGGATAAATACGGCACCTTTTTTATCCTGGCGATCATCGTCGCCATCTTTGGTTCGCTGTCACCAGAATATTTTCTGACCACCAATAATATTACCCAGATTTTTGTTCAAAGCTCCGTGACGGTATTGATCGGCATGGGCGAGTTTTTCGCTATCCTGGTCGCTGGTATCGACCTCTCGGTTGGCGCGATTCTGGCGCTTTCCGGTATGGTGACCGCCAAACTGATGTTGGCAGGTGTTGACCCGTTTCTCGCAGCGATGATTGGCGGTGTACTGGTTGGCGGCGCACTGGGGGCGATCAACGGCTGCCTGGTCAACTGGACGGGGCTACACCCGTTCATCATCACCCTTGGCACCAACGCGATTTTCCGTGGGATCACGCTGGTGATCTCCGATGCCAACTCGGTATACGGCTTCTCATTTGACTTCGTGAACTTCTTTGCCGCCAGCGTAATTGGGATACCTGTCCCCGTTATCTTCTCACTAATTGTCGCGCTCATCCTTTGGTTTCTGACAACGCGTATGCGGCTCGGGCGCAACATCTACGCACTGGGCGGCAACAAAAATTCGGCGTTCTATTCCGGGATTGACGTGAAATTCCACATCCTGGTGGTGTTTATCATCTCCGGTGTTTGTGCAGGTCTGGCAGGCGTCGTCTCAACTGCACGACTCGGTGCCGCAGAACCGCTTGCCGGTATGGGTTTTGAAACCTATGCCATTGCCAGCGCCATCATTGGCGGCACCAGTTTCTTCGGCGGCAAGGGGCGCATTTTCTCTGTGGTGATTGGCGGGTTGATCATCGGCACCATCAACAACGGTCTGAATATTTTGCAGGTACAAACCTATTACCAACTGGTGGTGATGGGCGGATTAATTATCGCGGCTGTCGCCCTTGACCGTCTTATCAGTAAGTAA[SEQ ID NO: 69]

[0236] In some embodiments, *E. coli* AlsC comprises 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% of the amino acid sequence shown in SEQ ID NO: 68. In some embodiments, *E. coli* AlsC comprises the amino acid sequence shown in SEQ ID NO: 68. SEQ ID NO: 68 is provided below.

[0237] MGFTTRVKSEASEKKPFNFALFWDKYGTFFILAIIVAIFGSLSPEYFLTTNNITQIFVQSSVTVLIGMGEFFAILVAGIDLSVGAILALSGMVTAKLMLAGVDPFLAAMIGGVLVGGALGAINGCLVNWTGLHPFIITLGTNAIFRGITLVISDANSVYGFSFDF VNFFAASVIGIPVPVIFSLIVALWFLTTRMRLGRNIYALGGNKNSAFYSGIDVKFHILVVFIISGVCAGLAGVVSTARLGAAEPLAGMGFETYAIASAIIGGTSFFGGKGRIFSVVIGGLIIGTINNGLNILQVQTYYQLVVMGGLIIAAVALDRLISK[SEQ ID NO: 68]

[0238] In some embodiments, the enzyme metabolizing allosugar is D-allokinase (AlsK) (Entrez gene ID: 948596; EcoCyc ID: EG11956; UniProt ID: P32718). In some embodiments, AlsK is E. coli AlsK. alsK The representative nucleotide sequence is shown in SEQ ID NO: 71, or is at least 90%, 95%, 95% or 99% identical to SEQ ID NO: 71, which is provided below.

[0239] ATGCAAAAACAGCATAACGTCGTAGCGGGCGTGGATATGGGGGCAACGCATATCCGCTTTTGTCTGCGGACAGCAGAAGGTGAAACGCTACACTGCGAAAAAAAGCGGACCGCAGAAGTCATTGCTCCCGGCCTGGTGTCGGGTATCGGCGAAATGATTGACGAGCAACTCAGGCGCTTTAACGCTCGCTGTCATGGTCTGGTGATGGGATTTCCGGCGCTGGTCAGTAAAGATAAACGCACCATTATTTCTACGCCTAACCTGCCGTTAACAGCGGCGGATTTATATGATCTCGCCGATAAGCTCGAAAATACGCTGAATTGTCCGGTTGAGTTTTCCCGCGACGTTAACCTGCAACTCTCCTGGGACGTAGTAGAAAACCGCCTTACGCAACAACTGGTTCTGGCGGCCTATCTCGGTACGGGGATGGGGTTCGCAGTGTGGATGAACGGTGCGCCGTGGACGGGTGCACACGGTGTGGCAGGCGAACTGGGTCATATCCCCCTGGGAGATATGACCCAACACTGCGCGTGTGGCAATCCTGGGTGCCTGGAAACCAATTGCTCTGGAATGGCGCTAAGACGCTGGTACGAACAACAGCCCCGAAATTACCCATTGCGCGATCTTTTCGTCCATGCGGAAAACGCCCCTTTCGTCCAGAGTCTGCTTGAAAACGCGGCACGGGCCATTGCCACCAGCATTAATCTGTTCGATCCCGATGCGGTGATCCTGGGCGGTGGCGTGATGGATATGCCCGCCTTCCCACGCGAGACTCTCGTTGCCATGACCCAAAAGTACCTGCGCCGTCCACTGCCGCATCAGGTCGTGCGCTTTATTGCCGCCTCATCTTCTGACTTTAATGGCGCTCAGGGTGCAGCAATATTGGCGCATCAACGTTTTTTGCCACAGTTCTGTGCTAAAGCCCCATGA [SEQ ID NO: 71]

[0240] In some embodiments, *E. coli* AlsK comprises 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% of the amino acid sequence shown in SEQ ID NO: 70. In some embodiments, *E. coli* AlsK comprises the amino acid sequence shown in SEQ ID NO: 70. SEQ ID NO: 66 is provided below.

[0241] MQKQHNVVAGVDMGATHIRFCLRTAEGETLHCEKKRTAEVIAPGLVSGIGEMIDEQLRRFNARCHGLVMGFPALVSKDKRTIISTPNLPLTAADLYDLADKLENTLNCPVEFSRDVNLQLSWDVVENRLTQQLVLAAYLGTGMGGFVWMNGAPW TGAHGVAGELGHIPLGDMTQHCACGNPGCLETNCSGMALRRWYEQQPRNYPLRDLFVHAENAPFVQSLLENAARAIATSINLFDPDAVILGGGVMDMPAFPRETLVAMTQKYLRRPLPHQVVRFIAASSSDFNGAQGAAILAHQRFLPQFCAKAP [SEQ ID NO: 70]

[0242] In some embodiments, the enzyme metabolizing allosugar is an HTH-type transcriptional regulator (AlsR) (Entrez gene ID: 948603; EcoCyc ID: G7821; UniProt ID: P0ACS7). In some embodiments, AlsR is *E. coli* AlsR gene. alsR The representative nucleotide sequence is shown in SEQ ID NO: 73 or SEQ ID NO: 84, or is at least 90%, 95%, 95% or 99% identical to SEQ ID NO: 73 or SEQ ID NO: 84, which is provided below.

[0243] ATGAGCCAGTCAGAGTTTGATTCAGCGCTTCCGAACGGTATAGGGTTAGCGCCTTACCTGCGAATGAAGCAGGAAGGAATGACAGAAAATGAAAGCCGCATCGTGGAGTGGTTACTCAAACCCGGTAACCTGAGTTGTGCACCCGCAATTAAAGATGTCGCAGAAGCTCTGGCGGTATCTGAAGCGATGATAGTTAAGGTATCAAAGCTGCTGGGGTTTAGCGGCTTTCGTAACTTACGCAGTGCGCTGGAAGATTATTTTTCTCAGTCAGAACAGGTATTGCCTTCCGAGTTGGCTTTTGATGAAGCGCCGCAGGATGTGGTGAATAAGGTATTTAACATCACTTTACGCACCATTATGGAAGGTCAGTCGATCGTCAACGTTGATGAGATCCACCGTGCCGCCCGCTTTTTCTATCAGGCCAGACAGCGGGATTTGTACGGTGCCGGAGGATCAAATGCTATCTGTGCTGATGTACAGCACAAGTTCTTGCGCATTGGCGTACGCTGTCAGGCCTATCCTGATGCTCACATCATGATGATGTCCGCTTCGTTGTTACAGGAAGGAGATGTTGTGCTGGTAGTGACCCATTCCGGGCGAACCAGTGATGTAAAAGCGGCCGTAGAACTGGCAAAAAAGAACGGGGCAAAGATTATTTGTATAACCCATAGCTACCATTCACCGATAGCGAAACTGGCCGATTATATTATTTGCTCACCAGCCCCGGAAACGCCGTTATTAGGTCGTAATGCCTCGGCAAGAATATTACAACTAACTTTGCTGGACGCTTTTTTTGTCTCTGTCGCCCAGCTCAACATTGAACAAGCTAATATTAATATGCAAAAAACCGGCGCAATTGTTGATTTCTTCTCACCAGGCGCGCTGAAATAA [SEQ ID NO:73]

[0244] atgAGCCAGTCAGAGTTTGATTCAGCGCTTCCGAACGGTATAGGGTTAGCGCCTTACCTGCGAATGAAGCAGGAAGGAATGACAGAAAATGAAAGCCGCATCGTGGAGTGGTTACTCAAACCCGGTAACCTGAGTTGTGCACCCGCAATTAAAGATGTCGCAGAAGCTCTGGCGGTATCTGAAGCGATGATAGTTAAGGTATCAAAGCTGCTGGGGTTTAGCGGCTTTCGTAACTTACGCAGTGCGCTGGAAGATTATTTTTCTCAGTCAGAACAGGTATTGCCTTCCGAGTTGGCTTTTGATGAAGCGCCGCAGGATGTGGTGAATAAGGTATTTAACATCACTTTACGCACCATTATGGAAGGTCAGTCGATCGTCAACGTTGATGAGATCCACCGTGCCGCCCGCTTTTTCTATCAGGCCAGACAGCGGGATTTGTACGGTGCCGGAGGATCAAATGCTATCTGTGCTGATGTACAGCACAAGTTCTTGCGCATTGGCGTACGCTGTCAGGCCTATCCTGATGCTCACATCATGATGATGTCCGCTTCGTTGTTACAGGAAGGAGATGTTGTGCTGGTAGTGACCCATTCCGGGCGAACCAGTGATGTAAAAGCGGCCGTAGAACTGGCAAAAAAGAACGGGGCAAAGATTATTTGTATAACCCATAGCTACCATTCACCGATAGCGAAACTGGCCGATTATATTATTTGCTCACCAGCCCCGGAAACGCCGTTATTAGGTCGTAATGCCTCGGCAAGAATATTACAACTAACTTTGCTGGACGCTTTTTTTGTCTCTGTCGCCCAGCTCAACATTGAACAAGCTAATATTAATATGCAAAAAACCGGCGCAATTGTTGATTTCTTCTCACCAGGCGCGCTGAAAtaa[SEQ ID NO:84]

[0245] In some embodiments, *E. coli* AlsR comprises 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% of the amino acid sequence shown in SEQ ID NO: 72. In some embodiments, *E. coli* AlsR comprises the amino acid sequence shown in SEQ ID NO: 72. SEQ ID NO: 72 is provided below.

[0246] MSQSEFDSALPNGIGLAPYLRMKQEGMTENESRIVEWLLKPGNLSCAPAIKDVAEALAVSEAMIVKVSKLLGFSGFRNLRSALEDYFSQSEQVLPSELAFDEAPQDVVNKVFNITLRTIMEGQSIVNVDEIHRAARFFYQARQRDLYGAG GSNAICADVQHKFLRIGVRCQAYPDAHIMMMSASLLQEGDVVLVVTHSGRTSDVKAAVELAKKNGAKIICITHSYHSPIAKLADYIICSPAPETPLLGRNASARILQLTLLDAFFVSVAQLNIEQANINMQKTGAIVDFFSPGALK[SEQ ID NO: 72]

[0247] In some embodiments, gene deletion includes non-frameshift deletion, frameshift deletion, or a combination thereof. In some embodiments, gene deletion can be achieved by insertion (e.g., non-frameshift insertion, frameshift insertion, or a combination thereof). In some embodiments, gene deletion includes nonsense mutations.

[0248] 2.3. Cells

[0249] This disclosure provides recombinant microorganisms. Any culturable microorganism is suitable for the compositions and methods described herein. In some embodiments, the microorganism is a bacterium. In some embodiments, the microorganism is selected from: Acetobacter acetobacter (… Aceiobacter aceti ), Achromobacterium spp. Achromobacter ), Acidophilus genus ( Acidiphilium Acinetobacter spp. Acinetobacter Madura Actinobacteria ( Actinomadura ), genus Actinomycetes ( Actinoplanes ), Agile thermophilic bacteria ( Aeropyrumpernix ), Agrobacterium ( Agrobacterium ), Alcaligenes ( Alkaligenes ), Pineapple comosus(M) Arthrobacter spp. Arthrobacter ), Bacillus alkalophilus ( Bacillus alcalophilus ), Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), Bacillus brevis ( Bacillus brevis ), Bacillus circularis ( Bacillus circularis ), Bacillus clausti ( Bacillus clausii ), Bacillus tarda ( Bacillus lentus ), Bacillus licheniformis ( Bacillus licheniformis ), Bacillus subtilis ( Bacillus soaking ), thermophilic steatobacterium ( Bacillus stearothermophilus Bacillus subtilis ( Bacillus subtle Bifidobacterium spp. Bifidobacterium ), Bacillus shortiflora ( Brevibacillus brevis Burkholderia cepacia (), Burkholderia cepacia ), Candida columnarum ( Candida cylindrica ), Charge papaya(L) Fiber microbes ( Cellulose microbiome ), Cephalosporium genus ( Cephalosporium ), amorphous chaetotrichum ( Chaetomium erraticum ), Fine-grained Chaetomium ( Chaetomium gracile Clostridium ( Clostridium Clostridium butyricum ( Clostridium butyricum Clostridium acetonebutanol ( Clostridium acetobutylicum Clostridium thermophilum ( ) Clostridium thermocellum Corynebacterium (Glutamic acid Corynebacterium) Corynebacterium (glutamic acid) ), High-efficiency Corynebacterium ( Corynebacterium efficiens ), Escherichia coli ( Escherichia to be cultivated ), Enterococcus spp. Enterococcus Erwinia chrysogenum ( Erwina chrysanthemum Staphylococcus spp. Glycobacterium ), Staphylococcus spp. Gluconacetobacter ), genus *Saltboxia* Halo arch ), specific humic mold ( Humicola insolens ), Kitarispora setada ( Kitasatospora setae ), Klebsiella spp. Klebsiella Klebsiella pneumoniae ( ) Klebsiella oxytoca ), Cochlea spp. Kocuria ), Milk milk Lactobacillus ( Lactobacillus ), Lactobacillus fermentum ( Lactobacillus fermentum ), Lactobacillus casei ( Lactobacillus sake Lactococcus spp. Lactococcus ), Lactococcus lactis ( Lactococcus lactis Leuconostoc ( ) Leuconostoc ), Methanogenic bacteria ( Methylocystis ), Sicilian methanophora ( Methanol Sicily ), organophilic methylbacterium ( Organophilic methanogen ), Methanobacterium brevicornu ( Methanobacterium bryantii ), moth microbacterium ( Microbacterium imperialis ), Micrococcus lysate ( Micrococcus lysodeikticus ), genus *Malus* Microlunate ), Java mold ( Mucorjavanicus ), Mycobacterium ( Mycobacterium ), genus *Lycoperdon* ( Myrothecium ), Nitrifying Bacteria ( Nitrobacter ), Nitrosomonas ( Nitrosomonas Nocardia ( ) Nocardia ), Papaya carica Pediococcus ( Pediococcus ), halophilic Pediococcus ( Pediococcus halophilus ), Paracoccus pantrophicus ( Paracoccus omnivorous ), Propionibacterium spp. Propionibacterium ), Pseudomonas spp. Pseudomonas ), Fluorescent Pseudomonas ( Pseudomonas fluorescens ), denitrifying Pseudomonas ( Pseudomonas denitrificans ), genus *Pomacea* Pyrococcus ), Vibrio parahaemolyticus ( Pyrococcusfuriosus ), Fireball bacteria ( Pyrococcus horikoshii Rhizobium ( ) Rhizobium ), Rhizopus oryzae ( Rhizomucor miehei ), microrhizobium ( Rhizomucor pusillus Lindt ), Rhizopus ( Rhizopus ), Dalborneella ( Rhizopus delemar ), Rhizopus japonicum ( Rhizopus japonicas ), Rhizopus spp. ( Rhizopus niveus ), Rhizopus oryzae ( Rhizopus rice ), Rhizopus oligosporus ( Rhizopus oligosporus ), Rhodococcus ( Rhodococcus ), Scroiina libertina Polysphingosine monocytogenes ( Sphingobacterium multivorum ), Sphingolipids ( Sphingomyelophytum ), Sphingomyelinus ( Sphingomyelomonas Streptococcus spp. Streptococcus Streptococcus thermophilus Y-1 ( Streptococcus thermophilus Y-1 Streptomyces ( Streptomyces ), Streptomyces griseus ( Streptomyces griseus Streptomyces limonene ( ), Streptomyces limonene ( Streptomyces lividans Streptomyces oryzae ( Streptomyces murineus ), Streptomyces rustii ( Streptomyces ruighinosus ), Streptomyces purpureus ( Streptomyces violaceoruber ), Streptomyces mohara ( Streptoverticillium mobaraense), Tetracoccus ( Tetragenococcus ), Thermomyces ( Thermae ), aerobic denitrifying bacteria ( Thiosphaera pantotropha ), spp. Trametes ), alginolytic Vibrio ( Vibrio alginolyticus Xanthomonas ( ) Xanthomonas ), Fermentation Monotrophs ( Zymomonas ) and motile fermentation monoclonal bacteria ( Zymomonus mobile In some embodiments, the microorganism is *Escherichia coli*. In some embodiments, the microorganism is *Bacillus subtilis*. In some embodiments, the microorganism is *Lactococcus lactis*.

[0250] In some embodiments, the *Escherichia coli* is selected from enterotoxigenic *Escherichia coli* (ETEC), enteropathogenic *Escherichia coli* (EPEC), enteroinvasive *Escherichia coli* (EIEC), enterohemorrhagic *Escherichia coli* (EHEC), urinary tract pathogenic *Escherichia coli* (UPEC), vemurotoxin-producing *Escherichia coli*, *Escherichia coli* O157:H7, *Escherichia coli* O104:H4, *Escherichia coli* O121, *Escherichia coli* O104:H21, *Escherichia coli* K1, and *Escherichia coli* NC101. In some embodiments, the *Escherichia coli* is *Escherichia coli* K12. In some embodiments, the *Escherichia coli* is *Escherichia coli* B. In some embodiments, the *Escherichia coli* is *Escherichia coli* C.

[0251] In some implementations, *Escherichia coli* is derived from strains selected from the following: NCTC 12757, NCTC 12779, NCTC 12790, NCTC 12796, NCTC 12811, ATCC 11229, ATCC 25922, ATCC 8739, DSM 30083, BC5849, BC 8265, BC 8267, BC 8268, BC 8270, 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.

[0252] In some implementations, *Escherichia coli* is derived from strains selected from the following: BC 4734 (O26:H11), BC4735 (O157:H-), BC 4736, BC 4737 (nd), BC 4738 (O157:H7), BC 4945 (O26:H-), BC 4946 (O157:H7), BC 4947 (O111:H-), BC 4948 (O157:H), BC 4949 (O5), BC 5579 (O157:H7), BC5580 (O157:H7), BC 5582 (O3:H), BC 5643 (O2:H5), BC 5644 (O128), BC 5645 (O55:H-), BC5646 (O69:H-), BC 5647 (O101:H9), BC 5648 (O103:H2), BC 5850 (O22:H8), BC 5851 (O55:H-), BC 5852 (O48:H21), BC 5853 (O26:H11), BC 5854 (O157:H7), BC 5855 (O157:H-), BC 5856 (O26:H-), BC 5857 (O103:H2), BC 5858 (O26:H11), BC 7832, BC 7833 (Oraw form:H-), BC 7834 (ONT:H-), BC 7835 (O103:H2), BC 7836 (O57:H-), BC 7837 (ONT:H-), BC 7838, BC 7839(O128:H2), BC 7840(O157:H-), BC 7841(O23:H-), BC 7842(O157:H-), BC 7843, BC 7844(O157:H-), BC 7845(O103:H2), BC 7846(O26:H11), BC 7847(O145:H-), BC 7848(O157:H-), BC 7849(O156:H47), BC 7850, BC 7851(O157:H-), BC 7852(O157:H-), BC 7853(O5:H-), BC 7854(O157:H7), BC 7855(O157:H7), BC 7856 (O26:H-), BC 7857, BC 7858, BC 7859 (ONT:H-), BC 7860 (O129:H-), BC 7861, BC 7862 (O103:H2), BC 7863, BC 7864 (O original form:H-), BC 7865, BC 7866 (O26:H-), BC7867 (O original form: H-), BC 7868, BC7869 (ONT: H-), BC 7870 (O113: H-), BC 7871 (ONT: H-), BC 7872 (ONT: H-), BC 7873, BC 7874 (O original form: H-), BC 7875 (O157: H-), BC 7876 (O111: H-), BC 7877 (O146: H21), BC 7878 (O145: H-), BC 7879 (O22: H8), BC 7880 (O original form: H-), BC 7881 (O145: H-), BC 8275 (O157: H7), BC 8318 (O55: K-: H-), BC 8325 (O157: H7), BC 8332(ONT) and BC 8333.

[0253] In some implementations, *Escherichia coli* is derived from strains selected from the following: BC 8246(O152:K-:H-), BC8247(O124:K(72):H3), BC 8248(O124), BC 8249(O112), BC 8250(O136:K(78):H-), BC8251(O124:H-), BC 8252(O144:K-:H-), BC 8253(O143:K:H-), BC 8254(O143), BC 8255(O112), BC 8256(O28a.e), BC 8257(O124:H-), BC 8258(O143), BC 8259(O167:K-:H5), BC8260(O128a.c.:H35), BC 8261(O164), BC 8262(O164:K-:H-), BC 8263(O164) and BC 8264(O124).

[0254] In some embodiments, *Escherichia coli* is derived from strains selected from the following: BC 5581 (O78:H11), BC5583 (O2:K1), BC 8221 (O118), BC 8222 (O148:H-), BC 8223 (O111), BC 8224 (O110:H-), BC8225 (O148), BC 8226 (O118), BC 8227 (O25:H42), BC 8229 (O6), BC 8231 (O153:H45), BC8232 (O9), BC 8233 (O148), BC 8234 (O128), BC 8235 (O118), BC 8237 (O111), BC 8238 (O110:H17), BC 8240 (O148), BC 8241(O6H16), BC 8243(O153), BC 8244(O15:H-), BC 8245(O20), BC 8269(O125a.c:H-), BC 8313(O6:H6), BC 8315(O153:H-), BC 8329, BC 8334(O118:H12) and BC 8339.

[0255] In some embodiments, *Escherichia coli* is derived from strains selected from the following: BC 7567 (O86), BC 7568 (O128), BC 7571 (O114), BC 7572 (O119), BC 7573 (O125), BC 7574 (O124), BC 7576 (O127a), BC 7577 (O126), BC 7578 (O142), BC 7579 (O26), BC 7580 (O26), BC 7581 (O142), BC 7582 (O55), BC 7583 (O158), BC 7584 (O-), BC 7585 (O-), BC 7586 (O-), BC 8330, BC 8550 (O26), BC 8551 (O55), BC 8552(O158), BC 8553(O26), BC 8554(O158), BC 8555(O86), BC 8556(O128), BC 8557(OK26), BC 8558(O55), BC 8560(O158), BC 8561(O158), BC 8562(O114), BC 8563(O86), BC 8564(O128), BC 8565(O158), BC 8566(O158), BC 8567(O158), BC 8568(O111), BC 8569(O128), BC 8570(O114), BC 8571(O128), BC 8572(O128), BC 8573(O158), BC 8574(O158), BC 8575(O158), BC 8576(O158), BC 8577(O158), BC 8578(O158), BC 8581(O158), BC 8583(O128), BC 8584(O158), BC 8585(O128), BC 8586(O158), BC 8588(O26), BC 8589(O86), BC 8590(O127), BC 8591(O128), BC 8592(O114), BC 8593(O114), BC8594(O114), BC 8595(O125), BC 8596(O158), BC 8597(O26), BC 8598(O26), BC 8599(O158), BC 8605(O158), BC 8606(O158), BC 8607(O158), BC 8608(O128), BC 8609(O55), BC 8610(O114), BC 8615(O158), BC 8616(O128), BC8617(O26), BC 8618(O86), BC 8619, BC 8620, BC 8621, BC 8622, BC 8623, BC 8624(O158) and BC 8625(O158).

[0256] In some implementations, Bacillus subtilis is derived from strain 168.

[0257] In some implementations, the lactococcus is derived from strain A12.

[0258] In some embodiments, the microorganism is a fungal cell. In some embodiments, the fungal cell is selected from the genus *Aspergillus*. Aspergillus Aspergillus nidus ( ) Aspergillus nidulans ), Aspergillus niger ( Black Aspargillus Aspergillus oryzae ( Aspargillus oryzae ), Aspergillus honey Aspergillus melleus ), powdery Aspergillus ( Aspergillus dusty Aspergillus spp. (Saito) Aspergillus saitoi Aspergillus oryzae ( ), soy sauce aspergillus ( Aspergillus sojae Aspergillus terreus () Aspergillus terreus Aspergillus pseudoterreus ( ) Aspergillus pseudoterreus ), Aspergillus Usami( Aspergillus usamii ), Candida folds ( White wrinkled ), Oriental Isaac yeast ( Issatchenkia oriental ), Kluyveromyces ( Kluyveromyces Kluyveromyces brittle-walled Kluyveromycesfragilis Kluyveromycin (lactic acid yeast) Kluyveromyces lactis ), Max Kluyveromycin ( Kluyveromyces marxianas ), Penicillium ( Penicillium ), Penicillium carmenensis ( Penicillium camemberti ), Penicillium citrinum ( Penicillium citrinum ), Emersonian penicillin ( Penicillium emersonii ), Penicillium loudi ( Penicillium roqueforti ), Palepurium ( Penicillum lilactinum ), Penicillium polychromatum ( Penicillum multicolor ), Red syringo yeast ( Rhodosporidium toruloides ), brewer's yeast ( Saccharomyces cerevisiae ), Saccharomyces cerevisiae ( Schizosaccharomyces pombe Trichoderma ( ) Trichoderma ), Trichoderma longifolia ( Trichoderma longibrachiatum Trichoderma reesei ( Trichoderma reesei ), green Trichoderma ( Trichoderma viride ), Penicillium spores ( Trichosporon penicillaium) Yeast extract ( Yarrowia lipolytica ) and Luvian conjugated yeast ( Zygosaccharomyces rouxii).

[0259] In some embodiments, the microorganism is a yeast cell. In some embodiments, the yeast cell is *Saccharomyces cerevisiae*.

[0260] 2.4. Exemplary Microorganisms

[0261] In some embodiments, this disclosure provides recombinant microorganisms that, compared to naturally occurring microorganisms, contain increased allose production. In some embodiments, the recombinant microorganisms comprise an exogenous epimerase, an exogenous isomerase, and an exogenous phosphatase. In some embodiments, the exogenous epimerase is allulose-6-phosphate 3-epimerase (AlsE). In some embodiments, the exogenous isomerase is allose-6-phosphate isomerase (RpiB). In some embodiments, the exogenous phosphatase is hexitol phosphatase A (HxpA). In some embodiments, the recombinant microorganism is a bacterium. In some embodiments, the bacterium is *Escherichia coli*.

[0262] In some embodiments, this disclosure provides recombinant microorganisms that, compared to naturally occurring microorganisms, contain increased allose production. In some embodiments, the recombinant microorganisms comprise an exogenous epimerase, an exogenous isomerase, and an exogenous phosphatase. In some embodiments, the exogenous epimerase is allulose-6-phosphate 3-epimerase (AlsE). In some embodiments, the exogenous isomerase is allose-6-phosphate isomerase (RpiB). In some embodiments, the exogenous phosphatase is fructose-1-phosphate phosphatase (YqaB). In some embodiments, the recombinant microorganism is a bacterium. In some embodiments, the bacterium is *Escherichia coli*.

[0263] In some embodiments, this disclosure provides recombinant microorganisms that, compared to naturally occurring microorganisms, contain increased allosugar production. In some embodiments, the recombinant microorganism comprises the deletion of an exogenous epimerase, an exogenous isomerase, an exogenous phosphatase, and one, two, three, or four (4) or more genes. In some embodiments, the exogenous epimerase is allulose-6-phosphate 3-epimerase (AlsE). In some embodiments, the exogenous isomerase is allosugar-6-phosphate isomerase (RpiB). In some embodiments, the exogenous phosphatase is hexitol phosphatase A (HxpA). In some embodiments, the deleted genes are glucose-6-phosphate 1-dehydrogenase, phosphofructokinase-1, and mannose-6-phosphate isomerase. In some embodiments, the recombinant microorganism is a bacterium. In some embodiments, the bacterium is *Escherichia coli*.

[0264] In some embodiments, this disclosure provides recombinant microorganisms that, compared to naturally occurring microorganisms, contain increased allosugar production. In some embodiments, the recombinant microorganism comprises the deletion of an exogenous epimerase, an exogenous isomerase, an exogenous phosphatase, and one, two, three, or four (4) or more genes. In some embodiments, the exogenous epimerase is allulose-6-phosphate 3-epimerase (AlsE). In some embodiments, the exogenous isomerase is allosugar-6-phosphate isomerase (RpiB). In some embodiments, the exogenous phosphatase is fructose-1-phosphate phosphatase (YqaB). In some embodiments, the deleted genes are glucose-6-phosphate 1-dehydrogenase, phosphofructokinase-1, and mannose-6-phosphate isomerase. In some embodiments, the recombinant microorganism is a bacterium. In some embodiments, the bacterium is *Escherichia coli*.

[0265] In some embodiments, this disclosure provides microorganisms comprising recombinant polynucleotides, wherein the microorganisms contain increased allose production compared to naturally occurring microorganisms. In some embodiments, the recombinant polynucleotides comprise nucleotide sequences encoding exogenous epimerase, exogenous isomerase, and exogenous phosphatase. In some embodiments, the epimerase is allulose-6-phosphate 3-epimerase (AlsE). In some embodiments, the exogenous isomerase is allose-6-phosphate isomerase (RpiB). In some embodiments, the exogenous phosphatase is hexitol phosphatase A (HxpA). In some embodiments, the recombinant microorganism is a bacterium. In some embodiments, the bacterium is *Escherichia coli*.

[0266] In some embodiments, this disclosure provides microorganisms comprising recombinant polynucleotides, wherein the microorganisms contain increased allose production compared to naturally occurring microorganisms. In some embodiments, the recombinant polynucleotides comprise nucleotide sequences encoding exogenous epimerase, exogenous isomerase, and exogenous phosphatase. In some embodiments, the exogenous epimerase is allulose-6-phosphate 3-epimerase (AlsE). In some embodiments, the exogenous isomerase is allose-6-phosphate isomerase (RpiB). In some embodiments, the exogenous phosphatase is fructose-1-phosphate phosphatase (YqaB). In some embodiments, the recombinant microorganism is a bacterium. In some embodiments, the bacterium is *Escherichia coli*.

[0267] In some embodiments, this disclosure provides microorganisms comprising recombinant polynucleotides, wherein the microorganisms contain increased allose production compared to naturally occurring microorganisms. In some embodiments, the recombinant polynucleotides comprise nucleotide sequences encoding exogenous epimerase, exogenous isomerase, and exogenous phosphatase. In some embodiments, the exogenous epimerase is allulose-6-phosphate 3-epimerase (AlsE). In some embodiments, the exogenous isomerase is allose-6-phosphate isomerase (RpiB). In some embodiments, the exogenous phosphatase is hexitol phosphatase A (HxpA). In some embodiments, the recombinant polynucleotides also comprise nucleotides encoding exogenous isomerases. In some embodiments, the exogenous isomerase is allose-6-phosphate isomerase (RpiB). In some embodiments, the microorganisms also comprise a deletion of a first gene. In some embodiments, the first gene is glucose-6-phosphate 1-dehydrogenase. In some embodiments, the microorganisms also comprise a deletion of a second gene. In some embodiments, the second gene is phosphofructokinase-1. In some embodiments, the microorganism further includes the deletion of a third gene. In some embodiments, the third gene is mannose-6-phosphate isomerase. In some embodiments, the microorganism further includes the deletion of a fourth gene. In some embodiments, the fourth gene is deoxyribose-phosphoaldolase (DeoC). In some embodiments, in addition to the four deleted genes, the microorganism also includes the deletion of at least one gene selected from the following: D-allose importer ATP-binding protein (AlsA), D-allose-binding periplasmic protein (AlsB), D-allose transporter system permease protein (AlsC), D-allose kinase (AlsK), HTH-type transcription regulator (AlsR), fructose-1-phosphate phosphatase (YqaB), sugar phosphatase YbiV, hexitol phosphatase B (HxpB), sugar phosphatase YidA, or combinations thereof. In some embodiments, the recombinant microorganism further includes exogenous galactose:H + The recombinant microorganism contains a cotransporter protein (GalP) and glucokinase (Glk). In some embodiments, GalP is *E. coli* GalP and Glk is *E. coli* Glk. In some embodiments, the recombinant microorganism also contains an exogenous glucose-6-phosphate isomerase (Gpi). In some embodiments, the recombinant microorganism also contains a mutation in the gene encoding an enzyme of the phosphotransferase system (PTS). In some embodiments, the recombinant microorganism is a bacterium. In some embodiments, the bacterium is *Escherichia coli*. In some embodiments, the bacterium is *Bacillus subtilis*. In some embodiments, the bacterium is *Lactococcus lactis*.

[0268] In some embodiments, this disclosure provides microorganisms comprising recombinant polynucleotides, wherein the microorganisms contain increased allose production compared to naturally occurring microorganisms. In some embodiments, the recombinant polynucleotides comprise nucleotide sequences encoding exogenous epimerase, exogenous isomerase, and exogenous phosphatase. In some embodiments, the exogenous epimerase is allulose-6-phosphate 3-epimerase (AlsE). In some embodiments, the exogenous isomerase is allose-6-phosphate isomerase (RpiB). In some embodiments, the exogenous phosphatase is fructose-1-phosphate phosphatase (YqaB). In some embodiments, the recombinant polynucleotides further comprise nucleotides encoding the exogenous isomerase. In some embodiments, the exogenous isomerase is allose-6-phosphate isomerase (RpiB). In some embodiments, the microorganisms further comprise a deletion of a first gene. In some embodiments, the first gene is glucose-6-phosphate 1-dehydrogenase. In some embodiments, the microorganisms further comprise a deletion of a second gene. In some embodiments, the second gene is phosphofructokinase-1. In some embodiments, the microorganism further includes the deletion of a third gene. In some embodiments, the third gene is mannose-6-phosphate isomerase. In some embodiments, the microorganism further includes the deletion of a fourth gene. In some embodiments, the fourth gene is deoxyribose-phosphoaldolase (DeoC). In some embodiments, in addition to the four deleted genes, the microorganism also includes the deletion of at least one gene selected from the following: D-allose input ATP-binding protein (AlsA), D-allose binding periplasmic protein (AlsB), D-allose transport system permease protein (AlsC), D-allose kinase (AlsK), HTH type transcription regulator (AlsR), fructose-1-phosphate phosphatase (YqaB), sugar phosphatase YbiV, hexitol phosphatase B (HxpB), sugar phosphatase YidA, or combinations thereof. In some embodiments, the recombinant microorganism further includes exogenous galactose:H + The recombinant microorganism contains a cotransporter protein (GalP) and glucokinase (Glk). In some embodiments, GalP is *E. coli* GalP and Glk is *E. coli* Glk. In some embodiments, the recombinant microorganism also contains a mutation in the gene encoding an enzyme of the phosphotransferase system (PTS).

[0269] In some embodiments, the recombinant microorganism is a bacterium. In some embodiments, the bacterium is *Escherichia coli*. In some embodiments, the bacterium is *Bacillus subtilis*. In some embodiments, the bacterium is *Lactococcus lactis*.

[0270] In some embodiments, this disclosure provides recombinant microorganisms that, compared to naturally occurring microorganisms, contain increased allosugar production. In some embodiments, the recombinant microorganisms contain deletions of an exogenous epimerase, an exogenous isomerase, an exogenous phosphatase, an exogenous nuclease, sgRNA, and four (4) genes. In some embodiments, the exogenous epimerase is allulose-6-phosphate 3-epimerase (AlsE). In some embodiments, the exogenous isomerase is allosugar-6-phosphate isomerase (RpiB). In some embodiments, the exogenous phosphatase is hexitol phosphatase A (HxpA). In some embodiments, the four deleted genes are... zwf、pfkA、manA and deoC In some implementations, in addition to the four missing genes, the microorganism also contains at least one gene selected from... alsA, alsB, alsC, alsK, alsR, yqaB, ybiV, hxpB, yidA Or a combination of these gene deletions. In some implementations, sgRNA targets pfkB In some implementations, the recombinant microorganisms also contain exogenous galactose:H + The recombinant microorganism contains a cotransporter protein (GalP) and glucokinase (Glk). In some embodiments, GalP is *E. coli* GalP and Glk is *E. coli* Glk. In some embodiments, the recombinant microorganism also contains a mutation in the gene encoding an enzyme of the phosphotransferase system (PTS).

[0271] In some embodiments, the recombinant microorganism is a bacterium. In some embodiments, the bacterium is *Escherichia coli*. In some embodiments, the bacterium is *Bacillus subtilis*. In some embodiments, the bacterium is *Lactococcus lactis*.

[0272] In some embodiments, this disclosure provides recombinant microorganisms that, compared to naturally occurring microorganisms, contain increased allosugar production. In some embodiments, the recombinant microorganisms contain deletions of an exogenous epimerase, an exogenous isomerase, an exogenous phosphatase, an exogenous nuclease, sgRNA, and four (4) genes. In some embodiments, the exogenous epimerase is allulose-6-phosphate-3-epimerase (AlsE). In some embodiments, the exogenous isomerase is allosugar-6-phosphate isomerase (RpiB). In some embodiments, the exogenous phosphatase is fructose-1-phosphate phosphatase (YqaB). In some embodiments, the exogenous nuclease is dCas9. In some embodiments, the four deleted genes are... zwf、pfkA、manA and deoC In some implementations, in addition to the four missing genes, the microorganism also contains at least one gene selected from... alsA, alsB, alsC, alsK, alsR, ybiV, hxpB, yidA Or a combination of these gene deletions. In some implementations, sgRNA targets pfkB In some implementations, the recombinant microorganisms also contain exogenous galactose:H + The recombinant microorganism contains a cotransporter protein (GalP) and glucokinase (Glk). In some embodiments, GalP is *E. coli* GalP and Glk is *E. coli* Glk. In some embodiments, the recombinant microorganism also contains an exogenous glucose-6-phosphate isomerase (Gpi). In some embodiments, the recombinant microorganism also contains a mutation in the gene encoding an enzyme of the phosphotransferase system (PTS). In some embodiments, the recombinant microorganism is a bacterium. In some embodiments, the bacterium is *Escherichia coli*. In some embodiments, the bacterium is *Bacillus subtilis*. In some embodiments, the bacterium is *Lactococcus lactis*.

[0273] In some embodiments, this disclosure provides microorganisms comprising recombinant polynucleotides, wherein the microorganisms contain increased allosugar production compared to naturally occurring microorganisms. In some embodiments, the recombinant polynucleotide comprises a nucleotide sequence encoding an exogenous epimerase, a nucleotide sequence encoding an exogenous isomerase, a nucleotide sequence encoding an exogenous phosphatase, a nucleotide sequence encoding an exogenous isomerase, and a nucleotide sequence encoding a nuclease. In some embodiments, the exogenous epimerase is allulose-6-phosphate 3-epimerase (AlsE). In some embodiments, the exogenous isomerase is allosugar-6-phosphate isomerase (RpiB). In some embodiments, the exogenous phosphatase is hexitol phosphatase A (HxpA). In some embodiments, the exogenous nuclease is dCas9. In some embodiments, the microorganism also comprises a deletion of a first gene. In some embodiments, the first gene is... zwf In some embodiments, the microorganism also contains a missing second gene. In some embodiments, the second gene is... pfkA In some embodiments, the microorganism also contains the deletion of a third gene. In some embodiments, the third gene is... manA In some embodiments, the microorganism also contains the deletion of a fourth gene. In some embodiments, the fourth gene is... deoC In some implementations, in addition to the four missing genes, the microorganism also contains at least one gene selected from... alsA, alsB, alsC, alsK, alsR, pgm, yqaB, ybiV, hxpB, yidA Or a combination thereof, including gene deletion. In some embodiments, the microorganism also contains sgRNA. In some embodiments, the sgRNA targets... pfkB In some implementations, the recombinant microorganisms also contain exogenous galactose:H +The recombinant microorganism contains a cotransporter protein (GalP) and glucokinase (Glk). In some embodiments, GalP is *E. coli* GalP and Glk is *E. coli* Glk. In some embodiments, the recombinant microorganism also contains an exogenous glucose-6-phosphate isomerase (Gpi). In some embodiments, the recombinant microorganism also contains a mutation in the gene encoding an enzyme of the phosphotransferase system (PTS). In some embodiments, the recombinant microorganism is a bacterium. In some embodiments, the bacterium is *Escherichia coli*. In some embodiments, the bacterium is *Bacillus subtilis*. In some embodiments, the bacterium is *Lactococcus lactis*.

[0274] In some embodiments, this disclosure provides microorganisms comprising recombinant polynucleotides, wherein the microorganisms contain increased allosugar production compared to naturally occurring microorganisms. In some embodiments, the recombinant polynucleotides comprise nucleotide sequences encoding an exogenous epimerase, an exogenous isomerase, an exogenous phosphatase, an exogenous isomerase, and a nuclease. In some embodiments, the exogenous epimerase is allulose-6-phosphate 3-epimerase (AlsE). In some embodiments, the exogenous isomerase is allosugar-6-phosphate isomerase (RpiB). In some embodiments, the exogenous phosphatase is fructose-1-phosphate phosphatase (YqaB). In some embodiments, the microorganisms also contain a deletion of a first gene. In some embodiments, the first gene is... zwf In some embodiments, the microorganism also contains a missing second gene. In some embodiments, the second gene is... pfkA In some embodiments, the microorganism also contains the deletion of a third gene. In some embodiments, the third gene is... manA In some embodiments, the microorganism also contains the deletion of a fourth gene. In some embodiments, the fourth gene is... deoC In some implementations, in addition to the four missing genes, the microorganism also contains at least one gene selected from... alsA, alsB, alsC, alsK, alsR, pgm, ybiV, hxpB, yidA Or a combination thereof, including gene deletion. In some embodiments, the microorganism also contains sgRNA. In some embodiments, the sgRNA targets... pfkB In some implementations, the recombinant microorganisms also contain exogenous galactose:H +The recombinant microorganism contains a cotransporter protein (GalP) and glucokinase (Glk). In some embodiments, GalP is *E. coli* GalP and Glk is *E. coli* Glk. In some embodiments, the recombinant microorganism also contains an exogenous glucose-6-phosphate isomerase (Gpi). In some embodiments, the recombinant microorganism also contains a mutation in the gene encoding an enzyme of the phosphotransferase system (PTS). In some embodiments, the recombinant microorganism is a bacterium. In some embodiments, the bacterium is *Escherichia coli*. In some embodiments, the bacterium is *Bacillus subtilis*. In some embodiments, the bacterium is *Lactococcus lactis*.

[0275] 3. Methods for producing and generating microbial allosugar

[0276] This disclosure also provides methods for preparing and / or generating any of the microorganisms disclosed herein. One or more recombinant polynucleotides of this disclosure can be introduced into microorganisms using many recombinant techniques well known in the art, including but not limited to protoplast fusion, transfection, transformation, conjugation, and transduction. These techniques include conventional molecular biology techniques (e.g., recombinant techniques), microbiology, cell biology, and biochemistry, which are within the scope of the art. Further information on these techniques can be found in *Molecular Cloning: A Laboratory Manual, second edition* (Sambrook et al., 1989); *Oligonucleotide Synthesis* (Gait, ed., 1984); *Animal Cell Culture* (Freshney, ed., 1987); *Gene Transfer Vectors for Mammalian Cells* (Miller & Calos, eds., 1987); *Current Protocols in Molecular Biology* (Ausubelet et al., eds., 1987); *PCR: The Polymerase Chain Reaction* (Mullis et al., eds., 1994); and *Current Protocols in Immunology* (Coligan et al., eds., 1991).

[0277] 3.1. Recombinant polynucleotides

[0278] In some embodiments, the recombinant polynucleotides disclosed herein can be stably integrated into microbial chromosomes. In some embodiments, the recombinant polynucleotides disclosed herein are stably integrated into microbial chromosomes using homologous recombination, transposon-based chromosome integration, recombinase-mediated cassette exchange (RMCE; e.g., using the Cre-lox system), or integration plasmids (e.g., yeast integration plasmids). A variety of integration techniques suitable for a range of microorganisms are known in the art (see, e.g., Griffiths, AJF, Miller, JH, Suzuki, DT). et al. AnIntroduction to Genetic Analysis. 7 th (ed. New York: WH Freeman; 2000). In some embodiments, the recombinant polynucleotides disclosed herein are maintained in the recombinant microorganisms of this disclosure on an extrachromosomal plasmid (e.g., an expression plasmid or vector). A variety of extrachromosomal plasmids suitable for a range of microorganisms are known in the art, including but not limited to replication plasmids (e.g., yeast replication plasmids containing the autonomous replication sequence ARS), centromere plasmids (e.g., yeast centromere plasmids containing the autonomous replication sequence CEN), episome plasmids (e.g., 2-pm plasmids), and / or artificial chromosomes (e.g., yeast artificial chromosome YAC or bacterial artificial chromosome BAC).

[0279] 3.1.1. Carrier

[0280] In some embodiments, this disclosure provides vectors comprising 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 may include, but are not limited to, cloning vectors, expression vectors, shuttle vectors, plasmids, and cassettes. As used herein, the term "plasmid" refers to a circular double-stranded DNA construct used as a cloning and / or expression vector. In some embodiments, when introduced into microorganisms, plasmids may be extrachromosomal self-replicating genetic elements (e.g., episome plasmids). In some embodiments, plasmids may be integrated into the microbial chromosome. In some embodiments, vectors may direct the expression of coding regions operatively linked thereto, e.g., "expression vectors." These expression vectors allow the expression of exogenous polynucleotides and / or polypeptides in microorganisms. In some embodiments, vectors allow the integration of one or more polynucleotides into the genome of a microorganism.

[0281] In some embodiments, the vectors disclosed herein contain a promoter. In some embodiments, the vector is a bacterial or prokaryotic expression vector. In some embodiments, the vector is a yeast or fungal cell expression vector.

[0282] In some embodiments, the vector disclosed herein contains a nucleotide sequence in a single operon.

[0283] 3.1.2. Promoter

[0284] In some non-limiting embodiments, the recombinant polynucleotides disclosed herein include control sequences, enhancers, or promoters. For example, but not limited to, encoding... alsE Gene, hxpA Genes and / or yigL The nucleotide sequence of a gene can be operatively linked to control sequences, enhancers, or promoters.

[0285] As used herein, the term "promoter" refers to any nucleotide sequence under its control that regulates the initiation of transcription of a particular coding sequence. Biologically, promoters are not transcribed themselves, but rather coordinate the assembly of components that initiate the transcription of other nucleotide sequences. Furthermore, promoters can restrict this assembly and subsequent transcription to specific prerequisites. For example, but not limited to, promoters can allow transcription in response to one or more environmental, temporal, or developmental stimuli. Bacterial and fungal cells possess a variety of proteins that sense external or internal conditions and initiate signaling cascades that terminate at the binding of the protein to a specific promoter and the subsequent initiation of nucleic acid transcription under the promoter's control. In some embodiments, the promoter is endogenous. In some embodiments, the promoter is exogenous. In some embodiments, the promoter is artificially engineered for expression in a particular species.

[0286] In some embodiments, the promoter is a constitutive promoter. A constitutive promoter is a promoter that drives the expression of a nucleotide sequence continuously and uninterrupted in response to internal or external stimuli. Constitutive promoters are commonly used in recombinant engineering to ensure the continuous expression of the desired nucleotide sequence. Constitutive promoters result in robust amounts of nucleic acid expression and are therefore used in many recombinant engineering applications to achieve high levels of recombinant protein and enzymatic activity. Non-limiting examples of constitutive promoters included in this disclosure include the *E. coli* promoter. P spc , P bla , P RNAI , P RNAII , from rrnB of P 1 and P 2, and the λ phage promoter P L (Liang, ST et al. JMoi. Biol.292(1):19-37(1999)). In some implementations, the promoter is active during the stationary phase of the microorganism. Exemplary stationary phase promoters can be, for example, Shimada, et al. , Journal of Bacteriology , Nov. 2004, p. 7112–7122; Pletnev at el. , ACTA NATURAE | VOL. 7 Found in 2015 (4(27)).

[0287] In some 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 some embodiments, an inducible promoter drives graded levels of expression associated with the amount of stimulus. Non-limiting examples of stimuli for an inducible promoter include heat shock, the absence or use of an exogenous compound (e.g., sugar, metal, drug, or phosphate), salt or osmotic shock, oxygen, and biostimuli (e.g., growth factors or pheromones). Non-limiting examples of inducible promoters include the *E. coli* promoter. P lac , P taq , P tac , P T7 , P BAD and P Lacuv .

[0288] In some embodiments, the recombinant polynucleotide may contain multiple promoters. In some embodiments, the multiple promoters may be the same. For example, but not limited to, the recombinant polynucleotide may contain coding that is operatively linked to a first promoter. alsE The nucleotide sequence of the gene and the coding that is operatively linked to the second promoter. yigL The nucleotide sequence of the gene, wherein the first and second promoters are identical. For example, but not limited to, the recombinant polynucleotide may contain coding that is operatively linked to the first promoter. alsE The nucleotide sequence of the gene and the coding that is operatively linked to the second promoter. hxpA The nucleotide sequence of the gene, wherein the first and second promoters are identical. In some embodiments, the multiple promoters can be different. For example, but not limited to, the recombinant polynucleotide can contain coding that is operatively linked to the first promoter. alsE The nucleotide sequence of the gene and the coding that is operatively linked to the second promoter. yigLThe nucleotide sequence of the gene, wherein the first and second promoters are distinct. For example, but not limited to, the recombinant polynucleotide may contain coding that is operatively linked to the first promoter. alsE The nucleotide sequence of the gene and the coding that is operatively linked to the second promoter. hxpA The nucleotide sequence of the gene, wherein the first and second promoters are different. In some implementations, the promoter is... P LlacO1 Promoter. In some implementations, P LlacO1 The promoter contains the nucleotide sequence shown in SEQ ID NO: 42. In some embodiments, P LlacO1 The promoter consists of the nucleotide sequence shown in SEQ ID NO: 42. P LlacO1 The promoter is a promoter containing bacteriophage λ. P L The heterozygous regulatory region, in which the CI binding site is lacO 1 Replacement. Hybrid designs allow for suppression by LacI (Lac repressor, i.e., repressor) or strong initiation induced by IPTG.

[0289] In some implementations, the promoter is P LtetO1 Promoter. In some implementations, P LTETO1 The promoter contains the nucleotide sequence shown in SEQ ID NO: 43. In some embodiments, P LTETO1 The promoter consists of the nucleotide sequence shown in SEQ ID NO: 43.

[0290] In some implementations, the promoter is P T7 Promoter. In some implementations, P T7 The promoter contains the nucleotide sequence shown in SEQ ID NO: 44. In some embodiments, P T7 The promoter consists of the nucleotide sequence shown in SEQ ID NO: 44.

[0291] In some implementations, the promoter is P tet Promoter. In some implementations, P tet The promoter contains the nucleotide sequence shown in SEQ ID NO: 45. In some embodiments, Ptet The promoter consists of the nucleotide sequence shown in SEQ ID NO: 45.

[0292] In some implementations, the promoter is P gadB Promoter. In some implementations, P gadB The promoter contains the nucleotide sequence shown in SEQ ID NO: 46. In some embodiments, P gadB The promoter consists of the nucleotide sequence shown in SEQ ID NO: 46.

[0293] P LlacO1 Promoter nucleotide sequence: AATTGTGAGCGGATAACAATTGACATTGTGAGCGGATAACAAGATACTGAGCACATCAGCAGGACGCACTGACCGAATTCATTAAAGAGGAGAAAAGATATACC (SEQ ID NO: 42) P LtetO1 Promoter nucleotide sequence: tccctatcagtgatagagattgacatccctatcagtgatagagatactgagcacatcagcaggacgcactgaccgaattcattaaagaggagaaaggtacc (SEQ ID NO: 43) P T7 Promoter nucleotide sequence: taatacgactcactataggggaattgtgagcggataacaattcccctctagaaataattttgtttaactttaagaaggagatatacc (SEQ ID NO: 44) P tet Promoter nucleotide sequence: gttgacactctatcgttgatagagttattttaccactccctatcagtgatagagaaaagaattcaaaagatctaaagaggagaaaggatct(SEQ ID NO: 45) P gadB Promoter nucleotide sequence: GTAATAATTTTATAAATGCGTTCAAAATAATAATCAAGTACTAATAGTGATATTTTAAGGTCTGATTTTTACGTGATAATTCAGGAGACACAGAATGCGCATAAAAATAACAGCATAAAACACCTTACCACCACCCAAGAATTTCATATTGTATTGTTTTTCAATGAAAAAATATTA TTCGCGTAATATCTCACGATAAATAACATTAGGATTTTGTTATTTAAACACGAGTCCTTTGCACTTGCTTACTTTATCGATAAATCCTACTTTTTTAATGCGATCCAATCATTTTAAGGAGTTTAAAATGGATAAGAAGCAAGTGAATTCATTAAAGAGGAGAAAAGATATACC(SEQ ID NO: 46) In some implementations, the promoter is a stationary-phase promoter. As used herein, the term "stationary-phase promoter" refers to a promoter upstream of a gene that is transcribed during the stationary phase of microbial growth. The life cycle of an *E. coli* culture comprises five distinct phases: lag phase, logarithmic phase, stationary phase, death phase, and long-term stationary phase. The lag phase occurs when cells are inoculated into a culture medium and regulate their metabolic processes in response to their new environment. Cells then grow and divide rapidly, entering the logarithmic phase. At this time, enzymes associated with central carbon metabolism are most important, and the transcription of the corresponding genes is upregulated. Once cells sense environmental stressors (e.g., nutrient deficiency in the culture medium), their growth and division slow down, and the culture enters the stationary phase. The use of stationary-phase promoters prevents generative pathways from competing with central carbon metabolism for carbon flux during the logarithmic phase of growth (when cells require carbon for vigorous growth and division).

[0294] In some implementations, the stationary promoter is P gadB In some implementations, P gadB The promoter contains the nucleotide sequence shown in SEQ ID NO: 46 or SEQ ID NO: 53. In some embodiments, P gadB The promoter consists of the nucleotide sequence shown in SEQ ID NO: 53.

[0295] In some implementations, the stationary promoter is P cbpA2 In some implementations, P cbpA2The promoter contains the nucleotide sequence shown in SEQ ID NO: 54. In some embodiments, P cbpA2 The promoter consists of the nucleotide sequence shown in SEQ ID NO: 54.

[0296] In some implementations, the stationary promoter is P ihfA4 In some implementations, P ihfA4 The promoter contains the nucleotide sequence shown in SEQ ID NO: 55. In some embodiments, P ihfA4 The promoter consists of the nucleotide sequence shown in SEQ ID NO: 55.

[0297] In some implementations, the stationary promoter is P dps In some implementations, P dps The promoter contains the nucleotide sequence shown in SEQ ID NO: 56. In some embodiments, P dps The promoter consists of the nucleotide sequence shown in SEQ ID NO: 56.

[0298] GTAATAATTTTATAAATGCGTTCAAAATAATAATCAAGTACTAATAGTGATATTTTAAGGTCTGATTTTTACGTGATAATTCAGGAGACACAGAATGCGCATAAAAATAACAGCATAAAACACCTTACCACCACCCAAGAATTTCATATTGTATTGTTTTTCAATGAAAAAATATTAT TCGCGTAATATCTCACGATAAATAACATTAGGATTTTGTTATTTAAACACGAGTCCTTTGCACTTGCTTACTTTATCGATAAATCCTACTTTTTTAATGCGATCCAATCATTTTAAGGAGTTTAAAATGGATAAGAAGCAAGTCGAATTCATTAAAGAGGAGAAAGGTACCATG(SEQ ID NO: 53)

[0299] TTTGCAGTGCAACTAATTCCATGTATATTACTACCCATATATAGCGTCTATAAAATTTAATAAATAATGACGCCCTAGTTAAACTTAAAGTGCCTGGTTCAACTATCAAAAATCGCTCACCCTTTTTCACCTGTTTAAAATATGTTCAGCAACCCATCTTGATGGCGACCTCCTCTCCGCGATGATTTCAATAACATATTCTGTGTTGGCATATGAAATTTTGAGGATTACCCTACACTTATAGGAGTTACCTTACAGGGGTTCCTTCAATTTGTGTTGATTTACGCGAGATAACGCTCGAATTCATTAAAGAGGAGAAAGGTACCATG(SEQ ID NO: 54)

[0300] TATCCGAATGTAAGAAAGTTGGCGTAAATCAGGTAGTTGGCGTAAACTTATTTGACGTGTACCGCGGTAAGGGTGTTGCGGAGGGGTATAAAGAGCCTCGCCATAAGCCTGATCCTGCAAGATACCAGCCGTACACTCGAAGAAGAGGAGATTGCCGCTACCGTCGCCAAATGTGTAGAGGCATTAAAAGAGCGATTCCAGGCATCATTGAGGGATTGAACCTCGAATTCATTAAAGAGGAGAAAGGTACCATG (SEQ ID NO: 55)

[0301] TCATTGAATCTTTATTAGTTTTGTTTTTCACGCTTGTTACCACTATTAGTGTGATAGGAACAGCCAGAATAGCGGAACACATAGCCGGTGCTATACTTAATCTCGTTAATTACTGGGACATAACATCAAGAGGATATGAAATTCGAATTCATTAAAGAGGAGAAAGGTACCATG (SEQ ID NO: 56)

[0302] 3.1.3. Genetic markers

[0303] In some embodiments, the recombinant polynucleotides of this disclosure comprise genetic markers. These genetic markers allow selection of microorganisms having one or more desired polynucleotides (e.g., recombinant polynucleotides). In some embodiments, the genetic markers are selected from antibiotic resistance markers such as apramycin resistance, ampicillin resistance, kanamycin resistance, spectinomycin resistance, tetracycline resistance, neomycin resistance, chloramphenicol resistance, gentamicin resistance, erythromycin resistance, carbenicillin resistance, actinomycin D resistance, neomycin resistance, polymyxin resistance, zeocin resistance, and streptomycin resistance. In some embodiments, the genetic marker comprises a coding sequence for an antibiotic resistance protein (e.g., a β-lactamase for certain ampicillin resistance markers) and a promoter or enhancer element that drives expression of the microorganisms disclosed herein. In some embodiments, the microorganisms of this disclosure are grown under conditions that express the antibiotic resistance marker and confuse the microorganisms with resistance, thereby selecting microorganisms that successfully integrate the marker. In some embodiments, the genetic marker is an auxotrophic marker. In some embodiments, the auxotrophic marker is a gene involved in vitamin, amino acid, fatty acid synthesis, or carbohydrate metabolism. In some embodiments, the auxotrophic marker is a gene for synthesizing amino acids. In some embodiments, the auxotrophic marker is a gene for synthesizing glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, methionine, asparagine, glutamine, lysine, arginine, histidine, aspartic acid, or glutamic acid. In some embodiments, the auxotrophic marker is a gene for synthesizing adenosine, biotin, thiamine, leucine, glucose, lactose, or maltose. In some embodiments, the microorganisms of this disclosure are grown under conditions where the auxotrophic resistance marker is expressed in an environment or culture medium lacking the corresponding nutrient, thereby accumulating microbial growth (lacking the endogenous ability to produce nutrients), thereby selecting microorganisms that successfully integrate the marker.

[0304] 3.2. Gene deletion and reduced expression

[0305] In some embodiments, this disclosure also provides methods for introducing deletions of any of the genes or enzymes disclosed herein. These deletions can be generated by any suitable gene editing method. In some embodiments, deletions are generated by methods including homologous recombination, zinc finger nucleases, meganucleases, transcription activator-like effector nucleases (TALENs), clustered regular-interval short palindromic repeat (CRISPR) systems, or combinations thereof.

[0306] In some implementations, the deletion is generated by a CRISPR system. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system is a genome editing tool found in prokaryotic cells. When used for genome editing, the system comprises Cas9 (a protein capable of modifying DNA using crRNA as its guide), CRISPR RNA (crRNA containing RNA that Cas9 uses to guide it to the correct segment of host DNA and a region that binds to tracrRNA (typically in the form of a hairpin loop), which forms an active complex with Cas9), trans-activating crRNA (tracrRNA, which binds to crRNA and forms an active complex with Cas9), and an optional segment of a DNA repair template (DNA that guides the cellular repair process that allows the insertion of a specific DNA sequence). Multiple crRNAs and tracrRNAs can be packaged together to form a single guide RNA (sgRNA). This sgRNA can be linked to the Cas9 gene and made into a plasmid for transfection into cells. In some implementations, the CRISPR system includes a base editor. In some implementations, the CRISPR system includes a transposase / recombinase. In some embodiments, the CRISPR system includes prime editors. In some embodiments, the CRISPR system includes epigenetic regulators. In some embodiments, the CRISPR system includes a CRISPRoff system. Further details regarding the CRISPR system disclosed herein can be found in Anzalone et al., Nature biotechnology 38.7(2020): 824-844 and Nuñez et al ., Cell 184.9(2021): 2503-2519 and Jiang et al., Appl Environ Microbiol The contents of each entry are incorporated into the whole by reference in .2015 Apr;81(7):2506-14.

[0307] In some implementations, the deletion is generated by zinc finger nucleases. Zinc finger nucleases (ZFNs) are artificial restriction enzymes created by combining a zinc finger DNA-binding domain with a DNA-cutting domain. The zinc finger domain can be engineered to target specific DNA sequences and allow the zinc finger nuclease to target desired sequences within the genome. The DNA-binding domain of a single ZFN typically contains multiple individual zinc finger repeats, each capable of recognizing multiple base pairs. The most common method for generating novel zinc finger domains is by combining smaller zinc finger “modules” with known specificity. The most common cleavage domain in ZFNs is the nonspecific cleavage domain derived from the type IIs restriction endonuclease FokI.

[0308] In some implementations, deletions are generated by the TALEN system. Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific sequences of DNA. TALEN systems operate on almost the same principle as ZFNs. They are generated by combining a transcription activator-like effector DNA-binding domain with a DNA-cleaving domain. A transcription activator-like effector (TALE) consists of a 33-34 amino acid repeat motif with two variable positions that strongly recognize specific nucleotides. By assembling arrays of these TALEs, the TALE DNA-binding domain can be engineered to bind to the desired DNA sequence, thereby guiding the nuclease to cleave at a specific location in the genome.

[0309] In some implementations, the deletion is generated by a meganuclease. A meganuclease is an endonuclease that recognizes a double-stranded DNA site of approximately 12 to approximately 40 base pairs that appears only once in the genome. Meganucleases are among the most specific naturally occurring restriction enzymes. Meganucleases are also defined as molecular DNA scissors because they can replace, eliminate, or modify sequences in a highly targeted manner. Protein engineering allows modification of both their recognition and target sequences.

[0310] In some embodiments, this disclosure also provides methods for reducing the expression of any of the genes or enzymes disclosed herein. In some embodiments, reducing the expression of the genes and enzymes disclosed herein includes using oligonucleotides having a complementary sequence to the mRNA of the disclosed genes (e.g., zwf , manA , pfkA , pfkB (etc.). Non-limiting examples of these oligonucleotides include small interfering RNA (siRNA), short hairpin RNA (shRNA), and microRNA (miRNA). In some embodiments, these oligonucleotides can be combined with... zwfAt least a portion thereof is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the mRNA sequence. In some embodiments, these oligonucleotides may be identical to... zwf At least a portion of the mRNA sequence is identical. In some embodiments, these oligonucleotides may be identical to... pfkA At least a portion thereof is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the mRNA sequence. In some embodiments, these oligonucleotides may be identical to... pfkA At least a portion of the mRNA sequence is identical. In some embodiments, these oligonucleotides may be identical to... pfkB At least a portion thereof is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the mRNA sequence. In some embodiments, these oligonucleotides may be identical to... pfkB At least a portion of the mRNA sequence is identical. In some embodiments, these oligonucleotides may be identical to... rpiB At least a portion thereof is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the mRNA sequence. In some embodiments, these oligonucleotides may be identical to... rpiB At least a portion of the mRNA sequence is identical. In some embodiments, these oligonucleotides may be identical to... manA At least a portion thereof is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the mRNA sequence. In some embodiments, these oligonucleotides may be identical to... manA At least a portion of the mRNA sequence is identical. In some embodiments, the antisense nucleic acid, shRNA, miRNA, or siRNA molecule may contain DNA or atypical or non-naturally occurring residues, such as, but not limited to, phosphate thioester residues.

[0311] In some implementations, reducing the expression of the genes and enzymes disclosed herein may include using CRISPR, which can mutate coding sequences or promoters to reduce or eliminate the expression of gene products; or CRISPR may be used to target the genes disclosed herein, thereby reducing the expression of one or more genes. See, for example, Arroya-Olarte. et al. , Microorganisms, 2021 Apr; 9(4): 844;Zhang et al. , Front. Microbiol (31 March 2021).

[0312] In some embodiments, the reduced expression of the genes and enzymes disclosed herein includes the use of a CRISPR system. In some embodiments, the CRISPR system comprises Cas9. In some embodiments, Cas9 comprises 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% of the amino acid sequence shown in SEQ ID NO: 79. In some embodiments, Cas9 consists of the amino acid sequence shown in SEQ ID NO: 79. SEQ ID NO: 79 is provided below.

[0313]

[0314] In some embodiments, the reduced expression of the genes and enzymes disclosed herein includes the use of the CRISPRi system. The CRISPRi system silences genes at the transcriptional level and may have fewer sequence-specific off-target effects than RNAi. In some embodiments, the CRISPRi system comprises catalytically inactivated Cas9 (dCas9). dCas9 is a programmable transcription factor that can target a promoter via sgRNA, where it can function as a repressor. In some embodiments, dCas9 comprises 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% of the amino acid sequence shown in SEQ ID NO: 57. In some embodiments, dCas9 comprises the amino acid sequence shown in SEQ ID NO: 57. SEQ ID NO: 57 is provided below.

[0315]

[0316] In some embodiments, the nuclease (e.g., Cas9 or dCas9) is regulated by a promoter (e.g., as described in section 3.1.2). In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a stationary promoter. In some embodiments, the CRISPR / CRISPRi system includes a small guide RNA (sgRNA). In some embodiments, the sgRNA of the CRISPR / CRISPRi system targets a gene encoding an enzyme in a competitive pathway. For example, but not limited to, the sgRNA may target... zwf Gene, pgm Gene, pfkA Gene, pfkB Gene or ManA Genes. In some implementations, sgRNA can target any part of a gene. For example, but not limited to, sgRNA can target promoters, operons, or sequences encoding proteins.

[0317] In some embodiments, the CRISPR / CRISPRi system comprises a nuclease (e.g., Cas9 or dCas9) and sgRNA. In some embodiments, the nuclease (e.g., Cas9 or dCas9) is regulated by an inducible promoter. In some embodiments, the inducible promoter is... P tet In some implementations, sgRNA targets pfkB Genes. In some implementations, sgRNA targets pfkB Gene promoters.

[0318] 3.3. Transformation and Gene Editing

[0319] In some embodiments, this disclosure provides for the use of the plasmids and vectors disclosed herein for transformation. The vectors and plasmids disclosed herein can be transformed into cells using any system known in the art. For example, but not limited to, the microorganisms disclosed herein can be transformed by particle bombardment, chemical transformation, Agrobacterium-mediated transformation, nano-spike transformation, electroporation, and viral transformation.

[0320] In some embodiments, a variety of techniques can be used to introduce the vectors of this disclosure into microorganisms, including transformation, transfection, transduction, viral infection, gene gun, or Ti-mediated gene transfer. Non-limiting examples of these methods include calcium phosphate transfection, DEAE-glucan-mediated transfection, lipid transfection, and electroporation (see, for example, Davis, L., Dibner, M., Battey, I., 1986 “Basic Methods in Molecular Biology”; Gietz et al., Nucleic Acids Res. 27:69-74 (1992); Ito et al., J. Bacterol. 153:163-168 (1983); and Becker and Guarente, Methods in Enzymology 194:182-187 (1991)). In some embodiments, the transformed microorganisms are referred to as recombinant microorganisms.

[0321] In some embodiments, this disclosure provides methods for introducing exogenous proteins (e.g., nucleases), RNA (e.g., gRNA), and DNA (e.g., recombinant polynucleotides disclosed herein) into microorganisms. Several methods for achieving this goal have been previously described, including direct transfection or DNA conversion of protein and nucleotide sequences, followed by intracellular expression of RNA and protein (see, for example, Dicarlo, JE et al. “Genome engineering in Saccharomycescerevisiae using CRISPR-Cas systems.” Nucleic Acids Res (2013). doi:10.1093 / nar / gkt135; Ren, ZJ, Baumann, RG & Black, LW “Cloning of linear DNAsin vivo by overexpressed T4 DNA ligase: construction of a T4 phage hoc gene display vector.” Gene 195, 303-311 (1997); Lin, S., Staahl, BT, Alla, RK & Doudna, JA “Enhanced homology-directed human genome engineering by controlled timing of CRISPR / Cas9 delivery.” Elife 3, e04766 (2014)).

[0322] 3.4. Reorganization System

[0323] In some embodiments, this disclosure also provides homologous recombination systems for editing (e.g., insertion, deletion) in microorganisms. In some embodiments, the homologous recombination system may be native to the host cell or introduced into the cellular host. For example, but not limited to, the gene of the homologous recombination system may be introduced onto a plasmid, introduced onto a linear DNA fragment, introduced as RNA or a collection of RNAs and translated from or as a protein or a collection of proteins. In some embodiments, the method includes the recombinant polynucleotides disclosed herein. In some embodiments, the polynucleotide comprises sequences (e.g., left and right homologous arms) homologous to regions in a nucleic acid (e.g., genome, plasmid, etc.) such that the left and right homologous arms are separated by designed genetic editing (e.g., promoter, insertion, substitution, SNP, terminator, degron, tag sequence, degradation signal sequence, or deletion). In some embodiments, the recombinant polynucleotide comprises a genetic marker, a reverse-selectable genetic marker (e.g., SacB or PheS), and an origin of replication (e.g., R6K).

[0324] In some embodiments, recombinant polynucleotides containing homologous arms and sequences for genetic editing are introduced into microorganisms using any of the methods disclosed herein (e.g., transformation via electroporation, conjugation, etc.). In some embodiments, after transformation, the resulting transformants can be plated on a culture medium to select transformants expressing selectable genetic markers. Recombination of the plasmid containing the homologous arm with a target locus in the nucleic acid (e.g., genome, plasmid, etc.) can occur at one of two homologous sites targeted by the homologous arm present on the plasmid, and flanked by the designed genetic edit. In some embodiments, the resulting transformants are grown as colonies on a selective medium and can be selected and plated on a second type of selective medium (e.g., reverse selective medium). In some embodiments, the second type of selective medium allows selection of cells containing the desired genetic edit.

[0325] In some embodiments, the methods disclosed herein include using proteins from one or more recombination systems. The recombination system may be endogenous to a microorganism or exogenous. In some embodiments, proteins from one or more recombination systems may be introduced as nucleic acids (e.g., as plasmids, linear DNA, or RNA, or integrons) and integrated into the genome of a host cell, or stably expressed by an extrachromosomal element. In some embodiments, proteins from one or more recombination systems may be introduced as RNA and translated by a host cell. In some embodiments, proteins from one or more recombination systems may be introduced as proteins into a host cell. Non-limiting examples of recombination systems include the λ red recombination system, the RecET recombination system, the Red / ET recombination system, any homolog, or paralog of proteins from the λ red recombination system, the RecET recombination system, the Red / ET recombination system, or λ red-mediated recombination systems, or any combination thereof. Details of recombination systems from the RecET recombination system may be found in Zhang Y., Buchholz F., Muyrers JPP and Stewart AF, “A new logic for DNA engineering using recombination in…” E. coli ." Nature Genetics 20(1998) 123-128; Muyrers, JPP, Zhang, Y., Testa, G., Stewart, AF "Rapid modification of bacterialartificial chromosomes by ET-recombination." Nucleic Acids Res. 27(1999)1555-1557; Zhang Y., Muyrers JPP, Testa G. and Stewart AF "DNA cloning. by homologous recombination in E. coliAny of the references cited in 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, are incorporated herein by reference in their entirety.

[0326] 4. Methods for producing allosugar

[0327] This disclosure also provides methods for producing allose. Cell-free methods (e.g., in vitro synthesis) utilize thermodynamically unfavorable enzymatic reactions. In some embodiments, the methods of this disclosure for producing allose include culturing microorganisms (e.g., the microorganisms disclosed in Part 2) and purifying allose.

[0328] 4.1. Cell Culture

[0329] This disclosure provides methods for culturing the microorganisms disclosed herein. As used herein, "culturing" cells means introducing a suitable culture medium under appropriate conditions to promote cell growth. In some embodiments, culturing is performed using liquid or solid growth media. In some embodiments, culturing is performed under aerobic or anaerobic conditions based on the needs of the microorganisms and the desired metabolic state of the microorganisms. In some embodiments, culturing includes specific conditions such as temperature, pressure, light, pH, and cell density.

[0330] In some embodiments, the method of producing allosugar includes a culture medium for culturing recombinant bacteria. As used herein, "culture medium" refers to any composition or broth that supports the growth of the microorganisms disclosed herein. The culture medium can be liquid or solid. In some embodiments, the culture medium contains nutrients, salts, buffers, elements, and other compounds that support cell growth and viability. Furthermore, the culture medium may contain nitrogen sources, carbon sources, amino acids, carbohydrates, trace elements, vitamins, and minerals. In some embodiments, the culture medium contains a complex extract (e.g., yeast extract). In some embodiments, the culture medium is enriched to support rapid growth. In some embodiments, the culture medium is modified to support slower growth. In some embodiments, the culture medium contains an agent capable of inhibiting the growth of contaminating organisms or killing contaminating organisms (e.g., antibiotics). In some embodiments, the culture medium contains an agent capable of activating an inducible promoter or enzyme (e.g., IPTG). Non-limiting examples of culture media covered by this disclosure include M9 medium, Lysogeny Broth (LB), Terrific Broth (TB), and YT Broth. In some embodiments, the culture medium contains a substrate that is converted into allosugar by the recombinant microorganisms.

[0331] In some embodiments, the substrate is a sugar (e.g., glucose or fructose) that can be phosphorylated by bacteria via a kinase (e.g., hexokinase) and converted to fructose-6-phosphate. In some embodiments, the substrate is glucose. In some embodiments, glucose may be derived from cellulose, C5 sugar, hemicellulose, and / or xylose. In some embodiments, the substrate is a component of the culture medium. In some embodiments, the substrate is supplemented with the culture medium. In some embodiments, the substrate is continuously present in the culture medium. In some embodiments, the substrate is supplemented during the growth phase. In some embodiments, the substrate is supplemented during the stationary phase.

[0332] 4.2. Purification of allosugar

[0333] In some embodiments, the methods of this disclosure further include purifying allose produced by the microorganisms of this disclosure, for example, from cell cultures or cell culture media. Various methods known in the art can be used to purify products from microorganisms or microbial cultures. In some embodiments, one or more products can be purified continuously, for example, from continuous cultures. In some embodiments, one or more products can be purified individually from fermentation, for example, from batch or fed-batch cultures. Those skilled in the art will appreciate that the specific purification method used can be particularly dependent on the microorganism, culture conditions, and / or the specific product.

[0334] In some embodiments, purifying allosugar includes isolating or filtering microorganisms from cell culture media, isolating allosugar from the culture medium (e.g., by chromatography), concentrating water (e.g., by evaporation), and lyophilizing allosugar.

[0335] 5. Food products

[0336] This disclosure also provides a delivery system method for food products comprising D-allose prepared and / or generated from any of the microorganisms disclosed herein.

[0337] As used herein, the term "food product" includes any food product, such as those shown in 21 CFR 101.12. Non-limiting examples of such food products include frozen desserts, baked goods, fillings, nutritional drinks, beverages, salad dressings or similar condiments, sauces, frostings, puddings and custards, batters, etc. A variety of baked goods are disclosed in U.S. Patent No. 6,536,599, the disclosure of which is incorporated herein by reference in its entirety. Non-limiting examples of baked goods include cookies, cakes, rolls, pastries, biscuits, brownies, breads, bagels, etc. D-allose prepared and / or generated by any of the microorganisms disclosed herein is also suitable as a component in frozen foods.

[0338] In some embodiments, food products are prepared by mixing D-allose with any optional ingredients in an ingestible medium to form a homogeneous mixture. The final composition is prepared using standard methods and equipment generally known to those skilled in the art (e.g., the confectionery industry). According to the subject matter of this disclosure, useful equipment includes mixing equipment well-known in the art, and therefore the choice of specific equipment is obvious to those skilled in the art.

[0339] As used herein, “mixing,” such as “mixing D-allose with a food product,” refers to the process of mixing, adding, or combining a flavor composition with, or adding to, a finished product, or with some or all of the product’s components during product formation or some combination of these steps. When used in the context of mixing, the term “product” refers to the product or any of its components. The mixing step may include processes selected from the following steps: adding D-allose to a product, spraying D-allose onto a product, coating D-allose onto a product, suspending the product in D-allose, applying D-allose to a product, adhering D-allose to a product, encapsulating the product with D-allose, mixing D-allose with a product, and any combination thereof. D-allose can be a liquid, dry powder, spray, paste, suspension, and any combination thereof.

[0340] In some embodiments, this application relates to improved edible food products produced by the methods disclosed herein. In some embodiments, the food products can be produced by methods well known to those skilled in the art for producing edible products.

[0341] In some embodiments, D-allose prepared and / or generated by any of the microorganisms disclosed herein can be dissolved or dispersed in one of a number of known edible-acceptable liquids, solids or other carriers, such as water, fruit or vegetable juices, vinegar, marinades, beer, wine, natural water / fat emulsions (e.g., milk or condensed milk), whey or whey products, edible oils and shortenings, fatty acids, certain low molecular weight oligomers of propylene glycol, glycerides of fatty acids, and dispersions or emulsions of such hydrophobic substances in aqueous media, salts (e.g., sodium chloride), vegetable flour, solvents (e.g., ethanol), solid edible diluents (e.g., vegetable powder or flour), etc., and then combined with a precursor of an edible or pharmaceutical product, or applied directly to an edible or pharmaceutical product.

[0342] Those skilled in the art of preparing and marketing food products are fully aware of the many categories, subcategories, and types of edible compositions and use well-known and recognized terms in the art to refer to those edible compositions, while also engaging in the preparation and marketing of a variety of such edible compositions. A series of such technical terms are listed below, and particular consideration is given to the possibility that D-allose prepared and / or generated by any of the microorganisms disclosed herein can be used alone or in all reasonable combinations or mixtures therein to improve or enhance the flavor of the edible compositions listed below.

[0343] In some embodiments, food products mixed with D-allose prepared and / or generated from any of the microorganisms disclosed herein include, for example, wet soups, dehydrated and cooked foods, beverages, frozen foods, snacks, and condiments or condiment mixtures as described herein.

[0344] In some embodiments, D-allose prepared and / or generated by any of the microorganisms disclosed herein is mixed with one or more of the following: candies, chocolate candies, chocolate chips, countlines chocolates, self-mies / softlines chocolates, boxed assorted chocolates, standard boxed assorted chocolates, twist-up mini chocolates, seasonal chocolates, chocolates with toys, assorted candies, other chocolate candies, mints, standard mints, strong mints, hard candies, candy lozenges, gummy candies, jellies and chewing candies, toffee, caramel and nougat, medicinal candies, lollipops, licorice candies, other sugar candies, gum, chewing gum. gum, sweetened gum, sugar-free gum, functional gum, bubble gum, bread, packaged / industrial bread, unpackaged / artificial bread, pastries, cakes, packaged / industrial cakes, unpackaged / artificial cakes, biscuits, chocolate-coated biscuits, sandwich biscuits, filled biscuits, savory biscuits and crackers, bread substitutes, breakfast cereals, instant cereals, family breakfast cereals, cereals, muesli (milk breakfast), other instant cereals, children's breakfast cereals, hot cereals, ice cream, impulse ice cream. Cream), single-serving dairy ice cream, single-serving water ice cream, multi-pack dairy ice cream, multi-pack water ice cream, family-sized ice cream, family-sized dairy ice cream, ice cream desserts, bulk ice cream, family-sized water ice cream, frozen yogurt, handmade ice cream, dairy products, milk, fresh / pasteurized milk, whole milk fresh / pasteurized milk, semi-skimmed fresh / pasteurized milk, UHT milk, whole milk UHT milk, semi-skimmed UHT milk, skimmed UHT milk, goat milk, condensed milk / evaporated milk, regular condensed milk / evaporated milk, flavored milk, functional milk and other condensed milk, flavored milk beverages, dairy-only products. Flavored milk beverages, flavored milk beverages with fruit juice, soy milk, sour milk beverages, fermented milk beverages, coffee whitening agents, milk powder, flavored milk powder beverages, cream, cheese, processed cheese, spreadable processed cheese, non-spreadable processed cheese, unprocessed cheese, spreadable unprocessed cheese, hard cheese, packaged hard cheese, unpackaged hard cheese, yogurt, plain / natural yogurt, flavored yogurt, fruit yogurt, probiotic yogurt, drinkable yogurt, regular drinkable yogurt, probiotic drinkable yogurt, refrigerated and long-shelf-life desserts, dairy desserts, soy desserts, refrigerated snacks, fresh soft cheeses and quark cheese (fromage frais andQuark cheese, regular fresh soft cheese and quark cheese, flavored fresh soft cheese and quark cheese, savory fresh soft cheese and quark cheese, sweet and savory snacks, fruit snacks, potato chips / fries, puffed foods, tortillas / nachos, popcorn, pretzels, nuts, other sweet and savory snacks, snack bars, granola bars, breakfast bars, energy bars, fruit bars, other snack bars, meal replacement products, weight loss products, recovery drinks, ready-to-eat meals, canned ready-to-eat meals, frozen ready-to-eat meals, dry ready-to-eat meals, refrigerated ready-to-eat meals, premixed meals (dinner) Mixes), frozen pizza, chilled pizza, soup, canned soup, dehydrated soup, instant soup, chilled soup, UHT soup, frozen soup, pasta, canned pasta, dry pasta, chilled / fresh pasta, noodles, regular noodles, instant noodles, cup / bowl instant noodles, small packet 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 food, frozen food, frozen processed red meat, frozen processed poultry, frozen processed fish / seafood, frozen processed vegetables, frozen meat substitutes, frozen potatoes, oven-baked potato chips, other oven-baked potato products, non-oven-baked frozen potatoes, frozen Baked goods, frozen desserts, frozen ready meals, frozen pizzas, frozen soups, frozen noodles, other frozen foods, dried foods, dessert premixes, dry ready meals, dehydrated soups, instant soups, dry pasta, regular noodles, instant noodles, cup / bowl instant noodles, small packet instant noodles, refrigerated foods, refrigerated processed meats, refrigerated fish / seafood, refrigerated processed fish, refrigerated breaded fish, refrigerated smoked fish, refrigerated lunch packs, refrigerated ready meals, refrigerated pizzas, refrigerated soups, refrigerated / fresh pasta, refrigerated noodles, oils and fats, olive oil, vegetable oils and seed oils, cooking fats, butter, margarine, spreadable oils and fats, functional spreadable oils and fats, sauces, seasonings and condiments, tomato paste and tomato puree, broth / soup cubes, soup cubes, gravy powder Granules), stock and fond, herbs and spices, fermented sauces, bean sauces, pasta sauces, wet sauces, dry sauces / powdered premixes, ketchup, mayonnaise, regular mayonnaise, mustard, salad dressings, regular salad dressings, low-fat salad dressings, vinaigrettes, dips, pickled products, other sauces, seasonings and condiments, baby food, formula, standard formula, follow-up formula, toddler formula, hypoallergenic formula, pre-made baby food, dry baby food, other baby food, spreads, jams and preserves, honey, chocolate spread, nut spreads, and yeast-based spreads.

[0345] 5.1. Chewing gum

[0346] In some embodiments, D-allose prepared and / or generated from any of the microorganisms disclosed herein can be used in low-calorie chewing gum formulations and also in sugary chewing gum. Various details of chewing gum compositions are disclosed in U.S. Patent No. 6,899,911, the disclosure of which is incorporated herein by reference in its entirety. The chewing gum compositions of the subject matter of this disclosure follow the general pattern outlined below. Typically, chewing gum compositions generally contain a substantially anhydrous and water-insoluble chewable gum base portion, a water-soluble body portion, and a flavoring that is generally water-insoluble. The water-soluble portion dissipates along with a portion of the flavoring over a period of time during chewing. The gum base portion remains in the mouth during chewing. The insoluble gum base typically comprises elastomers, elastomer solvents, plasticizers, waxes, emulsifiers, and inorganic fillers. It also frequently contains plastic polymers, such as polyvinyl acetate, which acts to some extent as a plasticizer. Other plastic polymers that may be used include polyvinyl alcohol laurate, polyvinyl alcohol, and polyvinylpyrrolidone. Elastomers can include polyisobutylene, butyl rubber, isobutylene-isoprene copolymers, and styrene-butadiene rubber, as well as natural latexes such as chile. Elastomer solvents are typically resins, such as terpene resins. Plasticizers, sometimes called softeners, are typically fats and oils, including animal fats, hydrogenated and partially hydrogenated vegetable oils, and cocoa butter. Commonly used waxes include paraffin wax, microcrystalline waxes, and natural waxes such as beeswax and carnauba wax. Microcrystalline waxes, especially those with high crystallinity, can be considered thickeners or texture modifiers.

[0347] In some embodiments, the insoluble gum base comprises about 5% to about 95% of the chewing gum by weight. More preferably, the insoluble gum base comprises 10% to 50% of the chewing gum by weight, and most preferably about 20% to 35% of the chewing gum by weight. The gum base typically also contains a filler component. The filler component may be calcium carbonate, magnesium carbonate, talc, dicalcium phosphate, etc. The filler may comprise about 5% to about 60% of the gum base by weight. Preferably, the filler comprises about 5% to 50% of the gum base by weight.

[0348] The gum base typically also contains softeners, including glyceryl monostearate and glyceryl triacetate. The gum base may also contain optional ingredients such as antioxidants, colorants, and emulsifiers. The subject matter of this disclosure contemplates the use of any commercially acceptable gum base.

[0349] The water-soluble portion of chewing gum may also include softeners, sweeteners, flavorings, physiological cooling agents, and combinations thereof. Sweeteners typically function as fillers in chewing gum. Fillers typically comprise about 5% to about 95% of the chewing gum composition.

[0350] Softeners are added to chewing gum to optimize its chewability and texture. Softeners, also known in the art as plasticizers or phthalates, typically comprise about 0.5% to about 15% of the chewing gum. Softeners considered in the subject matter of this disclosure include glycerin, lecithin, and combinations thereof. Furthermore, aqueous solutions of sweeteners, such as those containing sorbitol, hydrogenated starch hydrolysate, corn syrup, and combinations thereof, can be used as both softeners and binders in chewing gum.

[0351] As mentioned above, D-allose prepared and / or generated by any of the microorganisms disclosed herein can be used in low-calorie chewing gum formulations. However, sugar-containing formulations are also within the scope of this invention. Sugar sweeteners generally include sugar-containing components commonly known in the chewing gum industry, including but not limited to, alone or in any combination of the following substances: sucrose, dextrose, maltose, dextrin, dry invert sugar, fructose, galactose, corn syrup solids, etc. D-allose prepared and / or generated by any of the microorganisms disclosed herein can also be used in combination with sugar-free sweeteners. Typically, sugar-free sweeteners include components that have a sweet taste but do not contain commonly known sugars, and include but not limited to, alone or in any combination of the following substances: sugar alcohols (e.g., sorbitol), hydrogenated isomaltulose, mannitol, xylitol, lactitol, erythritol, hydrogenated starch hydrolysate, maltitol, etc.

[0352] Depending on the desired specific sweetness release characteristics and shelf stability, coated or uncoated high-intensity sweeteners can be used in chewing gum compositions or applied to the coating of the core made from those chewing 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 compared to free aspartame. When using aspartame, free aspartame can also be added, and combinations of some free and encapsulated aspartame are preferred. Other high-intensity sweeteners that can be used in chewing gum cores are saccharin, sematrandilla, alicon, saccharin salt, sucralose, steviol glycosides (Stevia), and acesulfame potassium. Generally, the chewing gum composition will preferably contain about 0.5% to about 90% of a sweetener. Most typically, the sweetener will include at least one bulk sweetener and at least one high-intensity sweetener. Optional ingredients such as colorants, emulsifiers, and pharmaceutical agents may also be added as separate components of the chewing gum composition or as part of the gum base.

[0353] Aqueous syrups, such as corn syrup and hydrogenated corn syrup, can be used, especially if their water content is low. This can preferably be done by co-evaporating the aqueous syrup with a plasticizer (e.g., glycerol or propylene glycol) until the water content is less than 10%. Preferred compositions comprise hydrogenated starch hydrolysate solids and glycerol. Such syrups and methods of their preparation are described in detail in U.S. Patent No. 4,671,967.

[0354] A method for manufacturing chewing gum according to the subject matter of this disclosure involves sequentially adding multiple chewing gum ingredients to any commercially available mixer known in the art. After the ingredients are thoroughly mixed, the chewing gum is expelled from the mixer and shaped into a desired form, such as by rolling it into sheets and cutting it into sticks, extruding it into blocks, or casting it into pellets. Typically, the ingredients are mixed by first melting the gum base and adding it to the running mixer. The base may also be melted in the mixer itself. Colorants or emulsifiers, as well as syrups and a portion of the filler, may also be added at this point. A further portion of the filler may then be added to the mixer. The flavor system is typically added along with the final portion of the filler. If the flavor system is coated or otherwise modified to alter its release rate when incorporated into the delivery system, it is preferable to add the flavor system after the final portion of the filler has been added. The entire mixing process typically takes five to twenty minutes, but may sometimes require longer mixing times. Those skilled in the art will recognize that many variations of the above procedure can be followed.

[0355] If pellets or balls are formed, the chewing gum composition can be coated. The coating initially exists as a liquid syrup containing about 30% to about 80% or 85% sugar or sugar alcohol and about 15% or 20% to about 70% solvent (e.g., water). Typically, the coating process is carried out in conventional panning equipment. The chewing gum core sheet to be coated is placed in the panning equipment to form a moving substance.

[0356] The material or syrup that will ultimately form the coating is applied or distributed onto the gum core sheet. D-Allose can be added before, during, and after the syrup is applied to the gum core. Once the coating has dried to form a hard surface, additional syrup can be added to create multiple or multi-layered coatings. D-Allose can be added to any one of the coatings and / or layers or not.

[0357] In the sugar coating process, syrup is added to the gum core sheet at a temperature ranging from about 100℉ to about 240℉. Preferably, the syrup temperature is from about 140℉ to about 200℉. Most preferably, the syrup temperature should be kept constant throughout the process to prevent crystallization of the polyols in the syrup. The syrup can be mixed with, sprayed onto, poured onto, or added to the gum core sheet in any manner known to those skilled in the art.

[0358] In some embodiments, a soft coating is formed by adding a powder coating after a liquid coating. The powder coating may contain natural gum hydrolysate, maltodextrin, gelatin, cellulose derivatives, starch, modified starch, sugar, sugar alcohol, natural gum, and fillers such as talc and calcium carbonate.

[0359] Each component coating the chewing gum core can be applied in a single layer or multiple layers. Typically, multiple layers are obtained by applying a single coating, allowing the layer to dry, and then repeating the process. The amount of solids added in each coating step depends primarily on the concentration of the coating syrup. Any number of coatings can be applied to the chewing gum core sheet. Preferably, no more than about 75 layers of coating are applied to the chewing gum core. More preferably, fewer than about 60 layers of coating are applied, and most preferably, about 30 to about 60 layers of coating are applied. In any case, the subject matter of this disclosure contemplates applying an amount of syrup sufficient to produce a coated chewing gum product containing about 10% to about 65% coating. Preferably, the final product will contain about 20% to about 50% coating.

[0360] Those skilled in the art will recognize that, in order to obtain multiple coating layers, multiple pre-measured aliquots of the coating syrup can be applied to the gum core. However, the volume of the aliquots of syrup to be applied to the gum core can vary throughout the coating process.

[0361] Once the syrup coating is applied to the chewing gum core, the syrup is dried in an inert medium. A preferred drying medium includes air. Preferably, the drying air is forced to contact the wet syrup coating at a temperature ranging from about 70℉ to about 110℉. More preferably, the drying air is at a temperature ranging from about 80℉ to about 100℉. The invention also considers the drying air to have a relative humidity of less than about 15%. Preferably, the relative humidity of the drying air is less than about 8%.

[0362] Dry air can pass through and mix with the syrup-coated gum core in any manner commonly known in the art. Preferably, for large-scale operations, the dry air is blown over and around the syrup-coated gum core at a flow rate of about 2,800 cubic feet per minute. Lower flow rates will be used if smaller amounts of material are being processed, or if smaller equipment is being used. If flavoring is applied after the syrup coating has dried, the subject matter of this disclosure contemplates drying the flavoring with or without a drying medium.

[0363] The amount of D-allose used herein is generally dependent on preference, depending on factors such as the type of the final chewing gum composition, the individual flavor, the gum base used, and the desired strength of the flavoring. Therefore, the amount of D-allose can be varied to obtain the desired results in the final product, and such variations are within the capabilities of those skilled in the art without requiring excessive experimentation. In the chewing gum composition, D-allose prepared and / or generated by any of the microorganisms disclosed herein is generally present in an amount of about 0.02% to about 5%, preferably about 0.1% to about 2%, more preferably about 0.8% to about 1.8% by weight of the chewing gum composition.

[0364] 5.2. Sugar Candies

[0365] Another important aspect of the subject matter of this disclosure includes confectionery compositions incorporating D-allose prepared and / or generated by any of the microorganisms disclosed herein, and methods for preparing such confectionery compositions. The preparation of confectionery formulations is well known in the art. Confectionery is classified as either “hard” or “soft” confectionery. D-allose prepared and / or generated by any of the microorganisms disclosed herein can be incorporated into confectionery by mixing compositions of the subject matter of this disclosure into conventional hard and soft confectionery.

[0366] Hard candies can be processed and formulated using conventional methods. Typically, hard candies have a base consisting of a mixture of sugars and other carbohydrate fillers, which is maintained in an amorphous or vitreous state. Hard candies can also be sugar-free. Hard candies can also be low-calorie. This form is considered to be a solid syrup typically containing about 0.5% to about 1.5% water. By weight of the final composition, such a material typically contains up to about 92% sugar, up to about 55% corn syrup, and about 0.1% to about 5% water. The syrup component is typically prepared from sucrose and corn syrup, but may include other materials. In some embodiments, the syrup component includes D-allose prepared and / or generated by any of the microorganisms disclosed herein. Other ingredients, such as flavorings, sweeteners, acidifiers, colorings, etc., may also be added.

[0367] Such candies can be prepared using conventional methods, including but not limited to methods involving fire-cooked appliances, vacuum cookers, and scraped-surface cookers (also known as high-speed atmospheric cookers). Useful equipment according to the subject matter of this disclosure includes cooking and mixing equipment well-known in the field of confectionery manufacturing, and therefore the selection of specific equipment will be obvious to those skilled in the art.

[0368] Using a fire-cooking appliance involves a traditional method for manufacturing confectionery bases. In this method, a desired amount of carbohydrate filler is dissolved in water by heating an agent in a pot until the filler dissolves. Additional filler can then be added and the mixture is cooked until a final temperature of 145°C to 156°C is reached. The batch is then cooled and processed into a plastic-like substance for incorporation of additives such as flavorings, colorings, etc.

[0369] High-speed atmospheric pressure cookers utilize a heat exchanger surface, which involves spreading a candy film on the surface and heating the candy to 165°C to 170°C within seconds. The candy is then rapidly cooled to 100°C to 120°C and processed into a plastic-like substance that allows for the incorporation of additives such as flavorings, colorings, etc. In vacuum cookers, carbohydrate fillers are boiled to 125°C to 132°C, a vacuum is applied, and additional water is boiled away without further heating. When cooking is complete, the substance is semi-solid and has a plastic-like consistency. At this point, flavorings, colorings, and other additives are incorporated into the substance through conventional mechanical mixing operations.

[0370] The optimal mixing time required to evenly combine flavorings, colorings, and other additives during the standard manufacturing process of hard candies is determined by the time required to achieve a uniform distribution of the materials. Generally, a mixing time of 2 to 10 minutes has been found to be acceptable.

[0371] Once the confectionery material has been properly tempered, it can be cut into processable portions or shaped into desired forms. Various forming techniques can be used depending on the desired shape and size of the final product. A general discussion of the composition and preparation of hard candies can be found in HA Lieberman, Pharmaceutical Dosage Forms: Tablets, Volume 1 (1989), Marcel Dekker, Inc., New York, NY, pages 419–582, the disclosure of which is incorporated herein by reference.

[0372] Compressed candies contain specific materials and are structured under pressure. These candies typically contain up to about 95% sugar by weight of the composition, and contain typical tablet excipients such as binders and lubricants, as well as flavorings, colorings, etc. These candies can also be sugar-free.

[0373] Similar to hard candies, soft candies can be used in embodiments of the disclosed subject matter. The preparation of soft candies (e.g., nougat) involves conventional methods, such as a combination of two main components: (1) a high-boiling-point syrup, such as corn syrup, and (2) a relatively light-textured frappe, typically prepared from ovalbumin, gum arabic, gelatin, plant proteins (e.g., soy-derived compounds), unsweetened milk-derived compounds (e.g., milk proteins), and mixtures thereof. The frappe is typically relatively light and its density can range, for example, from about 0.5 g / cc to about 0.7 g / cc.

[0374] The high-boiling-point syrup or "bob syrup" used in soft candies is relatively viscous, denser than the whipped components, and typically contains a significant amount of carbohydrate filler. Generally, the final nougat composition is prepared by adding "bob syrup" to the whipped mixture under stirring to form the basic nougat mixture. Subsequently, other ingredients such as flavorings, additional carbohydrate fillers, colorings, preservatives, drugs, or mixtures thereof may be added under stirring. Soft candies can also be prepared as sugar-free. A general discussion of the composition and preparation of nougat candies can be found in BW Minifie, Chocolate, Cocoa and Confectionery: Science and Technology, 2nd edition, AVIPublishing Co., Inc., Westport, Conn. (1983), pp. 576–580, the contents of which are incorporated herein by reference.

[0375] Typically, the whipped ingredients are prepared first, and then the syrup ingredients are slowly added with stirring at a temperature of at least about 65°C, preferably at least about 100°C. The mixture of ingredients is continued to be mixed to form a homogeneous mixture, and then the mixture is cooled to a temperature below 80°C, at which point the flavoring can be added. The mixture is then further mixed for an additional period of time until it is ready to be removed and shaped into a suitable candy.

[0376] According to this disclosure, a certain amount of D-allose prepared and / or generated by any of the microorganisms disclosed herein can be incorporated into hard candies and soft candies. The exact amount of D-allose used generally depends on preference, which depends on factors such as the specific type of candy being prepared, the type of filler or carrier used, the type of flavoring used, and the desired intensity of breath freshness. Therefore, the amount of D-allose can be varied to obtain the desired results in the final product, and such variations are within the capabilities of those skilled in the art without requiring excessive experimentation. Typically, the amount of D-allose usually present in hard candies or soft candies is from about 0.001% to about 20% by weight, preferably from about 0.01% to about 15%, more preferably from about 0.01% to about 10%, and even more preferably from about 0.01% to about 5%, and even more preferably from 0.01% to about 0.5%.

[0377] The subject matter of this disclosure extends to methods for manufacturing improved confectionery. D-allose prepared and / or generated from any of the microorganisms disclosed herein can be incorporated into other conventional hard candy or soft candy compositions using standard techniques and equipment known to those skilled in the art. Useful equipment according to the subject matter of this disclosure includes mixing and heating equipment well-known in the confectionery manufacturing field; therefore, the selection of specific equipment will be obvious to those skilled in the art.

[0378] In this method, the composition is prepared by mixing D-allose along with other ingredients of the desired final composition into the confectionery composition. The other ingredients are typically incorporated into the composition based on the properties of the desired composition, as is well known to those skilled in the art. The final confectionery composition is prepared using methods commonly known in the food technology and pharmaceutical fields. The confectionery mixture can then be shaped into the desired confectionery shape.

[0379] D-allose prepared and / or generated from any of the microorganisms disclosed herein can be formulated with conventional ingredients that provide a variety of textures suitable for specific applications. Such ingredients can be in the form of hard and soft candies, compressed candies, toffee, nougat, chewing candies, chewing gum, etc., and center-filled candies (sweetened and sugar-free). Acceptable ingredients can be selected from a wide range of materials. Such materials include, but are not limited to, diluents, binders and adhesives, lubricants, disintegrants, fillers, humectants, buffers, and adsorbents. The preparation of such candy and chewing gum products is well known.

[0380] 5.3. Chocolate and Fillings

[0381] The subject matter of this disclosure is also used and / or incorporated in chocolate products, chocolate-flavored confectionery, and chocolate-flavored compositions. Chocolate also includes those chocolates containing crumb solids or solids made wholly or partially 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 entirety. A general discussion of the composition and preparation of chocolate confectionery can be found in BW Minifie, Chocolate, Cocoa and Confectionery: Science and Technology, 2nd edition, AVI Publishing Co., Inc., Westport, Conn. (1982), the disclosure of which is incorporated herein by reference.

[0382] As used herein, the term "chocolate" 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 a fat-like composition. This term is intended to include standardized or non-standardized compositions that comply with the Standards of Identity (SOI), Codex Alimentarius, and / or other international standards, as well as compositions that do not comply with the Standards of Identity or other international standards. This term includes dark chocolate, baked chocolate, sweet chocolate, bittersweet or semi-sweet chocolate, milk chocolate, buttermilk chocolate, skim milk chocolate, mixed dairy chocolate, white chocolate, sweet cocoa and vegetable fat coating, sweet chocolate and vegetable fat coating, milk chocolate and vegetable fat coating, vegetable fat-based coating, chocolate including white chocolate or coatings made with cocoa butter or vegetable fat, or combinations thereof, nutritionally modified chocolate-like compositions (chocolate or coatings made with low-calorie ingredients) and low-fat chocolate, aerated chocolate, complex coatings, non-standardized chocolate, and chocolate-like compositions, unless otherwise expressly stated.

[0383] For example, non-standardized chocolate is produced when nutritional carbohydrate sweeteners are partially or completely replaced; or when cocoa butter, cocoa butter substitutes, cocoa butter equivalents, cocoa butter extenders, cocoa butter substitutes, cocoa butter alternatives, or milk fat are partially or completely replaced; or when ingredients that mimic the flavor of milk, butter, or chocolate are added, or when other additions or deletions are made to the formulation that exceed FDA chocolate labeling standards, or a combination of the above. Chocolate compositions are those fat-based compositions that can be used as chocolate substitutes in applications such as coating, molding, or enrobing; for example, carob.

[0384] In the United States, chocolate is governed by uniformity standards established by the US Food and Drug Administration (FDA) under the Federal Food, Drug, and Cosmetic Act. The US has well-established definitions and standards for various types of chocolate. Non-standardized chocolate refers to chocolate whose composition does not fall within the scope of standardized chocolate regulations.

[0385] In some embodiments, the chocolate may contain D-allose prepared and / or generated by any of the microorganisms disclosed herein. Additionally, the chocolate may contain syrups / solids, invert sugar, hydrolyzed lactose, maple syrup, brown sugar, molasses, honey, sugar substitutes, etc. Nutrient carbohydrate sweeteners with varying degrees of sweetness intensity may be any of those commonly used in the art, including but not limited to sucrose (e.g., from sugarcane or sugar beets), glucose, fructose, lactose, maltose, glucose syrup solids, corn syrup solids, invert sugar, hydrolyzed lactose, honey, maple syrup, brown sugar, molasses, etc. Sugar substitutes may partially replace nutrient carbohydrate sweeteners. High-efficiency sweeteners include aspartame, cyclosulfonates, saccharin, acesulfame potassium, neohesperidin dihydrochalcone, sucralose, alitane, steviol glycoside sweeteners, glycyrrhizin, thomatose, etc., and mixtures thereof. Preferred high-efficiency sweeteners are aspartame, cyclosulfonates, saccharin, and acesulfame potassium. Examples of sugar alcohols may be any of those commonly used in the art, including sorbitol, mannitol, xylitol, maltitol, isomaltitol, lactitol, etc.

[0386] Chocolate may also contain fillers. The term "filler" as defined herein can be any of those commonly used in the art and includes polydextrose, cellulose and its derivatives, maltodextrin, gum arabic, etc.

[0387] Chocolate products may contain emulsifiers. Examples of safe and suitable emulsifiers may be any of those commonly used in the art, including lecithin derived from plant sources (e.g., soybean, safflower, corn, etc.), fractionated lecithin rich in phosphatidylcholine or phosphatidylethanolamine or both, monoglycerides and diglycerides, diacetyl tartrate of monoglycerides and diglycerides (also known as DATEM), monosodium phosphate derivatives of monoglycerides and diglycerides of edible fats or oils, sorbitan monostearate, hydroxylated lecithin, lactylated fatty acid esters of glycerol and propylene glycol, polyglycerol esters of fatty acids, propylene glycol monoesters and diesters of fats and fatty acids, or emulsifiers that may be approved for use in the soft candy category as defined by the U.S. FDA. Other emulsifiers that may be used include polyglycerol polyricinoleate (PGPR), ammonium phosphatidylcholine salts (e.g., YN), sucrose esters, oat extracts, etc., and any emulsifier found to be suitable for chocolate or similar fat / solid systems or any mixtures.

[0388] The term "chocolate-flavored confectionery" refers to food products that, in addition to being called "chocolate," have a chocolate flavor / aroma and contain cocoa fractions. These products are stable at ambient temperature for an extended period (e.g., more than one week) and are characterized as microbial shelf-stable under normal atmospheric conditions at 18–30°C. Examples include chocolate-flavored hard candies, chewables, chewing gum, etc.

[0389] The term "chocolate-flavored composition" refers to a chocolate-flavored composition that does not contain "chocolate," contains cocoa fractions, and has a chocolate flavor / aroma. Examples include chocolate-flavored cake mixes, ice cream, syrups, baked goods, etc. The term includes chocolate-flavored compositions (e.g., cakes, nougat, puddings, etc.) as well as compositions that do not have a chocolate flavor (e.g., caramel, etc.).

[0390] 5.4. Salty foods and other food products

[0391] In some embodiments, D-allose prepared and / or generated by any of the microorganisms disclosed herein is incorporated into the savory food product. In some embodiments, the savory food product is a food product having a savory flavor, said savory flavor including but not limited to, for example, spicy, peppery, dairy, vegetable, tomato, dill, meat, poultry, chicken, and reactive flavors added or generated during heating of the food product.

[0392] In some embodiments, D-allose prepared and / or generated by any of the microorganisms disclosed herein is incorporated into wet soup-type food products, which include wet / liquid soups regardless of concentration or container, including frozen soups. In some embodiments, soup-type food products refer to foods prepared from meat, poultry, fish, vegetables, grains, fruits, and / or other ingredients cooked in a liquid, which may include some or all of these ingredients in visible chunks. It can be clear (as broth) or viscous (as stew), smooth, thick, or blocky, ready-to-eat, semi-concentrated, or concentrated, and can be consumed hot or cold, as a first course or main course of a meal, or as a snack between meals (sipped like a beverage). Soup can be used as an ingredient in the preparation of other dietary components, ranging from broths (clear soups) to sauces (cream or cheese-based soups).

[0393] In some embodiments, D-allose prepared and / or generated by any of the microorganisms disclosed herein is incorporated into dehydrated and culinary food products, including (i) culinary aids, such as powders, granules, pastes, concentrated liquid products, including concentrated broths, broths, and broth-like products in the form of compressed cubes, tablets, or powders or granules, sold separately as a finished product or as an ingredient within a product; sauces and recipe mixtures (regardless of the technology used); (ii) dietary solution products, such as dehydrated and freeze-dried soups, including dehydrated soup mixtures, dehydrated instant soups, dehydrated ready-to-eat soups, ready-to-eat dishes prepared at dehydrated or room temperature, meals, and single-serving main courses, including pasta, potatoes, and rice; and (iii) meal embellishments. Product), such as: seasonings, marinades, salad dressings, salad ingredients, dips, breadcrumbs, batter mixtures, shelf-stable spreads, barbecue sauces, liquid formulation mixtures, concentrates, sauces or sauce mixtures, including salad formulation mixtures sold as finished products or as product ingredients, whether dehydrated, liquid or frozen.

[0394] In some embodiments, D-allose prepared and / or generated by any of the microorganisms disclosed herein is incorporated into meat food products. In some embodiments, meat food products include food products made by processing the edible residues of any dead animal (including birds, fish, crustaceans, shellfish, and mammals). Meat food products include, but are not limited to, prepared beef, lamb, pork, poultry, or seafood products. Examples of such meat food products include, for example, Bologna sausage, Frankfurt sausage, salami, luncheon meat, deli slices, bread, bacon, meatballs, fish strips, chicken strips, and ground meat, such as patties, meatballs, and hamburgers. Meat food products can be combined with meat-mimicking products. Meat-mimicking products include, for example, meat substitutes, meat analogues, soy burgers, soy Bologna sausage, soy Frankfurt sausage, soy salami, soy luncheon bread, soy bacon, and soy meatballs. Meat-mimicking products can be combined with meat food products.

[0395] In some embodiments, D-allose prepared and / or generated by any of the microorganisms disclosed herein is incorporated into snack food products. In some embodiments, snack products include any food that can be served as a convenient informal meal, including but not limited to sweet and savory snacks and snack bars. Examples of snacks include, but are not limited to, fruit snacks, potato chips / crisps, puffed foods, tortillas / cornflakes, popcorn, pretzels, nuts, and other sweet and savory snacks. Examples of snack bars include, but are not limited to, granola / muesli bars, breakfast bars, energy bars, fruit bars, and other snack bars.

[0396] In some embodiments, D-allose prepared and / or generated by any of the microorganisms disclosed herein is incorporated into frozen food products, including refrigerated or frozen food products such as, but not limited to, ice cream, impromptu ice cream, single-serving dairy ice cream, single-serving water ice cream, multi-pack dairy ice cream, multi-pack water ice cream, family-sized ice cream, family-sized dairy ice cream, ice cream desserts, bulk ice cream, family-sized water ice cream, frozen yogurt, artisanal ice cream, frozen ready-to-eat 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 baked goods, and frozen desserts.

[0397] 5.4. Drugs

[0398] D-allose prepared and / or generated from any of the microorganisms disclosed herein can also be in the form of a pharmaceutical. A non-limiting example of a pharmaceutical form is a suspension. Pharmaceutical suspensions can be prepared by conventional formulation methods. Suspensions may contain auxiliary materials used in the formulation of suspensions in the art. Suspensions of the subject matter of this disclosure may include preservatives, buffers, suspending agents, antifoaming agents, sweeteners, flavoring agents, coloring or decolorizing agents, solubilizers, and combinations thereof.

[0399] Flavoring agents, such as those well known to those skilled in the art, such as natural and artificial flavorings and peppermint (e.g., peppermint, menthol), citrus flavorings (e.g., orange and lemon), artificial vanilla, cinnamon and various fruit flavorings (alone and in combination), may be used in an amount of about 0.01% to about 5%, more preferably 0.01% to about 0.5% by weight of suspension.

[0400] The pharmaceutical suspensions of the subject matter of this disclosure can be prepared as follows: (i) mixing a thickener with water heated to about 40°C to about 95°C, preferably about 40°C to about 70°C, to form a dispersion (if the thickener is not water-soluble) or a solution (if the thickener is water-soluble); (ii) mixing D-allose prepared and / or generated from any of the microorganisms disclosed herein with water to form a solution; (iii) if desired, mixing a flavoring agent with the thickener-water mixture to form a homogeneous thickener-flavoring agent; (iv) combining and mixing a sweetener solution with the thickener-flavoring agent until homogeneous; and (v) mixing optional auxiliary materials (e.g., colorants, flavoring agents, decolorizing agents, solubilizers, defoamers, buffers, and additional water) with the mixture of step (iv) to form a suspension.

[0401] D-allose prepared and / or generated from any of the microorganisms disclosed herein may also be in a chewable form. Several considerations are important for achieving acceptable stability and quality, as well as good taste and mouthfeel, in chewable formulations. These considerations include the amount of active substance per tablet, the flavoring agent used, the compressibility of the tablet, and other characteristics of the composition. Chewable pharmaceutical confectionery is prepared using a procedure similar to that used for preparing soft candies. A general discussion of confectionery in tablet and chewable tablet forms can be found in HA Lieberman and L. Lachman, Pharmaceutical Dosage Forms: Tablets Volume 1, Marcel Dekker, InC, New York, NY (1989), pp. 367–418, the disclosure of which is incorporated herein by reference. In a typical procedure, a boiled sugar-corn syrup mixture is formed, to which a whipped mixture is added. The boiled sugar-corn syrup mixture can be prepared from a mixture of sugar and corn syrup in a weight ratio of about 90:10 to about 10:90. The sugar-corn syrup mixture is heated to a temperature above approximately 120°C to remove water and form a melt. A whipped material, typically prepared from gelatin, ovalbumin, milk protein (e.g., casein), and plant protein (e.g., soy protein), is added to a gelatin solution and rapidly mixed at ambient temperature to form an aerated, spongy substance. The whipped material is then added to the molten confectionery substance and mixed at a temperature of approximately 65°C to approximately 120°C until homogeneous. D-allose prepared and / or generated by any of the microorganisms disclosed herein can then be added to the homogeneous mixture while the temperature is lowered to approximately 65°C–95°C, followed by the addition of other ingredients, such as flavorings and colorings. The preparation is further cooled and formed into small pieces of the desired size.

[0402] In other pharmaceutical embodiments, a flavoring agent is incorporated into an ingestible topical medium, which may be in the form of mouthwash, rinsing solution, ingestible spray, suspension, teething gel, etc. Typical non-toxic ingestible media known in the pharmaceutical art can be used in the subject matter of this disclosure. Preferred ingestible media are water, ethanol, and water-ethanol mixtures. Water-ethanol mixtures are typically used in weight ratios of about 1:1 to about 20:1, preferably about 3:1 to about 20:1, and most preferably about 3:1 to about 10:1. The pH of the ingestible medium is typically about 4 to about 7, preferably about 5 to about 6.5. Ingestible topical media with a pH below about 4 generally irritate the ingestible cavity, while ingestible media with a pH above about 7 generally result in unpleasant taste.

[0403] Ingestible topical flavorings may also contain conventional additives typically used in those products. Conventional additives include compounds that provide fluoride, sweeteners, flavoring agents, coloring agents, humectants, buffers, and emulsifiers, provided that these additives do not interfere with the flavoring properties of the composition. The coloring agents and humectants listed above, and the amounts of these additives, may be used in ingestible topical compositions. Permissible flavorings (fragrances, flavorings) include those known to those skilled in the art, such as natural and artificial flavorings. Suitable flavorings include peppermint (e.g., peppermint), citrus flavorings (e.g., orange and lemon), artificial vanilla, cinnamon, various fruit spices (alone and in blends), etc. The amount of flavoring used in an ingestible topical composition is generally dependent on preference, depending on factors such as the type of the final ingestible composition, the individual flavor used, and the desired strength of the flavoring. Therefore, the amount of flavoring can be varied to achieve the desired results in the final product, and such variations are within the capabilities of those skilled in the art without requiring excessive experimentation. When used, the flavoring agent is typically used in amounts that can vary, for example, from about 0.05% to about 6% by weight of the ingestible local composition.

[0404] 5.5. Pet food products

[0405] D-allose prepared and / or generated from any of the microorganisms disclosed herein can be used in a variety of pet food products.

[0406] As used herein, the term "pet food" or "pet food product" refers to a product or composition intended for consumption by companion animals, such as cats, dogs, guinea pigs, rabbits, birds, and horses. For example, but not limited to, companion animals can be "domesticated" dogs, such as the Chinese Rural Dog (…). Canis lupus familiaris "Pet food" or "pet food products" includes any food, feed, snacks, food supplements, liquids, beverages, treats, toys (chewable and / or edible toys), meal substitutes, or meal replacements.

[0407] In some embodiments, D-allose prepared and / or generated from any of the microorganisms disclosed herein is added directly to the pet food product. In some embodiments, D-allose prepared and / or generated from any of the microorganisms disclosed herein may be added before, during, or after the formulation or packaging of the pet food product.

[0408] Non-limiting examples of suitable pet food products include wet food products, dry food products, semi-moist food products, pet food supplements (e.g., vitamins), pet beverage products, treats and sweets, and the pet food categories described herein.

[0409] In some embodiments, the pet food product is a dry food product. A dry or low-moisture nutritionally complete pet food product may contain less than about 15% moisture. In some embodiments, the pet food product is a wet food product. A wet or high-moisture nutritionally complete pet food product may contain more than about 50% moisture. In some embodiments, the pet food product is a nutritionally complete semi-moist food product. Semi-moist, such as semi-moist, semi-dry, soft-dry, soft-moist, or medium-moisture nutritionally complete pet food products, contain about 15% to about 50% moisture.

[0410] In some implementations, pet food products are pet food snack products. Non-limiting examples of pet food snack products include treat bars, pet chews, crunchy sweets, cereal bars, snacks, biscuits, and desserts.

[0411] 5.6. Delivery System

[0412] In some embodiments, D-allose may be incorporated into a delivery system for an edible composition. In some embodiments, the composition will contain another flavor or taste modifier, such as a salty, umami, bitter, astringent, and / or savory-umami enhancer. The delivery system may be liquid or solid, aqueous or non-aqueous. The delivery system is generally adapted to suit the needs of flavor compositions and / or edible compositions incorporating D-allose.

[0413] D-Allose can be used in liquid, dry, and / or solid form. When used in a dry form, a suitable drying method such as spray drying can be used. Alternatively, D-Allose can be encapsulated or absorbed into water-soluble materials, including but not limited to materials such as cellulose, starch, sugar, maltodextrin, gum arabic, etc. Practical techniques for preparing such dry forms are well known in the art and can be applied to the subject matter of this disclosure.

[0414] D-Allose can be used in many different physical forms well known in the art to provide an initial burst of flavor, taste, and / or texture; and / or a long-lasting sensation of flavor, taste, and / or texture. These physical forms are not limited to this; they include free forms such as spray-dried, powdered and beaded forms, encapsulated forms, and mixtures thereof.

[0415] In some embodiments, D-allose is encapsulated. Encapsulation materials and / or technologies can be selected to improve the stability of D-allose and / or the food product. In some embodiments, encapsulation materials and / or technologies are selected to modify the release characteristics of D-allose.

[0416] Suitable encapsulating materials may include, but are not limited to, hydrocolloids such as alginate, pectin, agar, guar gum, cellulose, etc.; proteins, polyvinyl acetate, polyethylene, cross-linked polyvinylpyrrolidone, polymethyl methacrylate, polylactic acid, polyhydroxyalkanoates, ethyl cellulose, polyvinyl acetate phthalates, polyethylene glycol esters, methacrylate-co-methyl methacrylate, ethylene-vinyl acetate (EVA) copolymers, etc., and combinations thereof. Suitable encapsulation techniques may include, but are not limited to, spray coating, spray drying, spray cooling, absorption, adsorption, inclusion complexation (e.g., generating fragrance / cyclodextrin complexes), coagulation, fluidized bed coating, or other methods that can be used to encapsulate components with encapsulating materials.

[0417] Delivery systems for encapsulating flavorings or sweeteners (e.g., D-allose) comprise a hydrophobic matrix of fat or wax surrounding a core of the sweetener or flavoring. The fat may be selected from any number of conventional materials, such as fatty acids, glycerides or polyglycerides, sorbitol esters, and mixtures thereof. Examples of fatty acids include, but are not limited to, hydrogenated and partially hydrogenated vegetable oils such as palm oil, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, soybean oil, cottonseed oil, sunflower oil, safflower oil, and combinations thereof. Examples of glycerides include, but are not limited to, monoglycerides, diglycerides, and triglycerides.

[0418] Useful waxes can be selected from natural waxes and synthetic waxes and mixtures thereof. Non-limiting examples include paraffin wax, petrolatum, carbon wax, microcrystalline wax, beeswax, carnauba wax, candelilla wax, lanolin, myrica wax, sugarcane wax, whale wax, rice bran wax, and mixtures thereof.

[0419] Fat and wax can be used alone or in combination, varying in amount from about 10% to about 70% by weight, and optionally from about 30% to about 60% of the encapsulation system. When used in combination, fat and wax are preferably present in a ratio of about 70:10 to 85:15, respectively.

[0420] Typical encapsulation compositions, flavoring or sweetener 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 entirety.

[0421] Liquid delivery systems may include, but are not limited to, systems having D-allose dispersions, such as in carbohydrate syrups and / or emulsions. Liquid delivery systems may also include extracts in which D-allose is dissolved in a solvent. Solid delivery systems may be produced by spray drying, spray coating, spray cooling, fluidized bed drying, absorption, adsorption, coagulation, complexation, or any other standard technique. In some embodiments, the delivery system may be selected to be compatible with or function in an edible composition. In some embodiments, the delivery system will include an oil-containing material, such as fat or oil. In some embodiments, the delivery system will include sugary fats, such as cocoa butter, cocoa butter substitutes, cocoa butter alternatives, or cocoa butter equivalents.

[0422] When used in a dry form, suitable drying methods such as spray drying can be used. Alternatively, D-allose can be adsorbed or absorbed onto a substrate, such as water-soluble materials like cellulose, starch, sugar, maltodextrin, gum arabic, etc., or it can be encapsulated. Practical techniques for preparing such a dry form are well known in the art.

[0423] Example

[0424] The subject matter of this disclosure can be better understood by referring to the following description. The following examples are merely exemplary and should not be considered as limiting.

[0425] Example 1 - Biosynthesis of allosugar from glucose

[0426] Escherichia coli naturally produces trace amounts of D-allose. In this embodiment, D-allose production is increased by overexpressing key genes, removing competing pathway genes, and optimizing production conditions.

[0427] Assessing the allosugar production capacity of Escherichia coli

[0428] Initially, the presence of an enzyme capable of producing allosugar in *E. coli* was tested. Allosugar production was tested in the production strain AL3601, described in Table 1 below. Cultures were grown at 30°C on M9P medium (M9 basal medium containing 5 g / L yeast extract) containing 10 g / L glucose, and supernatant samples were collected 24 hours after inoculation. Glucose concentration in the supernatant was analyzed using HPLC. Allosugar concentration in the supernatant was analyzed using HPAE-PAD. A series of gene knockout (KO) strains were constructed in AL3601. These KOs included Δ... pfkA、 Δ zwf Δ manA Δ deoC Δ pgm Δ alsABCEKR Δ hxpB Δ yqaB and Δ ybiVThese strains caused strains AL3755, AL4240, AL4290, AL4311, AL4328, AL4332, AL4373, AL4374, AL4446, AL4383 and AL4387 respectively (see Table 1). pfkA It encodes phosphofructokinase A (EC 2.7.1.11), which converts fructose-6-phosphate (F6P) to fructose-1,6-bisphosphate (F16BP) as part of the first key step in glycolysis. zwf It encodes glucose-6-phosphate dehydrogenase (Zwf) (EC 1.1.1.1.363), which converts glucose-6-phosphate (G6P) to 6-phosphate-D-gluconic acid-1,5-lactone as the first key step in the pentose phosphate pathway (PPP). manA It encodes mannose-6-phosphate isomerase (ManA), which catalyzes the reversible isomerization of mannose-6-phosphate and fructose-6-phosphate. deoC It encodes deoxyribose-phosphoaldolase, which catalyzes the reversible conversion of allose-6-phosphate to erythrose-4-phosphate and glycolaldehyde. pgm Enzymes that encode glycogen biosynthesis. alsABCEKR Enzymes encoding allosugar metabolism: alsA Encoding D-allose input ATP-binding protein; alsB Encoding D-allose-binding periplasmic protein; alsC Protein encoding the D-allose transport system permease; alsK Encoding D-allokinase; and alsR Encodes HTH-type transcriptional regulators. hxpA It encodes hexitol phosphatase A, which catalyzes the dephosphorylation of D-allose-6-phosphate. yqaB Encoding fructose-1-phosphate phosphatase (Entrez gene ID: 945776), which catalyzes the dephosphorylation of fructose-1-phosphate. ybiV It encodes a sugar phosphatase that catalyzes the dephosphorylation of D-allulose-6-phosphate. The KO strain showed increased allose production, indicating that *E. coli* possesses an enzyme capable of producing allose.

[0429] Table 1. List of strains

[0430] Determination of allosugar-producing enzymes

[0431] Next, we assume that fructose-6-phosphate (F6P) is an intermediate in the production of allosugar in *E. coli* because the absence of pfkA increases allosugar production. The production pathway begins with the natural assimilation of glucose into *E. coli* via a phosphotransferase system (PTS) or GalP / Glk, which converts glucose to glucose-6-phosphate (G6P) (Figure 1). G6P is then isomerized to F6P via glucose-6-phosphate isomerase (Gpi) (EC 5.3.1.9). F6P can be converted to allulose-6-phosphate via allulose-6-phosphate 3-epimerase (AlsE). P6P is then isomerized to allosugar-6-phosphate (A6P) by allosugar-6-phosphate isomerase (RpiB). Phosphatases can dephosphate allosugar-6-phosphate to free allosugar, which is then expelled from the cell. Allosugar production systems that use phosphorylation and dephosphorylation steps as the driving force should be more efficient than the pathways currently used in the industrial production of allosugar.

[0432] Literature review and genome mining identified isomerase candidates. Allosugar-6-phosphate isomerase (RpiB) has shown activity for allosugar. In addition, several phosphatase candidates were identified. YigL, YqaB, and HxpA have shown activity for allosugar-6-phosphate.

[0433] Plasmids were constructed (Table 2) to overexpress the gene under the inducible promoter PLAcO1. alsE, rpiB The plasmid was introduced into AL4332, and allosugar production was tested. Cultures were grown at 30°C on M9P medium containing 10 g / L glucose and induced with 1 mM IPTG. After 24 hours, the induced AL4332 / pAL2310 exhibited increased allosugar production.

[0434] Table 2. Plasmid List

[0435] Table 3.

[0436] Example 2 - Eliminating the innate ability of E. coli to produce D-allose

[0437] D-Allose is a rare monosaccharide that occurs naturally in low abundance. Due to its low-calorie properties and sucrose-like taste, D-Allose has the potential to be an ideal sugar substitute. D-Allose also exhibits unique properties and health benefits, making it applicable in various fields, including food and medical applications. D-Allose can be produced in vitro using two enzymatic steps: epimerization of D-fructose, followed by isomerization of the resulting D-allulose. Due to the reversible nature of the two reactions, the yield of this method is poor. *E. coli* was found to possess all the necessary enzymes to convert D-glucose to D-Allose via a thermodynamically favorable pathway through a series of phosphorylation-epimerization-isomerization-dephosphorylation steps. To increase the carbon flux toward D-Allose production, pathway genes were additionally expressed, and competing pathways were removed. Under in vitro conditions, the engineered strain achieved a yield of 56.4 g / L... -1 The titer was 0.65 g / L. -1 hr 1 The production of D-allose was achieved with a productivity of 4.4%.

[0438] Rare sugars are monosaccharides that are naturally occurring in low abundance[1]. However, they are widely used in the food and pharmaceutical industries because they can be non-nutritive or used as precursors for anticancer and antiviral drugs[2]. The rare sugar D-allose is a C-3 epimer of glucose with 80% of the sweetness of sucrose[3] and is metabolized to the least extent in mammals[4]. As a reducing sugar, D-allose has good food properties, such as being able to participate in Maillard reaction browning[5]. In fact, D-allose reacts with α-lactalbumin at a faster rate than D-fructose or D-glucose[6]. D-allose also exhibits cryoprotective effects[7], which can be used in pharmaceuticals and frozen food preservation.

[0439] D-allose also has clinically relevant health properties[5]. It has been shown to have anticancer and antitumor effects[8-11]. Cancer cells increase their glucose uptake to meet their dysregulated energy needs; D-allose reduces glucose uptake in cancer cells

[11] . D-allose has also been reported to have antioxidant and anti-inflammatory properties, and it can be used medically to minimize ischemia-reperfusion (I / R) injury

[12] , especially in the brain[13-16]. Other health benefits of D-allose include hypertension suppression

[17] and immunosuppression of allogeneic graft survival

[18] . Acute and subchronic toxicity studies in rats have demonstrated that D-allose is non-toxic

[19] .

[0440] These sensory, metabolic, physiological and safety properties have increased the demand for D-allose. As with other rare sugars, there is no cost-effective large-scale production system for D-allose; it has been extracted from the leaves of various plants, but in low yields[5].

[0441] Enzymatic pathways for the production of D-allose have been proposed, but these methods are not thermodynamically favorable, resulting in limited yields (approximately 10%) [20, 21]. D-allose can be enzymatically synthesized by isomerizing D-allulose to D-allose using L-rhamnose isomerase (L-RHI, EC5.3.1.14)

[22] or D-ribose-5-phosphate isomerase B (RpiB, EC5.3.1.6)

[23] . The predicted total conversion from D-fructose to D-allose is 2.4 kJ / mol. -1 ΔG'°( Figure 2A

[24] D-allulose is relatively expensive and therefore not an ideal raw material. This mixture of D-fructose, D-allulose and D-allose also presents purification difficulties.

[0442] D-allulose can be produced in industrially relevant microorganisms such as *Escherichia coli* using glycophosphorylation / dephosphorylation chemistry

[25] . Glycophosphorylation / dephosphorylation chemistry is naturally used for glucose uptake and metabolism

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

[27] . This disclosure demonstrates that this strategy can also be used for D-allose production. The predicted dephosphorylation of D-allose-6-phosphate to D-allose is a highly favorable response, with a predicted ΔG'm of -35.7 kJ mol at a typical physiological concentration of 1 mM. -1 ( Figure 2B

[24] . In order to utilize sugar phosphorylation / dephosphorylation chemistry in vitro, a source of phosphate such as ATP must be provided, which increases the production cost. Therefore, microbial production is used because no cofactor supply is required.

[0443] Escherichia coli can produce D-allose from D-glucose using only its natural enzymes. Figure 2CIn the D-allose pathway, D-fructose-6-phosphate (F6P) is epimerized to D-allulose-6-phosphate (P6P) by D-allulose-6-phosphate 3-epimerase (AlsE). The resulting P6P is further isomerized to D-allose-6-phosphate (A6P) by D-allose-6-phosphate isomerase / D-ribose-5-phosphate isomerase B (RpiB), which can then be dephosphorylated to D-allose by phosphatases. D-allose production is enhanced by additional expression of D-allose pathway genes and removal of competing pathways, including the pentose phosphate pathway (PPP), glycogen biosynthesis, glycolysis, D-mannose biosynthesis, D-allulose biosynthesis, and the D-allose salvage pathway. To further improve D-allose production, we used an inducer-free stationary promoter

[28] and regulated the expression of key pathway genes by designing a regulatory pathway operon. We used the galactose proton transporter GalP to improve D-glucose uptake

[29] , and then D-glucose was phosphorylated by the glucokinase Gluk

[30] .

[0444] Results and Discussion

[0445] a. Design of a thermodynamically favorable D-allose production pathway

[0446] The in vitro enzymatic method for D-allose is thermodynamically unfavorable

[21] ( Figure 2A Furthermore, the resulting mixture of monosaccharides requires expensive purification techniques [31-33]. Therefore, a D-allose biosynthetic pathway was developed, which utilizes the phosphorylation and dephosphorylation of sugars to generate a driving force in *E. coli* (…). Figure 2B A biosynthetic pathway for the production of D-allulose from D-glucose using phosphorylation / dephosphorylation chemistry has previously been established in E. coli

[25] . Branches of the D-allulose pathway were designed for D-allose. Instead of dephosphorylating P6P to D-allulose, P6P can be isomerized to A6P. Dephosphorylation of A6P will be the thermodynamic driver of D-allose production.

[0447] b. Elucidate the enzymes involved in D-allose production.

[0448] Initially, the natural ability of *E. coli* to produce D-allose was tested (Table 6), but it did not produce D-allose. Then, to increase the F6P library, the deletion... pfkA Strain 1 (AL3755, Table 4) was generated. F6P was converted to D-fructose 1,6-bisphosphate (D-fructose 1,6-bisphosphate) during glycolysis via phosphofructokinases A and B (pfkA and pfkB). Figure 2C ), and PfkA accounts for approximately 90% of phosphofructokinase activity [34, 35]. However, Δ pfkAThe strain does not produce D-allose.

[0449] AlsE can perform its reverse reaction to convert F6P to P6P

[25] . RpiB has previously been shown to isomerize A6P to P6P

[36] . The reverse reaction of this enzyme can be used to isomerize P6P to A6P (…). Figure 2C Escherichia coli possesses a variety of phosphatases with potential activity against A6P

[37] , and it is assumed that the natural expression of these phosphatases would be sufficient to dephosphate A6P to D-aloose. alsE and rpiB Genes are expressed by plasmids in Δ pfkA P in the strain LlacO1 Expression under the promoter. However, this strain does not produce D-allose ( Figure 3 ).

[0450] Additional expression of the phosphatase gene can convert A6P to D-allose. *E. coli* phosphatases with a broad range of sugar substrate activity were tested: hexitol phosphatase A (HxpA), hexitol phosphatase B (HxpB), sugar phosphatase YbiV, sugar phosphatase YidA, α-D-glucose-1-phosphate phosphatase YihX, phosphate-sugar phosphatase YigL, and fructose-1-phosphate phosphatase YqaB

[37] . To test the ability of each phosphatase to convert A6P to D-allose, the gene for each phosphatase was expressed along with... alsE and rpiB Together in P LlacO1 The expression plasmids (pAL2310, pAL2311, pAL2312, pAL2313, pAL2316, pAL2376, pAL2377, Table 5) were introduced under the promoter. The plasmids were then introduced into the Δ... pfkA The strain was supplemented with 10 g / L. -1 Glucose was grown in M9P for 24 hours. After 24 hours, it contained... hxpA and yqaB The strains containing plasmids (pAL2310 and pAL2313, Table 5) produced 1.15 g L each. -1 and 0.23 g L -1 D-allose ( Figure 3 Strains containing other phosphatase plasmids do not produce D-allose (). Figure 3 ).contain hxpB , yqaB , yidA and ybiV Strains (pAL2311, pAL2313, pAL2376, pAL2377, Table 5) showed increased D-allulose production, indicating that these phosphatases were able to dephosphorylate P6P. Therefore, hxpA was used for further improvement.

[0451] Table 4. List of strains.

[0452]

[0453] Table 5. List of plasmids.

[0454]

[0455] Table 6. Strains used in this example.

[0456]

[0457] Increasing D-allose production by eliminating competing pathways

[0458] To increase D-allose yield, the competing pathway was removed. Then, a product containing PLlacO1 was added. alsE-rpiB- hxpA The plasmid was introduced into these strains, and the yield of D-allose was measured.

[0459] First, it prevents the produced D-allose from being re-assimilated into metabolism. The body responsible for D-allose catabolism... alsABCEKR The operon

[38] is missing in strains 1 through 2 ( Figure 4 ). (Compared to 1.15 g L in strain 1) -1 D-Allose production: Strain 2 increased D-allose production to 1.88 g / L. -1 ( Figure 4 ).

[0460] G6P is converted to 6-phosphate-D-gluconic acid 1,5-lactone via glucose-6-phosphate dehydrogenase (Zwf) and transferred to the pentose phosphate pathway

[39] . In strain 2, the zwf gene encoding glucose-6-phosphate dehydrogenase was deleted to generate strain 3 ( Figure 4 Strain 3 further increased D-allose production to 2.4 g / L. -1 ( Figure 4 ).

[0461] D-mannose is a byproduct when the F6P pool in E. coli increases

[25] . The manA gene encodes a mannose-6-phosphate isomerase that isomerizes F6P to mannose-6-phosphate (M6P)

[40] , thereby diverting flux away from the D-allose production pathway. This pathway was missing in strain 3. manA Genes generate strain 4 ( Figure 4 ). manA The absence of D-allose did not increase D-allose production ( Figure 4 However, strain 4, which produces plasmids, consumes less glucose than the previous three strains, indicating efficient conversion of D-glucose.

[0462] Strain 4 lacks the encoding of deoxyribophosphoaldolase. deoC Genes are used to generate strain 5 ( Figure 4 The deoxyribose-phosphoaldolase can assimilate D-allose into central carbon metabolism (41). deoC The absence of D-allose did not increase D-allose production ( Figure 4 ).

[0463] Glucose can also be transferred from the D-allose pathway via glycogen biosynthesis. Glycogen is stored for use during starvation

[42] , which may be unnecessary during D-allose production. Phosphoglucose mutase (Pgm) converts glucose to glycogen biosynthesis by converting G6P to glucose-1-phosphate

[43] . Therefore, it is missing in strain 5. pgm Genes are used to generate strain 6 ( Figure 4 The absence of pgm slightly increased D-allose production (3.0 g / L). -1 , Figure 4 ).

[0464] Finally, the heterogeneity of E. coli phosphatases can transfer flux from the D-allose production pathway by dephosphorylating P6P

[25] . Hexitol phosphatase B (HxpB), phosphatase YbiV, phosphatase YidA, and fructose-1-phosphate phosphatase YqaB can dephosphorylate P6P to D-allose

[25] . Figure 3 ) Phosphatase genes were sequentially deleted. hxpB , yqaB , ybiV and yidA Strains 7, 8, 9 and 10 were generated respectively. Figure 4 The D-allose yields were similar in strains 7-10, but strain 9 showed the highest yield. Figure 4 Therefore, strain 9 (AL4387, Table 4) was used for further experiments.

[0465] Substrate channelization to reduce nonspecific dephosphorylation

[0466] D-allulose and D-allose generate competitive P6P

[25] . To reduce the generation of D-allulose, AlsE and RpiB were fused into the chimeric protein using flexible peptide linkers. Substrate channelization can be used to increase the flux from F6P to P6P, and then to A6P [44, 45]. Two types of linkers were used ((Gly-Ser-Gly)n (as disclosed in SEQ ID NO: 79, “(Gly-Ser-Gly)3”) and (Gly-Gly-Gly-Gly-Ser)n, n = 1, 3) (as disclosed in SEQ ID NO 78 and 79, “(Gly-Gly-Gly-Gly-Ser)1” and “(Gly-Gly-Gly-Gly-Ser)3”) (pAL2562-pAL2565, Table 5) [45, 46]. The effect of the linkers was tested in strain 9. However, none of the linkers decreased or increased D-allulose production. Figure 5 ).

[0467] Regulation of pathway enzyme expression

[0468] Regulating the expression of D-allose pathway genes to increase D-allose production. When multiple genes from the operon are expressed, the expression of downstream genes is lower than that of their upstream counterparts

[47] . To direct the flux of P6P toward A6P, we constructed a pathway containing P LlacO1 : rpiB-alsE-hxpA The plasmid (pAL2581, Table 5) makes it possible to obtain plasmids relative to P. LlacO1 : alsE-rpiB- hxpA rpiB is expressed more strongly.

[0469] Increasing hxpA The expression of D-allose was evaluated to assess its production. Natural hxpA Genes starting with the GTG codon result in lower translation compared to genes starting with the ATG codon

[48] . Using ATG ( hxpA (pAL2623, Table 5) Replace the start codon of hxpA.

[0470] Yields can also be increased through gene expression pathways under stronger promoters

[25] . Early stationary promoter P gadB The expression intensity was higher than that of the IPTG-induced promoter P LlacO1 It was 100 times stronger and increased D-allulose production

[25] . The effects of these modifications on D-allulose production were tested in AL4387 in combination of three factors (promoter, gene sequence and start codon) (pAL2580, pAL2609, pAL2624, Table 5). Figure 5Contains P gadB : rpiB-alsE-hxpA and P LlacO1 : alsE-rpiB-hxpA Strain 9 produced the most D-allose (3.1 g / L). -1 ()( Figure 6 ).

[0471] Utilizing GalP and Glk to supplement glucose infusion

[0472] Continuous input and phosphorylation of D-glucose are essential for the production of D-allose. Its limitations are... pfkA The absence of PTS results in a reduced pool of downstream metabolites of glycolysis, including phosphoenolpyruvate (PEP). PTS utilizes PEP to import extracellular D-glucose and phosphorylate it to intracellular G6P, and a reduction in the PEP pool can decrease glucose uptake [49, 50]. Furthermore, it has been shown that… pfkA In the absence of these cells, accumulated G6P and F6P may degrade ptsG mRNA

[51] .

[0473] The G6P pool is enhanced by alternative D-glucose input and phosphorylation mechanisms. GalP is a galactosomal proton transporter capable of inputting glucose

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

[30] . Figure 2C Built on P LlacO1 The promoter contains galP and glk The plasmid (pAL2264, Table 5) 25. pAL2264 was combined with pAL2310 (P LlacO1 : alsE-rpiB-hxpA ), pAL2609(P gadB : rpiB-alsE-hxpA ), pAL2623(P LlacO1 : alsE-rpiB-hxpA ) or pAL2624(P gadB : alsE-rpiB-hxpA This was introduced together with strain 9. The expected increase in glucose input

[25] will increase the glucose concentration supplemented to this disclosed strain to 40 g / L. -1 D-allose production was then measured 24 hours later (Figure 7).

[0474] galP-glk contains P gadB : alsE-rpiB-hxpA Expression in strain 9 resulted in the production of 10.59 g L. -1 D-Allose, yield 39.4% (Figure 7). In comparison, only P...gadB : alsE-rpiB-hxpA Strain 9 produced 4.25 g L. -1 D-Allose, yield 26.0% (Figure 7). Similarly, when expressing... galP-glk When containing P gadB : rpiB-alsE- hxpA Strain 9 also showed improved D-allose production (Figure 7). However, this strain was less effective than strains containing P. gadB : alsE-rpiB- hxpA Strain 9 produces more D-allose, which in the absence of galP-glk 6.1 g L was produced under the condition of expression. -1 D-allose, and in the presence of galP-glk 11.3 g L was produced under the condition of expression. -1 D-allose (33.9% and 41.4% yield, respectively) (Figure 7). Then, the product containing P... gadB : rpiB-alsE-hxpA and P LlacO1 : galP-glk Strain 9 was used for further production experiments.

[0475] Interestingly, when expressing galP-glk When containing P LlacO1 : alsE-rpiB-hxpA and P LlacO1 : alsE- rpiB-hxpA Strain 9 did not produce more D-allose (Figure 7). AlsE, RpiB, and HxpA in P LlacO1 Expression under the promoter is weaker than that under P gadB Promoter

[25] .

[0476] Allulose: Regulation of allulose production ratio

[0477] Incorporating different sugar substitutes into food or beverages can improve their sensory quality while minimizing their calorie content

[59] . Of particular interest is D-allulose, a zero-calorie sugar substitute that has been recognized as GRAS by the U.S. Food and Drug Administration

[60] . Its sweetness is estimated to be 70% that of sucrose and it is described as providing a refreshing sensation rather than bitterness

[61] .

[0478] To produce D-allulose and D-allose in different proportions, different native sugar phosphatase genes were expressed. Additional expression of the native phosphatase genes yqaB and hxpA from the plasmid led to the production of D-allulose and D-allose. Figure 3The plasmid pAL2310(P) was used. LlacO1 : alsE-rpiB-hxpA ) and pAL2313(P LlacO1 : alsE-rpiB-yqaB The strain was introduced into strain 9 (Table 4). The strain was supplemented with 10 g L... -1 Strain 9, containing pAL2310, was grown in M9P for 24 h. After 24 h, strain 9 produced 3.1 g L. -1 D-allose and 0.1 g L -1 D-Allulose, corresponding to a 27:1 ratio of D-allose:D-allulose ( Figure 7B Strain 9, containing pAL2313, produced 1.0 g L. -1 D-allose and 0.8 g L -1 D-Allulose, corresponding to a 1.3:1 ratio of D-allose to D-allulose ( Figure 7B These results indicate that the ratio of allose to allulose can be altered by differences in the substrate specificity of phosphatases.

[0479] D-Allose Production under High-Density Culture Conditions

[0480] To isolate growth and production, and to minimize the limitations of glucose availability on production, strain 9 containing the pAL2609 and pAL2264 plasmids (Table 5) was cultured under high cell density conditions. The strain was then grown to OD... 600 The cells were then induced with 1 mM IPTG and allowed to regrow for 30 minutes. Cell pelleting was then performed using a solution containing 40 g / L... -1 Glucose and 1 mM IPTG M9P resuspended to OD 600 Approximately 10. Every 24 h, remove 10% of the culture volume and use a solution containing 400 g L. -1 Replace the medium with M9P medium containing glucose and 1 mM IPTG.

[0481] Strain 9, containing pAL2609 and pAL2264, produced 56.4 g L after 120 hours. -1 D-Allosugar, net yield 29.7% ( Figure 8A Achieving 15.5 g L in 48-72 h. -1 sky -1 Peak productivity was 38.8%. Figure 8A The supplemented glucose was completely consumed during the 0 h–24 h, 24 h–48 h, and 48 h–72 h periods. Figure 8B Glucose consumption slowed down after 72 hours. Figure 8BThe consumption of glucose in the culture medium allows for easier purification of D-allulose during industrial production. After 96 hours, the D-allulose yield increased to 28.7 g / L. -1 Then reduce ( ) between 96 h and 120 h Figure 8A ). Measured g L in 24 h -1 D-Allose: g / L -1 The highest ratio of D-allulose (2.43:1) occurs when D-glucose is absent. Figure 8A ).

[0482] In this study, the natural metabolism of *E. coli* was rewired in a thermodynamically favorable manner to produce D-allose. alsE , rpiB , hxpA , galP and glk Gene expression, combined with the elimination of competing pathways, is used to direct carbon flux from D-glucose. Under in vitro conditions, the titer of D-allose produced after 120 hours was 56.4 g / L. -1 Peak productivity is 0.65 g / L -1 hr -1 The highest yield obtained was 41.4%, even exceeding that of one-step enzymatic methods for producing D-allose [52, 53]. This example demonstrates the strong potential of the bacteria of this disclosure to produce D-allose in high abundance.

[0483] method

[0484] Reagents. All enzymes used in the molecular cloning experiments were purchased from New England Biolabs (NEB). All synthesized oligonucleotides were synthesized by Integrated DNA Technologies. Sanger sequencing was provided by Genewiz. D-allulose was purchased from Sigma-Aldrich. D-glucose was purchased from Fisher Scientific. D-allose was purchased from ThermoScientific.

[0485] Strains and plasmids. All strains and plasmids used in this study are listed in Tables 6 and 7, respectively. All oligonucleotides are listed in Table 8. Plasmids for D-allose production were constructed using sequence and ligation-independent cloning (SLIC)

[54] . The constructed plasmids were validated by sequencing. Instructions for the construction of plasmids used in this study are detailed in Table 9.

[0486] Genomic modifications such as gene deletions and insertions were constructed using CRISPR-Cas9-mediated homologous recombination

[55] . Linear DNA repair fragments for gene deletions and insertions were constructed by PCR assembly amplification of genomic or plasmid DNA

[56] . Using pTargetF plasmid (Addgene # 62226) as a template, plasmids encoding sgRNAs for CRISPR-Cas9-mediated homologous recombination were constructed using Q5 site-directed mutagenesis (New England Biolabs). All genomic modifications were validated by sequencing. The guidelines for CRISPR-Cas9-mediated gene modifications used in this study are detailed in Table 10.

[0487] Table 7. Plasmids used in this embodiment.

[0488]

[0489] Table 8. Oligonucleotides used in this example.

[0490]

[0491] Table 9. Guidelines for plasmid construction.

[0492]

[0493] Table 10. Guidelines for CRISPR-Cas9-mediated gene deletion and insertion.

[0494]

[0495] Cultivation conditions Overnight cultures were grown at 37°C in 3 mL of Luria-Bertani (LB) medium containing appropriate antibiotics. The antibiotic concentrations were as follows: spectinomycin (50 μg / mL). -1 Ampicillin (200 μg / mL) -1 ), kanamycin (50 μg mL) -1 ), Gentamicin (3.75 μg mL) -1 The M9 basal medium consists of the following: 33.7 mM Na₂HPO₄, 22 mM KH₂PO₄, 8.6 mM NaCl, 9.4 mM NH₄Cl, 2 mM MgSO₄, 0.1 mM CaCl₂, and A5 trace metal mixture (2.86 mg / L). -1 H3BO3, 1.81 mg / L-1 MnCl2 4H2O, 0.079 mg L -1 CuSO4 5H2O, 49.4 μg L -1 Co(NO3)2 (6H2O), different concentrations of glucose, and appropriate antibiotics. M9P medium for D-allose production is supplemented with 5 g / L of... -1 The medium consisted of yeast extract and appropriate antibiotics in an M9 basal medium. No D-allose production was detected when the culture was grown in glucose-free M9P medium. The inducer concentrations were as follows: isopropyl-β-D-1-thiogalactoside (IPTG) (1 mM), ahydrotetracycline (aTc) (100 ng / mL). -1 ). OD 600 Measured using a Synergy H1 hybrid plate reader (BioTek Instruments, Inc.).

[0496] Production of D-allose. For routine cell density generation experiments, overnight culture was seeded at 1% in 3 mL of M9P medium. Cells were then grown at 37°C until the stated OD was reached. 600 Then, if necessary, induce with IPTG and aTc and grow at 30°C for 24 h.

[0497] To achieve high cell density production in M9P medium, overnight culture was seeded at 2% in 50 mL of M9P medium. Cells were then grown at 37°C until OD (dose-to-cell ratio) was reached. 600 Approximately 1. Then, if necessary, induce the culture with IPTG and aTc, and allow the culture to regrow for 30 minutes. Centrifuge the culture at 5,000 g for 15 min, and if necessary, resuspend in M9P medium with IPTG and aTc to the target OD. 600 The culture was grown at 30°C. Every 24 hours, 10% of the culture volume was removed and replaced with a solution containing 400 g L... -1 Replace the glucose in the M9P medium, and if necessary, replace it with IPTG and aTc.

[0498] HPLC analysis. D-glucose concentration was analyzed using HPLC (Shimadzu) equipped with a Rezex™ RCU-USP sugar alcohol column (Phenomenex) and a refractive index detector (RID) 10A. The mobile phase consisted of 100% MilliQ water. The injection volume was 1 µL, and the solution was injected at a rate of 0.5 mL / min. -1The sample was run at a flow rate of 7 min, with the column oven at 83°C and the RID cell at 40°C.

[0499] To prepare samples for HPLC analysis, 300 µL of culture was centrifuged at 17,000 g for 5 min. The supernatant was applied to a 96-well 0.2 µm PVDF hydrophilic membrane filter plate and centrifuged at 17,000 g for 2 min into 96 wells of polystyrene.

[0500] High-performance anion exchange chromatography (HPAE) analysis. D-allulose and D-allose concentrations were analyzed using an HPAE with pulsed amperometric detection (PAD) on an ICS-5000 system equipped with a CarboPac PA10 4x250mm (Thermo Fisher). The washing phase consisted of 200 mM NaOH in degassed MilliQ water at a flow rate of 0.8 mL / min. -1 The process was continued for 10 min. The equilibrium phase consisted of 100 mM NaOH in degassed MilliQ water at a flow rate of 0.8 mL / min. -1 The induction time was 25 min. The mobile phase consisted of 100 mM NaOH in degassed MilliQ water. The sample was injected in a 10 μL volume at a rate of 0.8 mL / min. -1 Run at the specified flow rate for 10 minutes. Maintain the column oven at 30°C.

[0501] To prepare samples for HPAE-PAD analysis, 300 µL of culture was centrifuged at 17,000 g for 5 min. The supernatant was diluted 100-fold and applied to a 96-well 0.2 µm PVDF hydrophilic membrane filter plate, and centrifuged at 17,000 g for 5 min into 96 wells of polystyrene.

[0502] Table 11. Figure 3 The report shows the yield and standard deviation of OD600.

[0503]

[0504] Table 12. Figure 4 The report shows the yield and standard deviation of OD600.

[0505]

[0506] Table 13. Figure 3 The report shows the yield and standard deviation of OD600.

[0507]

[0508] Table 14. Figure 6Standard deviations of the yields and OD600 reported in the medium.

[0509]

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[0572] Although the subject matter and advantages of this disclosure have been described in detail, it should be understood that various changes, substitutions, and modifications can be made to this document without departing from the spirit and scope of the invention as defined by the appended claims. Furthermore, the scope of this application is not intended to be limited to the specific embodiments of the processes, machines, manufactures, material compositions, means, methods, and steps described in the specification. As will be readily understood by those skilled in the art from the disclosure of the subject matter of this disclosure, existing or future existing processes, machines, manufactures, material compositions, means, methods, or steps that perform substantially the same functions or achieve substantially the same results as the corresponding embodiments described herein can be utilized based on the subject matter of this disclosure. Therefore, the appended claims are intended to include such processes, machines, manufactures, material compositions, means, methods, or steps within their scope.

[0573] Patents, patent applications, publications, product specifications and solutions cited in this application are incorporated herein by reference in their entirety for all purposes.

Claims

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

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

3. The recombinant microorganism according to claim 1 or 2, wherein the epimerase is *Escherichia coli* (…). E. coli )AlsE.

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

1.

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

1.

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

1.

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

8. The recombinant microorganism according to claim 1 or 7, wherein the phosphatase is Escherichia coli HxpA.

9. The recombinant microorganism according to claim 1, 7 or 8, wherein the phosphatase comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO:

62.

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

62.

11. The recombinant microorganism according to any one of claims 1 and 7-10, wherein the phosphatase consists of the amino acid sequence shown in SEQ ID NO:

62.

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

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

14. The recombinant microorganism according to claim 1 or 13, wherein the phosphatase is Escherichia coli YqaB.

15. The recombinant microorganism according to claim 1, 13 or 14, wherein the phosphatase comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO:

60.

16. The recombinant microorganism according to any one of claims 1 and 13-15, wherein the phosphatase comprises the amino acid sequence shown in SEQ ID NO:

60.

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

60.

18. The recombinant microorganism according to any one of claims 1-17, wherein the isomerase is allosugar-6-phosphate isomerase (RpiB).

19. The recombinant microorganism according to claim 18, wherein the RpiB is Escherichia coli RpiB.

20. The recombinant microorganism according to claim 18 or 19, wherein the isomerase comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO:

24.

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

24.

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

24.

23. The recombinant microorganism according to any one of claims 1-22, wherein the recombinant microorganism further comprises exogenous galactose:H + Cotransporter (GalP) and glucokinase (Glk).

24. The recombinant microorganism of claim 23, wherein the GalP is Escherichia coli GalP and the Glk is Escherichia coli Glk.

25. The recombinant microorganism of claim 24, wherein the GalP comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO: 38, and the Glk comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO:

40.

26. The recombinant microorganism of claim 25, wherein the GalP comprises the amino acid sequence shown in SEQ ID NO: 38, and the Glk comprises the amino acid sequence shown in SEQ ID NO:

40.

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

28. The recombinant microorganism according to any one of claims 1-27, wherein, compared with naturally occurring microorganisms, the recombinant microorganism further comprises a mutation in the gene encoding an enzyme of the pentose phosphate pathway.

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

30. The recombinant microorganism according to any one of claims 1-29, wherein, compared with naturally occurring microorganisms, the recombinant microorganism further comprises a mutation in a gene encoding an enzyme for glycolysis.

31. The recombinant microorganism according to claim 30, wherein the glycolytic enzyme is phosphofructokinase-1 (PfkA), phosphofructokinase-2 (PfkB), or pyruvate kinase (PykF).

32. The recombinant microorganism according to claim 30 or 31, wherein the glycolytic enzyme is phosphofructokinase-1 (PfkA).

33. The recombinant microorganism according to any one of claims 1-32, further comprising a mutation in the gene encoding an aldolase.

34. The recombinant microorganism according to claim 33, wherein the aldolase is deoxyribose-phosphoaldolase (DeoC).

35. The recombinant microorganism according to any one of claims 1-34, further comprising a mutation in at least one gene encoding a phosphatase.

36. The recombinant microorganism according to claim 35, wherein the gene encoding at least one phosphatase is selected from the genes of fructose-1-phosphate phosphatase (YqaB), sugar phosphatase YbiV, hexitol phosphatase B (HxpB), sugar phosphatase YidA, or combinations thereof.

37. The recombinant microorganism according to any one of claims 1-36, further comprising a mutation in at least one gene encoding an enzyme for allosugar metabolism.

38. The recombinant microorganism according to claim 37, wherein the gene encoding at least one enzyme of allosugar metabolism is selected from the genes of D-allosugar ATP-binding protein (AlsA), D-allosugar-binding periplasmic protein (AlsB), D-allosugar transport system permease protein (AlsC), D-allosugar kinase (AlsK), HTH-type transcription regulator (AlsR), or combinations thereof.

39. The recombinant microorganism according to any one of claims 1-38, further comprising a mutation in the gene encoding an enzyme of the mannose biosynthesis pathway.

40. The recombinant microorganism according to claim 39, wherein the enzyme in the mannose biosynthesis pathway is mannose-6-phosphate isomerase (ManA).

41. The recombinant microorganism according to any one of claims 1-40, further comprising a mutation in the gene encoding an enzyme of the phosphotransferase system (PTS).

42. The recombinant microorganism according to any one of claims 1-41, further comprising a mutation in the gene encoding an enzyme for glycogen biosynthesis, said enzyme for glycogen biosynthesis being selected from phosphoglucosuric enzyme (Pgm), UDP-glucose pyrophosphorylase, glycogen synthase, glycogen branching enzyme, and glycogen protein.

43. The recombinant microorganism according to claim 42, wherein the enzyme for glycogen biosynthesis is phosphoglucose mutase (Pgm).

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

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

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

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

48. The recombinant microorganism according to claim 46 or 47, wherein the epimerase is Escherichia coli AlsE.

49. The recombinant microorganism according to any one of claims 46-48, wherein the epimerase comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO:

1.

50. The recombinant microorganism according to any one of claims 46-49, wherein the epimerase comprises the amino acid sequence shown in SEQ ID NO:

1.

51. The recombinant microorganism according to any one of claims 46-50, wherein the epimerase consists of the amino acid sequence shown in SEQ ID NO:

1.

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

53. The recombinant microorganism according to claim 46 or 52, wherein the phosphatase is Escherichia coli HxpA.

54. The recombinant microorganism according to claim 46, 52 or 53, wherein the phosphatase comprises at least about 80% of the same amino acid sequence as the amino acid sequence shown in SEQ ID NO:

62.

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

62.

56. The recombinant microorganism according to any one of claims 46 or 52-55, wherein the phosphatase comprises the amino acid sequence shown in SEQ ID NO:

62.

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

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

59. The recombinant microorganism according to claim 46 or 58, wherein the phosphatase is Escherichia coli YqaB.

60. The recombinant microorganism according to claim 46, 58 or 59, wherein the phosphatase comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO:

60.

61. The recombinant microorganism according to any one of claims 46 or 58-60, wherein the phosphatase comprises the amino acid sequence shown in SEQ ID NO:

60.

62. The recombinant microorganism according to any one of claims 46 or 58-61, wherein the phosphatase comprises the amino acid sequence shown in SEQ ID NO:

60.

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

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

65. The recombinant microorganism according to claim 63 or 64, wherein the isomerase comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence shown in SEQ ID NO:

24.

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

24.

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

24.

68. The recombinant microorganism according to any one of claims 63-67, wherein the recombinant microorganism further comprises exogenous galactose:H + Cotransporter (GalP) and glucokinase (Glk).

69. The recombinant microorganism of claim 68, wherein the GalP is Escherichia coli GalP and the Glk is Escherichia coli Glk.

70. The recombinant microorganism of claim 69, wherein the GalP comprises at least about 80% of the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 38, and the Glk comprises at least about 80% of the same amino acid sequence as the amino acid sequence shown in SEQ ID NO:

40.

71. The recombinant microorganism of claim 70, wherein the GalP comprises the amino acid sequence shown in SEQ ID NO: 38, and the Glk comprises the amino acid sequence shown in SEQ ID NO:

40.

72. The recombinant microorganism according to any one of claims 46-71, wherein the recombinant microorganism further comprises exogenous glucose-6-phosphate isomerase (Gpi).

73. The recombinant microorganism according to any one of claims 46-72, wherein, compared with naturally occurring microorganisms, the recombinant microorganism further comprises a mutation in the gene encoding an enzyme of the pentose phosphate pathway.

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

75. The recombinant microorganism according to any one of claims 46-74, wherein, compared with naturally occurring microorganisms, the recombinant microorganism further comprises a mutation in a gene encoding an enzyme for glycolysis.

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

77. The recombinant microorganism according to claim 75 or 76, wherein the glycolytic enzyme is phosphofructokinase-1 (PfkA).

78. The recombinant microorganism according to any one of claims 46-77, further comprising a mutation in the gene encoding an aldolase.

79. The recombinant microorganism according to claim 78, wherein the aldolase is deoxyribose-phosphoaldolase (DeoC).

80. The recombinant microorganism according to any one of claims 46-79, further comprising a mutation in at least one gene encoding a phosphatase.

81. The recombinant microorganism according to claim 80, wherein the gene encoding at least one phosphatase is selected from the genes of fructose-1-phosphate phosphatase (YqaB), sugar phosphatase YbiV, hexitol phosphatase B (HxpB), sugar phosphatase YidA, or combinations thereof.

82. The recombinant microorganism according to any one of claims 46-81, further comprising a mutation in a gene encoding at least one enzyme for allosugar metabolism.

83. The recombinant microorganism according to claim 82, wherein the gene encoding at least one enzyme of allosugar metabolism is selected from the genes of D-allosugar ATP-binding protein (AlsA), D-allosugar-binding periplasmic protein (AlsB), D-allosugar transport system permease protein (AlsC), D-allosugar kinase (AlsK), HTH-type transcription regulator (AlsR), or combinations thereof.

84. The recombinant microorganism according to any one of claims 46-83, further comprising a mutation in the gene encoding an enzyme of the mannose biosynthesis pathway.

85. The recombinant microorganism according to claim 84, wherein the enzyme in the mannose biosynthesis pathway is mannose-6-phosphate isomerase (ManA).

86. The recombinant microorganism according to any one of claims 46-85, further comprising a mutation in the gene encoding an enzyme of the phosphotransferase system (PTS).

87. The recombinant microorganism according to any one of claims 46-86, further comprising a mutation in the gene encoding an enzyme for glycogen biosynthesis, said enzyme for glycogen biosynthesis being selected from phosphoglucosuric enzyme (Pgm), UDP-glucose pyrophosphorylase, glycogen synthase, glycogen branching enzyme, and glycogen protein.

88. The recombinant microorganism according to claim 87, wherein the enzyme for glycogen biosynthesis is phosphoglucose mutase (Pgm).

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

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

91. Microorganisms, which include: a) Recombinant polynucleotides encoding epimerases, isomerases, and phosphatases; b) Mutations in the genes encoding enzymes of the pentose phosphate pathway; c) Mutations in genes encoding enzymes that encode glycolysis; d) Mutations in the gene encoding aldolase; e) Mutations in genes encoding enzymes that facilitate the mannose biosynthesis pathway; f) optionally encoding a recombinant polynucleotide of GalP, Glk, or both; g) Optional reorganization of Gpi; and h) Mutations in genes that optionally encode enzymes of the phosphotransferase system.

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

93. The microorganism according to claim 91 or 92, further comprising a mutation of at least one allose metabolism enzyme.

94. Microorganisms, which include: a) Recombinant polynucleotides encoding allulose-6-phosphate 3-epimerase (AlsE), allulose-6-phosphate isomerase (RpiB), and hexitol phosphatase A (HxpA); b) Mutation of glucose-6-phosphate 1-dehydrogenase (Zwf); c) Mutations in phosphofructokinase-1 (PfkA); d) Mutation of deoxyribose-phosphoaldolase (DeoC); e) Mutation of mannose-6-phosphate isomerase (ManA); f) optionally encoding a recombinant polynucleotide of GalP, Glk, or both; g) Recombinant polynucleotides optionally encoding Gpi; and h) Mutations in genes that optionally encode enzymes of the phosphotransferase system.

95. The microorganism according to claim 94, further comprising at least one mutation selected from the genes of fructose-1-phosphate phosphatase (YqaB), sugar phosphatase YbiV, hexitol phosphatase B (HxpB), sugar phosphatase (YidA), ATP-binding protein (AlsA), D-allose-binding periplasmic protein (AlsB), D-allose transport system permease protein (AlsC), D-allose kinase (AlsK), HTH-type transcription regulator (AlsR), or combinations thereof.

96. Microorganisms, which include: a) Recombinant polynucleotides encoding allulose-6-phosphate 3-epimerase (AlsE), allulose-6-phosphate isomerase (RpiB), and fructose-1-phosphate phosphatase (YqaB); b) Mutation of glucose-6-phosphate 1-dehydrogenase (Zwf); c) Mutations in phosphofructokinase-1 (PfkA); d) Mutation of deoxyribose-phosphoaldolase (DeoC); e) Mutation of mannose-6-phosphate isomerase (ManA); f) optionally encoding a recombinant polynucleotide of GalP, Glk, or both; g) Recombinant polynucleotides optionally encoding Gpi; and h) Mutations in genes that optionally encode enzymes of the phosphotransferase system.

97. The microorganism according to claim 96, further comprising at least one mutation selected from the genes of: sugar phosphatase YbiV, hexitol phosphatase B (HxpB), sugar phosphatase (YidA), ATP-binding protein (AlsA), D-allose-binding periplasmic protein (AlsB), D-allose transport system permease protein (AlsC), D-allose kinase (AlsK), HTH-type transcription regulator (AlsR), or combinations thereof.

98. Microorganisms, which include: a) A recombinant polynucleotide encoding allulose-6-phosphate 3-epimerase (AlsE); b) Recombinant polynucleotide encoding allosugar-6-phosphate isomerase (RpiB); c) Recombinant polynucleotide encoding hexitol phosphatase A (HxpA); d) Mutation of glucose-6-phosphate 1-dehydrogenase (Zwf); e) Mutations in phosphofructokinase-1 (PfkA); f) Mutations in deoxyribose-phosphoaldolase (DeoC); g) Mutation of mannose-6-phosphate isomerase (ManA); h) optionally encodes a recombinant polynucleotide of GalP, Glk, or both; i) optional recombinant polynucleotides encoding Gpi; and j) Mutations in genes that optionally encode enzymes of the phosphotransferase system.

99. The microorganism according to claim 98, further comprising at least one mutation selected from the genes of fructose-1-phosphate phosphatase (YqaB), sugar phosphatase YbiV, hexitol phosphatase B (HxpB), sugar phosphatase (YidA), ATP-binding protein (AlsA), D-allose-binding periplasmic protein (AlsB), D-allose transport system permease protein (AlsC), D-allose kinase (AlsK), HTH-type transcription regulator (AlsR), or combinations thereof.

100. Microorganisms, which include: a) A recombinant polynucleotide encoding allulose-6-phosphate 3-epimerase (AlsE); b) Recombinant polynucleotide encoding allosugar-6-phosphate isomerase (RpiB); c) Recombinant polynucleotide encoding fructose-1-phosphate phosphatase (YqaB); d) Mutation of glucose-6-phosphate 1-dehydrogenase (Zwf); e) Mutations in phosphofructokinase-1 (PfkA); f) Mutations in deoxyribose-phosphoaldolase (DeoC); g) Mutation of mannose-6-phosphate isomerase (ManA); h) optionally encodes a recombinant polynucleotide of GalP, Glk, or both; i) optional recombinant polynucleotides encoding Gpi; and j) Mutations in genes that optionally encode enzymes of the phosphotransferase system.

101. The microorganism according to claim 100, further comprising at least one mutation selected from the genes of: sugar phosphatase YbiV, hexitol phosphatase B (HxpB), sugar phosphatase (YidA), ATP-binding protein (AlsA), D-allose-binding periplasmic protein (AlsB), D-allose transport system permease protein (AlsC), D-allose kinase (AlsK), HTH-type transcription regulator (AlsR), or combinations thereof.

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

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

104. The microorganism according to any one of claims 1-103, wherein the microorganism is *Escherichia coli*, *Bacillus subtilis*, or... Bacillus subtilis ) or Lactococcus lactis ( Lactococcus lactis ).

105. A method for producing allosugar, comprising culturing the microorganism according to any one of claims 1-104 under conditions suitable for converting a substrate into allosugar.

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

107. A method for preparing a food product containing allosugar, comprising: a) Culturing the microorganisms of any one of claims 1-104 under conditions suitable for converting the substrate into allosugar; as well as b) Mix the allosugar with one or more food products to form a food product containing allosugar.

108. The method of claim 107, wherein the food product is a beverage, yogurt, ice cream, baked goods, or nutrition bars.

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