Microorganisms for the production of d-tagatose

Abstract

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

Description

MICROORGANISMS FOR THE PRODUCTION OF D-TAGATOSECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 537,140, filed September 7, 2023,. the content of which is incorporated by reference in their entirety, and to which priority is claimed.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on September 5, 2024, is named 081906-1460213 253710PC SL, and is 148,479 bytes in size.TECHINICAL FIELD

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

[0004] Current industrial D-tagatose production is through challenging multi-step, in vitro enzymatic synthesis. One method comprises hydrolysis of lactose to galactose and glucose, and conversion of galactose to tagatose via L-atabinose isomerase (EC 5.3.1,4) at high temperature. Major drawbacks to this m vitro synthesis include that multiple separation and purification processes are required, and the isomerization of galactose to tagatose is thermodynamically unfavorable. Another method comprises conversion of D-fructose-6-phosphate (F6P) to D- tagatose-6-phosphate (T6P) using fructose 1,6-bisphosphate aldolase (EC 4.1.2, 13), followed by dephosphorylation to D-tagatose by an unspecified phosphatase, In vivo this pathway suffers from inhibition by regulatory agents within the celt / « vitro, this requires the addition of expensive cofactors and other enzymes. Thus, there is a need for novel methods and compositions for the production of D-tagatose.SUMMARY

[0005] In one aspect, the present disclosure relates to a recombinant microorganism comprising an exogenous epimerase, wherein the recombinant m icroorganism produces aft increased amoun t of tagatose as compared to a naturally occurring microorganism,

[0006] In some embodiments, the epimerase is a tagatose- 1 ,6-bisohosphate aldolase subunit Gather a ketose biphosphate aldolase subunit KbaZ. In some embodiments, the epimerase comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQID NO: 58. In some embodiments, the epimerase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58.

[0007] In some embodiments, the epimerase is an E. coli KbaZ. In some embodiments, the epimerase comprises an amino acid sequence at least about 80% identical to the amino add sequence sei forth in SEQ ID NO: 60. In some embodiments, the epimerase comprises or consists of the amino acid sequen ce set forth in SEQ ID NO: 60.

[0008] In some embodiments, the microorganism further comprises a recombinant phosphatase. In some embodiments, the phosphatase is a hexitol phosphatase A ( HxpA). In some embodiments, the phosphatase is an E. colt HxpA. In some embodiments, the phosphatase comprises an amino acid sequence at least, about 80% identical to the amino acid sequence set forth in SEQ ID NO: 62. In some embodiments, the phosphatase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62.

[0009] In some embodiments, the recombinant microorganism further comprises exogenous galactose.'H*' symporter (GalP) and glucokinase (Glk). In some embodiments, the GalP is an K call GalP and the Glk is E. call Glk. In some embodiments, the GalP comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 38 and the Glk comprises an amino acid sequence at. least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 40, In some embodiments, the GalP comprises the amino acid sequence set forth in SEQ ID NO: 38 and the Glk comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0010] In some embodiments, the recombinant microorganism further comprises a mutation of a gene encoding for an enzyme of the pentose phosphate pathway compared to a. naturally occurring microorganism. In some embodiments, the enzyme of the pentose phosphate pathway is glucose- 6-phosphate 1 -dehydrogenase (Zwf). In some embodiments, the recombinant microorganism further comprises a mutation of a gene encoding for an enzyme of the glycolysis compared to a naturally occurring microorganism.

[0011] In some embodiments, the enzyme of the glycolysis is phosphofiuctokinase-1 (PfkA), phosphofrtictokinase-2 (PfkB), or pyruvate kinase (PykF). In some embodiments, the enzyme of the glycolysis is phosphofructokinase- 1 (PfkA)

[0012] In some embodiments, the recombinant microorganism further comprises a mutation of a gene encoding for an enzyme that converts ftuctose-6-phosphate to psicose-6-phosphate. In some embodiments,, the enzyme that converts fructose-6-phosphate to psicose-6-phosphate is D- allulose-6-phosphate 3-ephnerase (AlsE). In some embodiments, the recombinant microorganism further comprises a mutation of a gene encoding for an enzyme of mannose biosynthesis pathway.In some embodiments, the enzyme of mannose biosynthesis pathway is mamtose-6-phosphate isomerase (ManA).

[0013] In some embodiments, the recombinant microorganism further comprises a mutation of a gene encoding for an enzyme of carbohydrate metabolism. In some embodiments, the enzyme of carbohydrate metabolism is D-tagatose-1 ,6-bisphosphate aldolase subunit (GatY). In some embodiments, the enzyme of carbohydrate metabolism is D-tagatose- 1,6- bisphosphate aldolase subunit (KbaY),

[0014] In some embodiments, the recombinant microorganism further comprises a mutation of a gene encoding for an enzyme of glycogen biosynthesis selected from phosphoglucomutase ( Pgm),UDP-ghicose pyrophosphorylase, glycogen synthase, glycogen branching enzyme, glycogenin, or combinations thereof. In some embodiments, the enzyme of glycogen biosynthesis is phosphog I ncorn utase (Pgm).

[0015] In another aspect, the present disclosure relates to a microorganism comprising a recombinant polynucleotide encoding an epimerase, wherein expression of the epimerase and the phosphatase results in an increased production of tagatose as compared to a microorganism lacking the recombinant polynucleotide.

[0016] In some embodiments, the epimerase is a tagatose- 1 ,6-bisohosphate aldolase subunit GatZ or a ketose biphosphate aldolase subunit KbaZ, In some embodiments, the epimerase is an E. call GatZ. In some embodiments, the epimerase is an E cali KbaZ. .

[0017] In some embodiments, the microorganism further comprises a recombinant phosphatase. In some embodiments, the phosphatase is a hexitol phosphatase A (HxpA). In some embodiments, the phosphatase is an E. coli HxpA,

[0018] In some embodiments, the microorganism further comprises a mutation of a t least one gene. In some embodiments,, the at least one gene is selected from glucose-6-phosphate 1 - dehydrogenase (cuy). phosphofructokinase- 1 (p / fel), phosphofructokinase-2 (ftfkB), pyruvate kinase ( / n4F), D-alluIose-6-phosphaie 3 -epimerase (aZvE), mannose-6-phosphate isomerase (manA), phosphoglucomutase (pgm), D-tagatose-l,6-bisphosphate aldolase subunit (gn / k), D- tagatoseri ,6-bisphosphate aldolase subunitor a combination thereof.

[0019] In some embodiments, the the at least one gene is glucose-6-phosphate 1 -dehydrogenase (cwf), phosphofructokinase-l (pjkA), D-ailulose-6-phosphate 3-epimerase (aZsfe'), mannose-6- phosphate isomerase (manA), phosphoglucomutase (pgm), D-tagatose- 1 ,6-bisphosphate aldolase subunit (ga / fo, D-tagatose-I ,6~bisphosphate aldolase subunit (kbaY'k or a combination thereof.

[0020] In some embodiments, the mutation is a deletion. In some embodiments, the mutation reduces or eliminates expression or activity of the enzyme. In some embodiments, the microorganism is E. coli, Bacillus sub fills at Eacloeoccus lactis.

[0021] In one aspect; the present disclosure also relates to a method for producing tagatose comprising culturing the microorganism disclosed herein under conditions suitable for converting a substrate to tagatose. In some embodiments, the substrate comprises glucose.

[0022] Additionally; the present, disclosure relates to methods of making a food product comprising tagatose, the method comprising culturing the microorganism disclosed herein under conditions suitable for convening a substrate to tagatose; and admixing the tagatose with one or more foods to form a food comprising tagatose. In some embodiments, the food product is a beverage, yogurt, ice cream, a baked good, or a nutritional bar.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figures 1 A and IB illustrate a pathway for the biosynthetic production of D-tagatose. Glucose is imported and phosphorylated to glucose-6-phosphate (G6P) by the phosphotransferase system (PTS) or GalP / Glk. G6P is then isomerized to fructose-6-phosphate (F6P) by Glucose-6- phosphate isomerase. F6P is epimerized to tag.atose-6-phosphate by a tagatose- 1,6-blsohosphate aldolase subunit GatZ or a ketose biphosphate aldolase subunit KbaZ, which is then dephosphorylated to free tagatose by native phosphatases. Finally, free tagatose can diffuse across the cell membrane into the supernatant. Competing pathways include the pentose phosphate pathway, catalyzed by gIucose-6-phosphate dehydrogenase (Zwfj, glycolysis, which is catalyzed by phosphofmctokinases A and B (PfkA and B), the conversion of F6P to psicose-6-phosphate, catalyzed by D~allulose-6-phosphate 3-epimerase, and the mannose biosynthesis pathway, catalyzed by mannose-6 -phosphate isomerase (ManA).

[0024] Figure 2 illustrates tagatose production capability of E. colL The ceils were grown in M9.P media (M9 minimum media with 5 g / L yeast extract) with 10 g / L glucose at 30 *C for 24 hrs.

[0025] Figure 3 illustrates gene supplementation experiment for D-tagatose production. The gatZ gene was deleted in AL4330, creating AL4386. Two plasmids, pAL252l (empty) and pAL2490 (PLlacOl :gatZ) (Table 2), were constructed as negative control and to express gatZ respectively under an inducible promoter, PLlacOl . Cells were grown in M9P media with 10 g / L glucose at 30“C and induced with 1 mM IPTG.

[0026] Figure 4 illustrates comparison of endogenous phosphatases for D-tagatose production. Seven plasmids, pAL2490, pAL2491, pAL292, pAL2493, pAL2494, pAE2495, pAL2496, were constructed to additionally express hxpA, hxpB, ybiV, yidA, yigL, yihX or yqaB along with gatZ under an inducible promoter, PLlacOl. AL4330 with the phosphatase plasmid was cultured in MOP media with 10 g / L glucose at 30 °C and induced with I mM IPTG.

[0027] Figures 5A-5C illustrate strategies for the biosynthesis of D-tagatose. Figure 5A shows izumoring strategy for D-tagatose production. Figure 5B shows phosphorylation and dephosphorylation strategies for D-tagatose. Figure 5C shows the proposed biosyntheticproduction of D-tagatose in coti. Deleted steps are outside the Tagatose Production Module box. Additionally expressed steps are in the Tagatose Production Module box. PTS, the phosphotransferase system; GatZ, putative tagatose- 1 , 6-bisphosphate aldolase 2 chaperone; KbaZ, putative tagatose- 1 , 6-bisphosphate aldolase 1 chaperone; HxpA, hexitol phosphatase A;Zwf, NADP+-dependent glucose-6-phosphate dehydrogenase; Pgm, phosphoglucomutase; PfkA, 6-phosphofructokinase 1 ; PfkB. 6-phosphofructokinase 2; AlsE, D-allulose-6-phosphate 3- epimerase; ManA, manaose-6-phosphate isomerase; GatY, tagatose- 1 , 6-bisphosphate aldolase 2; KbaY, tagatose- 1 , 6-bisphosphate aldolase 1.

[0028] Figures 6A-6D illustrate D-tagatose production capability of A eafi. Cells were grown in M9P media with 10 g I,"1glucose at 37 °C to ODwi -0.4, then grown al 30 "C for 24 h. At ODw -0.4, I mM IPTG was added. Figure 6A shows D-tagatose production in AL 1050 (MG 1655 + lacl tetR specR), AL3755 (AL 1050 with Ap / fcf) and AL4240 (AL 1050 with A / ? / fc4 Acp / ) (Table 4). Figure 6B shows effect of each epimerase gene deletion on D-tagatose production in AL4240. Figure 0C shows D-tagatose production in AL4424 (AL 1050 with bp / kA tewf AgafZ) with and without additional expression of gufZ (Tables 4 and 5). Figure 6D shows effect of additional expression of each phosphatase gene and gmZ on D-tagatose production in AL4424 (AL 1050 with ΔpfA Δzwf ΔgafZ), Error bars indicate s.d. (a ~ 3 biological replicates).

[0029] Figures 7 A and 7B i llustrate modulating expression of genes for D-tagatose production. Cells were grown in M9P media with 10 g L-1glucose at 37 °C to-0.4, then grown at 30 ”C for 24 h. At OD600-0.4, I tnM IPTG was added when required. Figure 7 A shows each candidate epimerase gene was expressed along with bxpA under .ZTncO! promoter (Table 5) in AL4424 (AL1050 with Ayr / M Ant / AgmZ, Table 4). Figure 7B shows either gatZ or kbaZ and hxpA were expressed under A.wn or / Lade. The start codon of hxpA was changed from GTG toErrors indicate s.d. (n ~ 3 biological replicates).

[0030] Figure 8 shows the effect of gene deletions on D-tagatose production. Cells were grown in M9P media with 10 g L-1glucose to ODw -0.4 at 37 °C, then grown at 30 "C for 24 h. Errors indicate s.d, (n - 3 biological replicates).

[0031] Figures 9A-9F illustrate high ceil density D-tagatose production. Cultures were grown in M9P media with 40 g L-1(Figures 9A-9C) and 15 g L-1(Figures 9D-9F) glucose concentration at 37 °C. until an OD§w of -0,4-0.6. Induced with l.mM IPTG if necessary and grown for a further 30 min. Cultures were then spun down and resuspended in M9P media with 40 g L-1(Figures 9A- 9C) and 15 g L-1(Figures 9D-9F) glucose, induced with 1 mM IPTG if necessary, to an OD^oo of -10 and grown at 30 °C. Each day M9P .media with 40 g L4(Figures 9A-9C) and 15 g Lu(Figures 9D-9F) glucose was added to the production media. Figures 9A and 9D show D-tagatoseproduction. Figures 9B and 9E show D-fructose production. Figures 9D and 9F show D-mannito! production. Error bars indicate s.d. (n = 3 biological replicates).

[0032] Figure 10 shows effect of gene deletion on D-mannose and D~psicose production. Cells were grown in M9P media with 10 g L-1glucose to OD600~0.4 at 37 °C, then grown at 30°C for 24h. Each strain was transformed with pAL2606gaZkxpA). Error bars indicate s.d. (n - 3 biological replicates).

[0033] Figure 1 1 shows glucose consumption of AL4534 at high cell density condition. Glucose consumption of AL4534 with pAL2606 or pAL2607. Cultures were grown in M9P media with 40 g L-1glucose at 37 *C until an OD§ooCultures were then spun down and resuspended in M9P media with 40 g L-1glucose to an OD«» of *-10 and grown at 30 "'C for 24 h. Error bars indicate s.d. (n = 3 biological replicates).DETAILED DESCRIPTION

[0034] The market for rare sugars as a foodstuff] dietary supplement, and health aid is expanding. Among rare sugars, D-tagatose has attracted particular attention. However, the current method of producing D- tagatose is costly, inefficient, and thermodynamically unfavorable, limiting its potential for wide-spread use. Significantly, the present disclosure addressed various obstacles in D-tagatose production including thermodynamic barriers, limited yield, the requirement for purified enzymes, and the addition of cofactors. Another significant finding disclosed herein was that E. coli naturally possesses a thermodynamically favorable path way for D-tagatose production al lowing the improvement of D-tagatose production by increasing the expression of native genes and eliminating competing pathways without the introduction of heterologous genes. The presently disclosed subject matter facilitates the industrial-scale production of D-tagatose without the need for costly enzyme purification or difficult feedstock and product separation,

[0035] The present disclosure is based, in part, on the discovery that microorganisms including specific genetic modifications (e,g., gene deletion) can be made for producing low-calorie sugars. In certain embodiments, the low-calorie sugar is tagatose. For clarity and not by way of limitation, the detailed description of the presently disclosed subject matter is divided into the following subsections:1. Definitions;2. Microorganisms Producing Tagatose;3. Methods for Producing and Generating Microorganisms;4. Methods for Producing Tagatose; and5. Food Products.1. Definitions

[0036] The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the methods and compositions of the invention and how to make and use them,

[0037] As used herein, the use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and "one or more than one.”'

[0038] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about’’ can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fbId, and more preferably within 2-fbId, of a value.

[0039] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The present disclosure also contemplates other embodiments “comprising,” “consisting of”, and “consisting essentially of” the embodiments or elements presented herein., whether explicitly set forth or not,

[0040] As used herein, the term “microorganism” refers to any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria, or eukarya, the latter including yeast and filamentous fungi, protozoa, algae, or higher Protista. In certain embodiments, the term includes prokaryotic or eukaryotic cells or organisms having a microscopic size including, but without any limitation, bacteria, archaea, and eubacteria of all species as well eukaryotic microorganisms such as yeast and fungi. In certain embodiments, the term microorganism includes cells that can be cultured for the production of a chemical (e.g„ sugar). In certain embodiments, the microorganism is a prokaryotic microorganism. In certain embodiments, the prokaryotic microorganism is a bacterium.

[0041] As used herein, the terms “bacterium.” “bacteria,” or “eubacteria,” refers to a domain of prokaryotic organisms. In certain embodiments, bacteria include gram-negative bacteria, grampositive bacteria, proteobacteria, cyanobacteria, spirochetes, and related species, planctomyces, bacteroides, chlamydia, green sulfur bacteria, green non-sulfur bacteria, radioresistant micrococci, and thermotoga and Thermosipho thermophiles.

[0042] As used herein, the term “gram-negative bacteria” includes cocci, nonenteric rods, and enteric rods. The genera of gram-negative bacteria include, for example, and without any limitation, A’eiwerra, Spirillum, Pasteurella, Brucella, Yersinia, Francisella, / iaemophilus, Bordetella, Ksoherichia, Sa / mrw / ta, Shigella, Klebsiella, Proteus, Vibrio, Pseudomonas, .Bacteroides, Acetobacfer, Aerobacter, Agrobacterium, Asotobacter, Spirilla, Serratia, Vibrio, Rhizobium, Chlamydia, Rickettsia, Treponema, and Fusobacterium.

[0043] As used herein, the term “gram-positive bacteria” includes cocci, nonsportdating rods, and sporulating rods, The genera of gram-positive bacteria include, for example, and without any limitation, Actinomyces, Bacillus, Clostridium, Corynebacterium, Prysipelothrix, Lactobacillus, Listeria, Mycobacterium, .Myxococcus, Nacardta, Staphylococcus, Streptococcus, and StreptomyCes.

[0044] As used herein, the term “recombinant microorganism' ’ refers to a microorganism that contains one or more recombinant polynucleotides.

[0045] The term “exogenous."as used herein, refers to molecules that are not naturally found in and / or produced by a given yeast, bacterium, organism, microorganism, or cell in nature. The term “endogenous,” as used herein, refers to molecules that are naturally found in and / or produced by a given yeast, bacterium, organism, microorganism, or cell in nature.

[0046] The term “nucleic acid molecule,” “nucleotide sequence,” or “polynucleotide,” as used herein, refers to a single or double-stranded covalently-linked sequence of nucleotides in which the 3’ and 5’ ends on each nucleotide are joined by phosphodiester bonds. The nucleic acid molecule can include deoxyribonudeotide bases or ribonucleotide bases and can be manufactured synthetically in vitro or isolated from natural sources,

[0047] As used herein, “recombinant polynucleotide” refers to a polynucleotide wherein the exact nucleotide sequence of the polynucleotide is foreign to (i.e., not naturally found in) a given host. In certain embodiments, a recombinant polynucleotide sequence is naturally found in a gi ven host, but in an unnatural (e,g., greater than or less than expected) amount, or additionally if the sequence of a polynucleotide cornprises two or more subsequences that are not found in the same relationship to each other in nature. For example, but without any limitation, a recombinant polynucleotide could have two or more sequences from unrelated polynucleotides or from endogenous nucleotides arranged to make a new polynucleotide. In certain embodiments, the present disclosure provides the introduction of a recombinant polynucleotide into a microorganism, wherein the polynucleotide encodes for a polypeptide that is not normally found in the microorganism. With reference to the microorganism’s genome, then, the polynucleotide sequence that encodes the polypeptide is recombinant or heterologous.

[0048] A "gene,” as used herein, refers to a DMA region (including exons and introns) encoding a gene product, as well as all DNA regions which regulate the production of the gene product, whether or not. such regulatory sequences are adjacent io coding and / or transcribed sequences, hi certain nondimiting embodiments, a gene includes promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions.

[0049] The terms “polypeptide,” “peptide,” "amino acid sequence” and “protein,” used interchangeably herein, refer to a molecule formed from the linking of at least two amino acids. The link between one amino acid residue and the next is an amide bond and is sometimes referred to as a peptide bond. A polypeptide can be obtained by a suitable method known in the art, including isolation from natural sources, expression in a recombinant expression system, chemical synthesis, or enzymatic synthesis. The terms can apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a cotresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0050] The term “amino acid,” as used herein, can be naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g, , hydroxyproline, gamma- carboxyglutamate, and O~phosphoserine. Amino acid analogs and derivatives can refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, Le. , a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e,g. , homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfbniutn. Such analogs can have modified R groups (dg., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics mean chemical compounds that have a structure that is different from the general chemical structure of amino acid, but that function in a manner similar to a naturally occurring amino acid. Non-limiting examples of amino acids include tryptophan, phenylalanine, histidine, glycine, cysteine, alanine, tyrosine, serine, methionine, asparagine, leucine, asparagine, threonine, isoleucine, proline, glutamic acid, aspartic acid, hydroxyl proline, arginine, cystine, glutamine, lysine, valine, ornithine, taurine, and combinations thereof.

[0051] The term “isolated,” as used herein, refers to a material that is removed from at least one component with which it is naturally associated (e.g., removed from its original environment).

[0052] As used herein, the terms “reduce” and “reduction” refer to a measurable lessening of an end-point (e.g., enzymatic activity, production of compound, expression of a protein) by at least about 10%, at least about 50%, at least about 75%, or at least about 90%. In certain embodiments, the reduction can be from about 10% to about 100%.

[0053] As used herein, the term “increase,” “elevate” and “elevation” refers to a measurable augmentation of an end-point (e.g., enzymatic activity, production of compound, expression of a protein) by at least about 10%, at least about 50%, at feast about 75%, or at least about 90%. In certain embodiments, the increase can be from about 10% to about 100%. In certain embodiments, the increase can be at least about 10-fold, about 100-fold, or about 1000-fold or more. In certain embodiments the increase can be about 100-fold or more, about 1000-fold or more, or about 10,000-fold or more.

[0054] As used herein, the term “isomerase” refers to any enzyme of EC class 5 that catalyzes geometric or structural changes within one molecule. According to the type of isomeric change they catalyze, they can be called racemases or epimerases (EC subclass 5.1); cis - hans - isomerases (EC subclass 5.2); intramolecular oxidoreductases (EC subclass 5.3); intramolecular transferases (mutases) (EC subclass 5.4); or intramolecular lyases (EC subclass 5.5); other isomerases are placed hi EC subclass 5.99.

[0055] As used herein, the term “epimerase” refers to a class of enzymes that catalyze the in version of asymmetric groups in a substrate with several centers of asymmetry.

[0056] The term “dehydrogenase,” as used herein, refers to any enzyme that catalyzes the removal of hydrogen atoms (e.g., dehydrogenation) in biological reactions. Dehydrogenases occur in many biochemical pathways and are involved in driving the electron transport chain reactions of cell respiration. In certain embodiments, dehydrogenases work in conjunction with the hydrogen- accepting coenzymes NAD and FAD,

[0057] As used herein, the term “phosphatase” refers to a class of enzymes that catalyze the removal of a phosphate group from an organic compound. In certain embodiments, the phosphatase catalyzes the removal of a phosphate group from a sugar. In certain embodiments, the sugar is a hexose,

[0058] The term “aldolase,” as used herein, refers to enzymes of the sub-subclass EC 4.1.2 (i.e. , the aldehyde-lyases) catalyzing aldol condensations and their reversal.

[0059] Techniques for determining nucleic acid and amino acid sequence identity are known in the art. Typically, suc h techniques inc lude determining the nucleotide sequence of the mRN A for a gene and / or determining the amino acid sequence encoded thereby and comparing these sequences to a second nucleotide or amino acid sequence. Genomic sequences can also be determined and compared in this fashion. In general, identity refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their percent identity. The percent identi ty of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100, Unless indicated otherwise, percent identity is determined for two sequences when compared and aligned for maximum correspondence over a comparison window or designated region as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters. &<.?, e.g,, the NCBI web site at ncbi.nlm.nih.gov / BLAST. For example, BLASTN and BLAST? can be used using the following default parameters: genetic code=standard; filier=noue; strand- both; cutotT=60; expect=10; Matrix“BLOSUM62; Descriptions-50 sequences; sort by-HIGH SCORE; Databases:=non-redundant. GenBank+EMBLTDIJBJ+PDB+GenBank CDS translations +Swrss protein r Spupdate-tPIR. Details of these programs can be found on the GenBank website.

[0060] A “mutation” in a gene can include, for example, nucleotide changes, deletions of one or more nucleotides (which can include the entire coding sequence and / or promoter or other regulatory sequences) and insertions of one or more nucleotides, which can occur in the coding sequence of the gene or its regulatory components (e.g., the gene’s promoter). Mutations can include, for example, mutations that reduce or eliminate function (e.g., nonsense mutations) as well as alterations of the genome that reduce or knockout expression of a gene product.

[0061] The present disclosure provides genetically engineered .microorganisms. In certain embodiments, the presently disclosed, microorganisms can produce an increased amount of D- tagatose, e.g., an increased amount compared to a control microorganism that is naturally- occurring.

[0062] D-Tagatose is a natural but rare monosaccharide. It is a ketohexose that has the same empirical formula as common monosaccharides such as glucose and fructose. It is epimeric w ithfructose at the 4-position. Its enantiomer, L-tagatose, is unknown in nature but has been synthesized. D-tagatose has the foltowing formula:

[0063] D-Tagatose is 92% as sweet as sucrose (e.g., table sugar), but it has only about one-third of the nutritional energy (e.g., calorie) value. As a result. D-tagatose can be used as a replacement for sucrose and artificial sweeteners.

[0064] The present disclosure provides genetically engineered microorganisms that have increased production of tagatose as compared to naturally-occurring microorganisms. Figures I and 5C illustrate the biochemical pathways modulated in exemplary microorganisms of the present disclosure. In these exemplary niicroo.rganisms, glucose is imported and phosphorylated to ghicose-6-phosphate (G6P) by the phosphotransferase system (PTS) or GalP / Glk. G6P is then isomerized to fructose-6-phosphate (F6P) by Glucose-6-phosphate isomerase. Next, F6P is epimerized to tagatose-6-phosphate by the tagatose- 1 ,6-bisphosphate aldolase subunit GatZ and / or ketose biphosphate aldolase subuni t KbaZ which is then dephosphorylated to free tagatose by native phosphatases. Finally, free tagatose can diffuse across the cell membrane into the supernatant. In some embodiments, the presently disclosed microorganisms also include gene editing (e.g., knockout) of certain competing pathways including the pentose phosphate pathway, catalyzed by glucose-6-phosphate dehydrogenase (Zwf); glycolysis, which is catalyzed by phosphofructokinases A and B (PfkA and PfkB); conversion of fructose-6-phosphate to psicose- 6-phosphate, catalyzed by allulose~6~phosphate 3-epimerase (AlsE); and the mannose biosynthesis pathway, catalyzed by mannose-6-phosphat.e isomerase (Man A).22, . fogaftw Production fazyao

[0065] In certain embodiments, the presently disclosed microorganisms include overexpression of at least one gene encoding an enzyme catalyzing reactions for the production of tagatose. In certain embodiments, the presently disclosed microorganisms include a recombinant polynucleotide encoding at least one enzyme catalyzing reactions for the production of tagatose. In certain embodiments, the enzyme is an epimerase, for example an epimerase that converts fructose-6-phosphate (F6P) to tagatose-6-phosphate. Non-limiting examples of epi merase include tagatose- 1 ,6-bisphosphate aldolase, ketose biphosphate aldolase, methy lmalonyl-CoA epimerase, UDPgalactose 4-epimerase, UDPglucose 4-epimerase, UDPg Incur onate 4-epimerase, UDPglucuronate 5 ’-epimerase, ribose~5-phosphate epimerase, GDPmannose 3,5-epimerase, h- ribulosephosphate 4-epimerase, UDP-N-acetylglucosamine 2-epimerase, UDP-N- acetylglucosamine 4-epimerase, UDPgalactose 4-epimerase, UDPglucose 4-epimerase, UDPglucuronate 4-epimerase, UDPglucuronate 5 ’-epimerase, GDPmannose 3,5-epimerase, methylmalonyl-GoA epimerase, ribose- 5 -phosphate epimerase, and UDP-N-acetylglucosamine 2- epimerase.

[0066] In certain embodiments, the epimerase is the tagatose- 1,6-bisphosphate aldolase subunit GatZ (UniProt No. P0C8J8). GatZ catalyzes the reversible epimerization of D~tagatose-6- phosphate to D-fructose-6-phosphate. In certain embodiments, GatZ is an £„ co / z GatZ. In certain embodiments,GatZ. In certain embodiments, GatZ comprises an amino acid sequence that is at least about 80%,at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 58. In certain embodiments, GatZ comprises the amino acid sequence set forth In SEQ ID NO: 58. In certain embodiments, GatZ consists of the amino acid sequence set forth in SEQ ID NO: 58. SEQ ID NO: 58 is provided below:

[0067] In certain embodiments, the gene ga / Z is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the nucleotide sequence set forth in SEQ ID NO: 59. In certain embodiments, gatZ comprises the nucleotide sequence set forth in SEQ ID NO: 59, In certain embodiments, gatZ consists of the nucleotide sequence set forth in SEQ ID NO: 59. SEQ ID NO: 59 is provided below:

[0068] In certain embodiments, the epimerase is the ketose biphosphate aldolase subunit KbaZ (UniProt No. P0C8K0). KbaZ catalyzes the reversible epimerization of D~tagatose-6-phosphate to D-faictose-6-phosphate. In certain embodiments, KbaZ is an E. eoli KbaZ. In certain embodiments, KbaZ comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about98%, at least about 99%, or at least about 100% identical to the amino acid, sequence set forth in SEQ ID NO: 60, In certain embodiments, KbaZ comprises the amino acid sequence set forth in SEQ ID NO: 60. In certain embodiments, KbaZ consists of the amino acid sequence set forth in SEQ ID NO: 60. SEQ ID NO: 60 is provided below:

[0069] In certain embodiments, the gene kbaZ is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the nucleotide sequence set forth in SEQ ID NO: 61, In certain embodiments, kbaZ comprises the nucleotide sequence set forth in SEQ I D NO: 6.1. In certain embodiments, kbaZ consists of the nucleotide sequence set forth in SEQ ID NO: 61, SEQ ID NO: 61 is provided below:

[0070] In certain embodiments, the enzyme is a phosphatase, for example a. phosphatase that dephosphorylates tagatose-6- phosphate to free tagatose. In certain embodiments, the phosphatase catalyzes the removal of a phosphate group from a sugar. In certain embodiments, the sugar is a hexose.

[0071] In certain embodiments, the phosphatase is a Hexitol phosphatase A (HxpA) (UniProt No. P 77625). HxpA catalyzes the dephosphorylation of D-allose-6-phosphate. In certain embodiments, HxpA is an E. colt HxpA. In certain embodiments, HxpA comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 62. In certain embodiments, HxpA comprises the amino acid sequence set forth in SEQ ID NO: 62. In certain embodiments, HxpA consists of the amino acid sequence set forth in SEQ ID NO: 62. SEQ ID NO: 62 is provided below:

[0072] In certain .embodiments, the gene hxpA is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the nucleotide sequence set forth in SEQ ID NO: 63. In certain embodiments, hxpA comprises the nucleotide sequence set forth in SEQ ID NO: 63. In certain embodiments, hxpA consists of the nucleotide sequence set forth in SEQ ID NO: 63. SEQ ID NO: 63 is provided below:

[0073] In certain embodiments, the phosphatase is a Hexitol phosphatase B (HxpB) (UniProt No. P77247 or UniProt No. Q7ADF8). HxpB catalyzes the dephosphorylation of D-pslcose-6- phosphate. In certain embodiments, HxpB iscoU HxpB. In certain embodiments, HxpB comprises an amino acid sequence that is at least about 80%. at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, HxpB comprises the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, HxpB consists of the amino acid sequence set forth in SEQ ID NO: 3. SEQ ID NO: 3 is provided below:

[0074] In certain embodiments.. HxpB comprises the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments. HxpB consists of the amino acid sequence set forth in SEQ ID NO: 4. SEQ ID NO: 4 is provided below:

[0075] In certain embodiments, the gene hxpB is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the nucleotide sequence set. forth in SEQ ID NO: 5. In certain embodiments, AxpB comprises the nucleotide sequence set forth in SEQ ID NO: 5. In certain embodiments, hxpB consists of the nucleotide sequence set forth in SEQ ID NO: 5. SEQ ID NO: 5 is provided below:

[0076] In certain embodiments, one or more transporter that transports glucose into the cell can be expressed (i.e., overexpressed) in the cell, thereby increasing glucose in the ceil. In some embodiments, one or both of galactose:H+symporter (GalP) and glucokinase (Glk) are expressed in the microorganism. For example, in some embodiments, GalP transports glucose into the cell where it is phosphorylated to glucose-6-phosphate by Glk and assimilated into central carbon metabolism.

[0077] In certain embodiments, the galactose:H+symporter (GalP) is as described in EG 12148 (EcoCyc) or P0AEP1 (UniProt). In certain embodiments, GalP is an E. coli GalP. In certain embodiments, GalP comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, GalP comprises the amino acid sequence set forth inSEQ ID NO: 38. In certain embodiments, GalP consists of the amino acid sequence set forth inSEQ ID NO: 38. SEQ ID NO: 38 is provided below:

[0078] In certain embodiments, the gene gd / P is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the nucleotide sequence set forth in SEQ ID NO: 39. In certain embodiments, galP comprises the nucleotide sequence set forth in SEQ ID NO: 39. In certain embodiments, galP consists of the nucleotide sequence set forth in SEQ ID NO: 39. SEQ ID NO: 39 is provided below:

[0079] In certain embodimen ts, the glucokinase (Glk) is as described in EG 12957 (EcoCyc) or P0A6V8 (iJniProt). In certain embodiments, Glk is an E. coli Glk. In certain embodiments, Glk comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 40. Incertain embodiments, Glk comprises the amino acid sequence set forth in SEQ ID NO: 40. In certain embodiments, Glk consists of the amino acid sequence set forth in SEQ ID NO: 40. SEQ ID NO: 40 is provided below;GVFIAGGIVPRFLEFFKASGFRAF.FEDKGREKEYVHDI PVYI;i VHDIMFGLLGSGAH LRQT LGHIL (SEQ ID RO : 40 }

[0080] In certain embodiments, the gene g / A* is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the nucleotide sequence set forth in SEQ ID NO: 41. In certain embodiments, glk comprises the nucleotide sequence set forth in SEQ ID NO; 41, In certain embodiments, glk consists of the nucleotide sequence set forth in SEQ ID NO: 41. SEQ ID NO: 41. is provided below:GTGCCGCATTTGAAGATAAAGGGCGCTTTAAAGAATATGTCCATGATATTCCGGTGTATCTCAT CGTGQATGAGAATG CGGGC GTTCTGGGTTC CGGTGGAC ATTT AGGC CAGACGTT AGGTCACATT CTGtaa ( SEQ ID NO : 41 )

[0081] Without being bound by any theory, the inventors of the present disclosure believe that any enzyme performing similar function, to the enzyme described above can be used in the presently disclosed microorganisms. For example, but without any limitation, the presently disclosed microorganism can include any enzyme that catalyzes the reversible epimerization of D-fructose-6-phosphate to D-tagatose-6-phosphate. In another non-limiting example, the presently disclosed microorganism can include any enzyme that dephosphorylates D~tagatose-6- phosphate to free tagatose.2.2. Competes / Q / feq rv

[0082] In certain embodiments, the presently disclosed microorganisms include a mutation of one or more gene encoding one or more enzymes regulating biochemical pathways that can reduce theproduction of tagatose. In certain embodiments, the presently disclosed microorganisms include a reduced expression of a gene encoding enzymes regulating biochemical pathways that can reduce the production of tagatose. Physiologically, cells catalyze sugars through the pentose phosphate pathway and glycolysis to produce energy (e.g,, ATP), The inventors of the present disclosure discovered that deletion or reduced expression of gene encoding enzymes of certain metabolic pathways results in increased tagatose production,

[0083] In certain embodiments, the presently disclosed microorganisms include a mutation of a gene encoding an enzyme that converts fructose- 6-phosphate (F6P) to psicose-6-phosphate. In certain embodiments, the presently disclosed microorganisms include a reduced expression of a gene encoding an enzyme that converts fructose-6-phosphate (F6P) to psicose-6-pliosphate. In certain embodiments, the enzyme that converts fructose -6-phosphate (F6P) to psicose-6- phospliate is D-allulose-6-phosphate 3-epimerase (AlsE) (UniProt No, P32719), In certain embodiments, AlsE is an A, co / i AlsE. A representative nucleotide sequence of the gene ai$£ is set forth in SEQ ID NO: 2 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 2, SEQ ID NO: 2 is provided below:

[0084] In certain embodiments, £ co / i AlsE comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, al least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments. E. coli AlsE comprises the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO: 1 is provided below:

[0085] In certain embodiments, the presently disclosed microorganisms include a mutation of a gene encoding an enzyme of the pentose phosphate pathway. In certain embodiments, the presently disclosed microorganisms include a reduced expression of a gene encoding an enzymeof the pentose phosphate pathway. In certain embodiments, the enzyme of the pentose phosphate pathway is selected from the group consisting of glucose-6-phosphate dehydrogenase, 6- phosphogluconolactonase, phosphogluconate dehydrogenase, phosphopentose isomerase, phosphopentose epimerase, transketolase, and tiansaldolase. In certain embodiments, the enzyme of the pentose phosphate path way is glucose-6-phosphate dehydrogenase (Zwf) (Entrez Gene ID: 946370; EcoCyc ID: EG 11221; IJniProt ID: POAC53), Zwf catalyzes the oxidation of glucose-6» phosphate to 6-phosphogluconolactone. In certain embodiments, Zwf is an E. coli Zwf A representative nucleotide sequence of the geneis sei forth in SEQ ID NO: 6 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 6. SEQ ID NO: 6 is provided below;

[0086] hi certain embodiments, E. coli Zwf comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 20. In certain embodiments, E. coli Zwf comprises the amino acid sequence set forth in. SEQ ID NO: 20. SEQ ID NO: 20 is provided below;

[0087] In certain embodiments, Zwf is a Bacillus subfilis Zwf A representative amino acid sequence of Bacillus subtllis Zwf is found as P54547 (Uniprot) / BSU23850 (KEGG) or as set forth in SEQ ID NO: 10 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 10. SEQ ID NO: 10 is provided below:

[0089] In certain embodiments, Zwf is a Laciacoccus lacfis Zwf. A representative amino acid sequence of laclacoccus / act A Zwf is found as GenBank HE92651.1 : glucose-6-phosphatedehydrogenase. EC 1.1.1 .49, or as set forth in SEQ ID NO: 11 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 11. SEQ ID NO: 11 is provided below:

[0090] A representati ve nucleotide sequence of a Lactococriis tocrts CM / gene is set forth in SEQ ID NO: 33 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 33. SEQ ID NO: 33 is provided below:

[0091] In certain embodiments, the presently disclosed microorganisms include a mutation of a gene encoding an enzyme of the glycogen biosynthesis. In certain embodiments, the presently disclosed microorganisms include a reduced expression of a gene encoding an enzyme of the glycogen biosynthesis. In certain embodiments; the enzyme of the glycogen biosynthesis is selected from the group consisting of phosphoglueomutase (Pgm), UDP-glucose pyrophosphorylase, glycogen synthase, glycogen branching enzyme, and glycogenin. In certain embodiments, the enzyme of the glycogen biosynthesis is phosphoglueomutase (Pgm) (EntrezGene ID: 946370; EcoCyc ID: EG12I44; Uuiprot ID: P36938). Pgm (EC 5.4.2.21 is an enzyme that transfers a phosphate group on an a-D-glucose monomer from the I to the 6 position in the forward direction or the 6 to the 1 position in the reverse direction. In certain embodiments, Pgm is an Zt. coll Pgm. A representative nucleotide sequence of genepgw is set forth in SEQ ID NO: 47 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 47. SEQ ID NO: 47 is provided below:

[0092] In certain embodiments, E. coll Pgm comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 48, hi certain embodiments, E. coll Pgm comprises the amino acid sequence set forth in SEQ ID NO: 48. SEQ ID NO: 48 is provided below:

[0093] In certain embodiments, Pgm is a Bacillus subtilis Pgm. A representative nucleotide sequence of gene pgm is set forth in SEQ ID NO: 49 or at least 90%, 95%, 95%. or 99% identical to SEQ ID NO; 49, SEQ ID NO: 49 is provided below:

[0094] In certain embodiments, Bacillus xiAfiOs Pgm comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 50, In certain embodiments. Bacillus xtibtilfc Pgm comprises the amino acid sequence set forth in SEQ ID NO; 50, SEQ ID NO; 50 is provided below:

[0095] In certain embodiments, Pgm is a Laciococctts lactis' Pgm. A representative nucleotide sequence of genepgw is set forth in SEQ ID NO: 51 or at least 90%, 95%, 95%. or 99% identical to SEQ ID NO: 51 . SEQ ID NO: 51 is provided below:

[0096] In certain embodiments, Lacfc / cocciis lactis Pgm comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 52. In certain embodiments, Lacfocacct / s lactis Pgm comprises the amino acid sequence set forth in SEQ ID NO; 52,. SEQ ID NO: 52 is provided below:

[0097] In certain embodiments, the presently disclosed microorganisms include a mutation of a gene encoding an enzyme of glycolysis, hi certain embodiments, the presently disclosed microorganisms include a reduced expression of a gene encoding an enzyme of the glycolysis. In certain embodiments, the enzyme of the glycolysis is selected from the group consisting of phosphofructokinase A, phosphofructokinase B, fructose-biphosphate aldolase, triosephosphate isomerase, glyceraldehyde-3 -phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, and pyruvate kinase. In certain embodiments, the enzyme of glycolysis is phosphofructokinase B (PfkB). In certain embodiments, the enzyme of glycolysis is pyruvate kinase. In certain embodiments, the enzyme of glycolysis is phosphofructokinase A (PfkA) (Entrez Gene ID: 948412; EcoCyc ID: EG10699; UniProt ID: P0A796). PfkA catalyzes the phosphorylation of D-fructose-6-phosphate to fructose 1 ,6-bisphosphate by ATP, the first committing step of glycolysis. In certain embodiments, PfkA is an E. coli PfkA. A representative nucleotide sequence of geneis set forth in SEQ ID NO: 7. SEQ ID NO: 7 is provided below:

[0098] In certain embodiments, E. coli PfkA comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%. or at least about 100% identical to the amino acid sequence sei forth in SEQ ID NO: 21 . In certain embodiments, E. coli PfkA comprises the amino acid sequence set forth in SEQ ID NO: 21. SEQ ID NO: 21 is provided below:

[0099] In certain embodiments, PfkA is a Bacillus subtifis PfkA. A representative amino acid sequence of Bacillus wbtilis PfkA is found as 034529 (Uniprot) I BSU29190 (KEGG) or as set forth in SEQ ID NO: 12 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 12. SEQ ID NO: 12 is provided below:

[0100] A representative nucleotide sequence of a Bacillus subtilis geneis set forth in SEQID NO: 27 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 27. SEQ ID NO: 27 is provided below: atgaaacgaataggggtattaacgagcggcggggattccocgggaatgaacgcagoagttcgcg cagtagtcagaaaagcga betateatgaegttgaagt ttacggtatttacaacggatacgcggg attgatcagcggaaagattgaaaagcttgaactcggatcagtaggcgatattatacatcgtgga gggactaagctttatacggcgagatgtcctgaattcaaaacagttgaaggccgtgaaaaagggagggtgcgaaaaaattaacggaacacgggtttccatgtgtaggtgtaccgggtacaattgataat gacattccgggcactgattttacaatcggtttcgatacagctttaaatacagtaattgaogcaa ttgataagattcgcgatacagcgacttctcatgaacgtacatatgtaatcgaagtaatgggccg tcatgccggcgatatcgcattgtgggccggtcttgcagggggcgcagaatcgatcttaatccct gaggcagactatgacatgcacgaaatcattgcccgcttaaaacgcggccacgaacgcggcaaga agcacagtattattattgttgccgaaggtgtaggcagcggtgttgaattcgggaaacgcattga agaagaaacaaatcttgaaactagggtatctgtattgggccatatccagcgcggaggttctccg agtgctgctgaccgtgtgttggcaagccgtctcggcgcatatgcagttgaactgctgcttgaag gaaaaggcggacgctgtgtaggtatacaaaacaataagcttgtagaccatgatattatagaaat acttgagacaaaacacacagttgagcaaaacatgtatcagctttcaaaagaactgtctatctaa ( SEO ID NO : 27 )

[0101] J Ih certain embodiments, PfkA is a / .uctococcirt Zattfzs PfkA. A representative amino acid sequence of faciocvccw tact A PfkA is found as GenBank: WP JJ03131080.1 : ATP- dependent 6- phosphoftuctokinase, EC2.7..1.11 , or as set forth in SEQ ID NO: 13 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 13, which is provided below:

[0102] A representative nucleotide sequence of a Zzrc / ococcn.s- kictis gene p / kd is set forth in SEQ ID NO: 32 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 32. SEQ ID NO: 32 is provided below:

[0103] In certain embodiments, PfkB is an Zi. coli PfkB. A representative nucleotide sequence of gene pfiB is set forth in SEQ ID NO: 22. SEQ ID NO: 22 is provided below:

[0104] In certain embodiments, Zf. coZz PfkB comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 23. In certain embodiments, E. coil PfkB comprises the amino acid sequence set forth in SEQ ID NO: 23. SEQ ID NO: 23 is provided below:

[0105] In certain embodiments, the presently disclosed microorganisms do not include a deletion or a reduced expression of hexokinase. In certain embodiments, the presently disclosed microorganisms do not include a deletion, a disruption, or a reduced expression of glucokinase. In certain embodiments, the presently disclosed microorganisms do not include a deletion or a reduced expression of glucose-6-phosphate isomerase.

[0106] In certain embodiments, the presently disclosed microorganisms include a mutation of a gene encoding an enzyme of the allose degradation pathway. In certain embodiments, the presently disclosed microorganisms include a reduced expression of a gene encoding an enzyme of the allose degradation pathway. In certain embodiments, the enzyme of the allose degradation pathway is allose-6-phosphate isomerase (RpiB) (Entrez Gene ID: 948602; EcoCyc ID: EG 11827; UniProt ID; P37351). RpiB catalyzes the interconversion of ribufose~5~P and ribose~5~P, as well the interconversion of D-alIose-6-phosphate (A1I6P) and D-allulose-6-phosphate. In certain embodiments, RpiB is an E. coli RpiB. A representative nucleotide sequence of gene rpiB is set forth in SEQ ID NO: 8 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 8. SEQ ID NO: 8 is provided below:TCCACATTACGCCAGTCAAGTCGCACTGGCTGTTGCTGGCGGAGAGGTTGATGGCGGGATTTTC

[0107] In certain embodiments, E. colt RpiB comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 24, In certain embodiments, E. &Ei RpiB comprises the amino acid sequence set forth in SEQ ID NO: 24. SEQ ID NO: 24 is provided below:MKK I A FGC D H VGF I LKEE I V RE LVERGVE vt DEGE WS SERE E Y P H ¥ A S QVALA VAG GEVDGG I A

[0109] A representative nucleotide sequence of jfac / tfasgene rp / 8 is set forth in SEQ IDNO: 28 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 28. SEQ ID NO: 28 is provided below: a t qa a aq t ag c ca 1 t gc a t eg g a t c a bag a qg cgttcacattcgaaatgaaatcaaagagttaa t g g a a g a a t t q c a aa 11 g a a t a ta 11 qa t a t g g g c t g t g a c t g c g g c a g c g g c t c t g t c g a 11 a tccggattatgcttttccggtgqccgaaaaagtggttagcggcgaagttgacagaggcatt tta atttgcgggacaggcatcggcatgagcatttccgctaataaagtaaaagggattcgc tgcgcgc tggcgcacgataccttcagcgcgaaggcgacgagggagcataatgacacaaacatcc ttgcgat gggtgaacgggtgatcggacctggtttggctcgggaaatcgcaaaaatctggctgactactgag tttaccgggggaagacaccaaacgcgtattggaaaaatctccgattatgaagagaaaaacctgt ag ( SEQ I D NO : 28 )

[0110] In certain embodiments, RpiB is a Lactocvccus lactin RpiB. A representative amino acid sequence of gene rpiB is ribose~5~pbosphaie isomerase, EC5.3.1 .6 or is set forth in SEQ ID NO: 15 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 15, which is provided below.

[0111] A representative nucleotide sequence of a Eactocvccttx factis gene fpi8 is set forth in SEQ ID NO: 34 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 34, SEQ ID NO: 34 is provided below:

[0112] In certain embodiments, the presently disclosed microorganisms include mutation of a gene encoding an enzyme of the mannose biosynthesis pathway. In certain embodiments, the presently disclosed microorganisms include a reduced expression of a gene encoding an enzyme of the mannose biosynthesis pathway. In certain embodiments, the enzyme of the mannose biosynthesis pathway is mannose~6~phosphate isomerase (Man A) (Entrez Gene ID: 944840; EcoCyc ID; EG 10566; UniProt ID: P00946). ManA is involved in the synthesis of the GDP- mannose and dolichol-phosphate-mannose required for a number of critical mannosyi transfer reactions. ManA also catalyzes the interconversion of fructose-6-phosphate and mannose-6- phosphate, In certain embodiments, ManA is an E. coli ManA. A representative nucleotide sequence of gene mcmA is set forth In SEQ ID NO: 9 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 9; which is provided below.

[0113] In certain embodiments, K ctBi ManA comprises an ammo acid sequence that is at feast about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about .100% identical to the amino acid sequence set forth in SEQ ID NO: 25. In certain embodiments, £comprises the amino acid sequence set forth in SEQ ID NO: 25. SEQ ID NO: 25 is provided below:

[0114] In certain embodiments, ManA is a Baci / hj.'i .w / rtife ManA. A. representative amino acid sequence of ManA is found at 031646 (Uniprot) / BSD 12020 (KEGG) or is set forth in SEQ ID NO: 16 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 16.

[0115] A representative nucleotide sequence of a BaeBfusgene manA is set forth in SEQ ID NO: 29 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 29. a t ga cga c t ga a c eg t t a 1 t 111 a aa gc at ga 11 t c a a a g a a a g a a 111 g gg go gg ga cc g a 1 t tagctgattttggctataccattccgtcacaacgaacaggggagtgctgggcttttgccgcgca tcaaaatggtcaaagcgttgttcaaaacggaatgtataaggggttcacgctcagcgaattatgg gaacatcacagacatttattcggacagcttgaaggggaccgtttccctctgcttacaaaaatat tagatgctgaccaggacttatctgttcaggtgcatccgaatgatgaatatgccaacatacatga aaacggtgagcttggaaaaacagaatgctggtacattattgattgccaaaaagatgccgagatt atttatggccacaatgcaacaacaaaggaagaactaactaccatgatagagcgtggagaatggg atgagctcttgcgccgtgtaaaggtaaagccgggggattttttctatgtgccaagcggtactgt tcatgcgattggaaaaggaattcttgctttggagacgcagcagaactcagacacaacctacaga ttatatgattatgaccgaaaagatgcagaaggcaagctgcgcgagcttcatctgaaaaagagca ttgaagtgatagaggtcccg tct at tccagaacggca tacagt tea ccatgaacaaattgaggatttgcttacaacgacattga ttgaatgcgcttacttttcgg tggggaaatggaacttatcaggatcagcaagc ttaaagcagca aaaaccattccttcttatcagtgtgattgaaggggagggccgtatgatctctggtgagtatgtc[OU 6] In certain embodiments,optionally in combination with deletion ofone or both of the following are also deleted.

[0117] (,i) yvj' / of Bacillus subtilis. A representative amino acid sequence of Yvyl is found at P3984 l(lJniprot) Z BSU35790 (KEGG) or is set forth in SEQ ID NO: 17 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 17. SEQ ID NO: 17 is provided below;

[0118] A representative nucleotide sequence of BaciBtrt swZfofe gene yen / is set forth in SEQ ID NO: 30 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 30. SEQ ID NO: 30 is provided below:

[0119] (ii) gffi-wF of fiaci / lus' SHMHS. A representative amino acid sequence of GrnuF is found at 00551 1 (Uniprot) / BSU05870 (KEGG) or is set forth, in SEQ ID NO: 18 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 18, SEQ ID NO: IS is provided below:

[0120] A representative nucleotide sequence of a BaOZZus suZfoZb gene gmuFis set forth in SEQ ID NO: 31 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 31. SEQ ID NO: 31 is provided below: atgacgcatccattatttttagagcctgtctttaaagaaagactatggggagggacgaagcttc gtgacgcttttggctacgcaataccctcacaaaaaacaggtgagtgctgggccgtttctgcaca tgcccatggctcgtcgtctgtaaaaaatggcccgctggcaggaaagacacttgatcaagtatggaaagatcatccagagatattcgggtttccggatggtaaggtgtttccgctgctggtaaagctgc tggacgccaatatggatctctccgtgcaagtocatoctgatgatgattatgoaaaactgoacga aaatggcgaccttggtaaaacggagtgctggtatatcattgattgcaaagatgacgccgaacta attttgggacatcatgcaagcacaaaggaagagttcaaacaacgaatagaaagcggtgattgga acgggctgctgaggcgaatcaaaatcaagccaggagatttcttttatgtgccaagcggtacact ccatgctttatgtaagggaaccottgtccttgaaatcoagcaaaaotctgataoaacatatogc gtatacgattatgaocgctgtaatgaccagggccaaaaaagaactcttcatatagaaaaagcca tggaagtcataacgatacogcatatcgataaagtgcatacaccggaagtaaaagaagttggtaa cgctgagatcattgtttatgtgcaatcagattatttctcagtgtaoaaatggaagattagcggc cgagctgcttttccttcatatcaaacctatttgctggggagtgttctgagcggatcaggacgaa tcataaataatggtattcagtatgaatgcaatgcaggctcacactttattctgcctgcgcattt tggagaatt tacaatagaaggaacat.gtgaa” tcatgatatctcatcctt.aa (SEQ IE NO <31

[0121] J In certain embodiments, ManA is a lacfocoecuv / ae / 'is ManA. A representative ammo acid sequence of LactococcMs Zacfe ManA is mannose-6-phosphate isomerase, EC5.3.1 .8 or is set forth in SEQ ID NO: 19 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 19. SEQ ID NO: 19 is provided below:

[0122] A representative nucleotide sequence of a Laciococaus tact A gene rnrnta is set forth in SEQ ID NO: 35 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 35. SEQ ID NO: 35 is provided below:

[0123] In certain embodiments, the presently disclosed microorganisms include a mutation of a gene encoding an enzyme of the carbohydrate metabolism. In certain embodiments, the presentlydisclosed microorganisms include a reduced expression of a gene encoding an enzyme of the carbohydrate metabol ism .

[0124] In certain embodiments, the enzyme of the carbohydrate metabolism is D-tagatose- 1 ,6- bisphosphate aldolase subunit (GatY) (UniProt ID: P0C8J6). GatY catalyzes the reversible aldol condensation of dihydroxyacetone phosphate (DHAP or glycerone-phosphate) with glyceraldehyde 3-phosphate (G3P) to produce tagatose 1 ,6-bisphosphate (TBP). In certain embodiments, GatY is an E, coli GatY. A representative nucleotide sequence of gene gtf / F is set forth in SEQ ID NO: 81 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 81. In certain embodiments, £ coli GatY comprises an amino acid sequence that, is at least about 80%, at least about 85'%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 82. In certain embodiments, E, coli GatY comprises the amino acid sequence set forth in SEQ ID NO: 82. SEQ ID NO: 81 and SEQ ID NO: 82 are provided below:

[0125] In certain embodiments, the enzyme of the carbohydrate metabolism is D-tagatose-1.,6- bisphosphate aldolase subunit (KbaY) (UniProtlD: P0AB74). KbaY catalyzes the reversible aldol condensation of dihydroayacetone phosphate (DHAP or glycerone-phosphate) with glyceraldehyde 3-phosphate (G3P) to produce tagatose 1,6-bisphosphate (I BP). In certain embodiments, KbaY is an E. coli KbaY. A representative nucleotide sequence of gene £ / wF is set forth in SEQ ID NO: 83 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 83. In certain embodiments. £ call KbaY comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 84. In certain embodiments, £. eoii KbaY comprises the amino acid sequence set forth in SEQ ID NO: 84, SEQ ID NO: 83 and SEQ ID NO: 84 are provided below

[0126] In certain embodiments, die deletion of a gene comprises a non-frameshift deletion, a frameshift deletion, or a combination thereof. In certain embodiments, the deletion of a gene can be achieved by an insertion (e.g., a non-ftameshift insertion, a frameshift insertion, or a combination thereof). In certain embodiments, the deletion of a gene comprises a nonsense mutation.2.3. Cei / s

[0127] The present disclosure provides recombinant microorganisms. Any culturable microorganism is suitable for use in the compositions and methods described herein. In certain embodiments, the microorganism is a bacterium. In certain embodiments, the microorganism is selected from the group consisting of BeGebacier ami. Achromobacter, rtddip / nfem Acfeefebaefer. Actinomadura, Actinopktnes, Aerapyrtrnipemix, Aprabaciertiiin. / hcahgenes, Aminas coniasns (M), Artfirobacter, Baci / teafcafop / tite. ifacdhix amytoJifnc^jciens, BodZ / ns brevis, Baeiifes circiiiars, BacH / MS ciausii, Bacriius ferities, BaciJfes irchcriifirniis, Bacillus macerans, Bacdhis sti&irothemtophikiS; Bacillus subtil®, Bifidobacterium, Brevibacillus brevis, Burkhokferia cepacia, Candida cylitidracea, Carica papaya (L), (felfefesimicrobiwn. Cephufosporium, Chaetomium erraticum, Chaetomium gracile. Clatfridium, (latiridium bfeyCcimi, Clostridium acetobufylicum, Cfesiridium fherffioee / hnn, Corynebacterinm fgbdamicum), Corynebacteriutn efficieas, Escherichia coll, Eiileracocciis, Erwina chrysanth&ni, Gliconobaeier. Gluconaeetobacter, Hafearcula, Humicolainsolcms, .Kitasafospvra setae. Klebsiella, Klebsiella axytoca, A7 / c«mr, Lactlacfis, Lactobacillus. Lactobacillus fermenfum, Lactobacillus .sate, Laciocaccuri factococeus lactis. Leuconostoc, Melhyhriystis, Melhanolobus siciliae. Methanogenium arganophilum. Methanobacterium bryantii. Micrishacterium imperials, Micrococcus lysodeikticiri, Microhmalas, MucorjaWHicus, Mycobacterium, Myrothecmm, Nitrobacter, Mllrosomonas, Mocanfia, Z7j / ?aro cartea, / WkKwcw. Pediococcus halophihts, Paracoccus pantotrophus, Prvpiombacteri^ Pseudomonas,horikoshii, Rhizobium, Rhicomucor miehei, Rhteomucor pusillus Lindt, Rhisopus, Rhizopus delentar. Rhisopusjaponicas, Rhisopus niveus. Rhkopus oryzae, Rhizopus oligospoms. Rhodoeocats, Scfawma libertina, Sphingobacterium nndtivortmi, Sphingpbium, Sphingomonas, Streptococcus. Streptococcus thermophilus Y~L Streptomyces, Streptomyces griseus, Streptomyces livtdans, Streptomyces murinus, Streptomyces ruhiginosus. Streptomyces vio / aceomber, Streptovertfcillium mobaraense, TePagenococcus. Ehemus. Ihiosphuem pantotropha, Tfametes, Fibrin algbmlyiictu', Xamhomottas, Zymomanas, and Zymtmwwus mobilis. In certain embodiments, the microorganism is Escherichia coll (E. coli}. In certain embodiments, the microorganism is bacillus sublilis. In certain embodiments, the microorganism is Lactecocctis lactis.

[0128] In certain embodiments, the E. coli is selected from the group consisting of Enterotoxigenic E. call (ETEC), Enteropathogenie E. coli (EPEC). Enteroinvasive £'. coli (EIEC), Entefohemorrhagic E. coli (EHEC), Uropathogeaic Zi. coli (UPEC), Verotoxin-producing E. coli, E. co / / O157:H7, A, m / z O104:H4, E. coZ / Oi2E £Uo / i O104:H2L £ coll KI, and£ coli. NC10L In certain embodiments, the Zi. coli is E. coli K12. In certain embodiments, the E. coli is E. coli B. In certain embodiments, the A. call is E. coli C.

[0129] In certain embodiments, the Zi. coli is derived from a strain selected from the group consisting of NC'I'C 12757, NCTC 12779, NCTC 12790, NCTC 12796, NCTC 12811, ATCC 11229, ATCC 25922, ATCC 8739, DSM 30083, BC 5849, BC 8265, BC 8267, BC 8268, BC 8270, BC 8271, BC 8272, BC 8273, BC 8276, BC 8277, BC 8278, BC 8279, BC 8312, BC 8317, BC 8319, BC 8320, BC 8321, BC 8322, BC 8326, BC 8327, BC 8331, BC 8335, BC 8338, BC 8341, BC 8344, BC 8345, BC 8346, BC 8347, BC 8348, BC 8863, and BC 8864,

[0130] in certain embodiments, the E, coli is derived from a strain selected from the group consisting of BC 4734 (026:11.1 1), BC 4735 (O157;H-J, BC 4736 , BC 4737 (n.d.), BC 4738 (O157:H7), BC 4945 (O26:H~), BC 4946 (O157:H7), BC 4947 (O111H-), BC 4948 (O157:H)3BC 4949 (05), BC 5579 (01.57:117), BC 5580 (0157:117), BC 5582 (03:11), BC 5643 (02:115), BC 5644 (0128), BC 5645 (O55:H-), BC 5646 (069:H-), BC 5647 (Olfrl:H9), BC 5648 (O103:H2), BC 5850 (O22:H8), BC 5851 (O55:H-), BC 5852 (O48:H21), BC 5853 (O26:H11)SBC 5854 (Ol57:H7), BC 5855 (O157:H-), BC 5856 (O26:H-), BC 5857 (0103:H2), BC 5858(O26:H11), BC 7832, BC 7833 (() raw form:H-), BC 7834 (ONT:H~), BC 7835 (0103:02), BC 7836 (O57:H-), BC 7837 (ONT:H-), BC 7838, BC 7839 (O128:H2), BC 7840 (O157:H~), BC 7841 (023:11-), BC 7842 (O157:H-), BC 7843, BC 7844 (0157:0-), BC 7845 (0103:02), BC 7846 (026:011), BC 7847 (O145:H-), BC 7848 (0157:0-), BC 7849 (0156:047), BC 7850, BC 7851 (0157:0-), BC 7852 (0157:0-), BC 7853 (05 :H-), BC 7854 (O157:H7), BC 7855 (O157:H7), BC 7856 (O26:H-), BC 7857, BC 7858, BC 7859 (0NT:H-), BC 7860 (0129:0-), BC 7861 , BC 7862 (0103:02), BC 7863, BC 7864 (O raw fbrm:O-), BC 7865, BC 7866 (026:0- ), BC 7867 (O raw fornrH-), BC 7868, BC 7869 (ONT.H-), BC 7870 (OH3:H-), BC 7871 (0NT:H-), BC 7872 (ONT:H-), BC 7873, BC 7874 (O raw tat'H-), BC 7875 (O157:H-), BC 7876 (0.11 1.1H-), BC 7877 (0146:021), BC 7878 (O145:H-), BC 7879 (022:08), BC 7880 (O raw farm:H-), BC 7881 (0145:0-), BC 8275 (O157:H7), BC 8318 (O55:K-:H-), BC 8325 (0157:07), BC 8332 (ONT), and BC 8333.

[0131] In certain embodiments, the £. co / z is derived from a strain selected from the group consisting of BC 8246 (0152:K-:H-), BC 8247 (OI24:K(72):O3), BC 8248 (0124), BC 8249 (0112), BC 8250 (O136:K(78):H-), BC 8251 (0124:0-), BC 8252 (O144:K-:H-), BC 8253 (O143:K:H-), BC 8254 (0143), BC 8255 (011.2), BC 8256 (028a. e), BC 8257 (0124:0-), BC 8258 (0143), BC 8259 (O167:K-:H5), BC 8260 (0128a. c.:O35), BC 8261 (0164), BC 8262 (O164-.K-. H-), BC 8263 (0164), and BC 8264 (0124),

[0132] In certain embodiments, the Zr. <%» / / is derived from a strain selected from the group consisting of BC 5581 (078:011), BC 5583 (O2:K1), BC 8221 <0118), BC 8222 (O148:H-), BC 8223 (0.1 1.1), BC 8224 (0110:11-), BC 8225 (0148), BC 8226 (01 18), BC 8227 (025:1142), BC 8229 (06), BC 8231 (0153:045), BC 8232 (09), BC 8233 (0148), BC 8234 (0128), BC 8235 (011.8), BC 8237 (011 1 ), BC 8238 (01 10:017), BC 8240 (0148), BC 8241 (O6H16), BC 8243 (0153), BC 8244 (015:0-), BC 8245 (020), BC 8269 (O125a.c:H-), BC 8313 (06:H6), BC 8315 (O153:H-), BC 8329, BC 8334 (01 18:012), and BC 8339.

[0133] In certain embodiments, thecoU is derived from a strain selected from the group consisting of BC 7567 (086), BC 7568 (0128), BC 7571 (01 14), BC 7572 (01 19), BC 7573 (0125), BC 7574 (0124), BC 7576 (0127a), BC 7577 (0126), BC 7578 (0.142), BC 7579 (026), BC 7580 (0K26), BC 7581 (0142), BC 7582 (055), BC 7583 (0158), BC 7584 (0-), BC 7585 (O-kBC 7586 (O-), BC 8330, BC 8550 (026), BC 8551 (055), BC 8552 (0158), BC 8553 (026), BC 8554 (0158), BC 8555 (086), BC 8556 (0128), BC 8557 (OK26), BC 8558 (055), BC 8560 (0158), BC 8561 (0158), BC 8562 (0114), BC 8563 (086), BC 8564 (01281 BC 8565 (0158), BC 8566 (0158), BC 8567 (0158), BC 8568 (011 1), BC 8569 (0128), BC 8570 (0114), BC 8571 (0128), BC 8572 (0128), BC 8573 (0158), BC 8574 (0158), BC 8575 (0158), BC 8576 (0158), BC 8577 (0158), BC 8578 (0158), BC 8581 (0158), BC 8583 (0128), BC 8584 (0158),BC 8585 (0128), BC 8586 (0158), BC 8588 (026), BC 8589 (086), BC 8590 (0127), BC 8591 (0128), BC 8592 (0114), BC 8593 (O114), BC 8594 (0114), BC 8595 (0125), BC 8596 (0158), BC 8597 (026), BC 8598 (026), BC 8599 (01.58), BC 8605 (0158), BC 8606 (0158), BC 8607 (0158), BC 8608 (0128), BC 8609 (055), BC 8610 (01 14), BC 8615 (0158), BC 8616 (0128), BC 8617 (026), BC 8618 (086), BC 8619, BC 8620, BC 8621 , BC 8622, BC 8623, BC 8624 (0158), and BC 8625 (0158).

[0134] In certain embodiments, the B. subd / is is derived from Strain 168,

[0135] In certain embodiments, the I.. lactis is derived from Strain A12.

[0136] In certain embodiments, the microorganism is a fungal cell. In certain embodiments, the fungal cell is selected from the group consisting of Aspergillus, Aspergillus uiduhus, Aspeugillus itiger, Aspurgillus oryze, Aspergillus melleus, Aspergillus pulvendenius, Aspergillus saitoi,mgosa, Issatchenkia oriental®, Kluyveromyces, Khryveromycesfragilis. Khtyveromyces laciis. Kluyveromyces marxicmos, Penicil&un, Penieiilium camemberti, Peuicillium ciJriuum, Perucillium emersonii, Peuicillium roqueforti, Peuicil / um lilaedmmt, Penidllum multicolor, Rhodosporidiumlipolytica, and Zygasaccbaramyces rowed

[0137] In certain embodiments, the microorganism is a yeast cell. In certain embodiments, the yeast cell is Saceharomyecs cerevisiae.2.4. .Exemplary uncruorgauisms

[0138] In certain embodiments, the present disclosure provides a recombinant microorganism comprising an increased production of D-tagatose as compared to a naturally occurring microorganism. In certain embodiments, the recombinant microorganism comprises an exogenous epimerase. In certain embodiments, the exogenous epimerase is a tagatose-1,6- bisphosphate aldolase subunit GatZ and / or ketose biphosphate aldolase subunit KbaZ. In certain embodiments, the recombinant microorganism comprises an exogenous phosphatase. In certain embodiments, the exogenous phosphatase is hexitol phosphatase A (HxpA). In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is E. coli.

[0139] In certain embodiments, the present disclosure provides a recombinant microorganism comprising an increased production of D-tagatose as compared to a naturally occurring microorganism. In certain embodiments, the recombinant microorganism comprises an exogenous epimerase and a deletion of one, two, three, or four (4) genes. In certain embodiments, the exogenous epimerase is tagatose-1.6-bisphosphate aldolase subunit GatZ and / or ketosebiphosphate aldolase subunit KbaZ. In certain embodiments, the exogenous phosphatase is hexitol phosphatase A (H.xpA). In certain embodiments, the four deleted genes are ghicose-6- phosphate 1 -dehydrogenase, phosphofTuctokinase-l , D-alhdose-6-phosphate 3-epimerase, and mannose-6-phosphate isomerase. In certain embodiments, the recombinant microorganism further comprises an exogenous phosphatase. In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is £. colL

[0140] In certain embodiments, the present disclosure provides a microorganism comprising a recombinant polynucleotide, wherein the microorganism comprises an increased production of D- tagatose as compared to a naturally occurring microorganism, In certain embodiments, the recombinant polynucleotide comprises a nucleotide sequence encoding an exogenous epimerase. In certain embodiments, the exogenous epimerase is tagatose- 1 ,6-bisphosphafe aldolase subunit GatZ and / or ketose biphosphate aldolase subunit KbaZ. In certain embodiments, the recombinant polynucleotide further comprises a nucleotide sequence encoding an exogenous phosphatase. In certain embodiments, the exogenous phosphatase is hexitol phosphatase A. (HxpA). In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is E. coli.

[0141] In certain embodiments, the present disclosure provides a microorganism comprising a recombinant polynucleotide, wherein the microorganism comprises an increased production of D- tagatose as compared to a naturally occurring microorganism. In certain embodiments, the recombinant polynucleotide comprises a nucleotide sequence encoding an exogenous epimerase. In certain embodiments, the exogenous epimerase is tagatose- 1 ,6-bisphosphate aldolase subunit GatZ and / or ketose bi phosphate aldolase subunit KbaZ, In certain embodiments, the recombinant polynucleotide further comprises an exogenous phosphatase. In certain embodiments, the exogenous phosphatase is hexitol phosphatase A (HxpA), In certain embodiments, the microorganism further comprises a deletion of a first gene. In certain embodiments, the first gene is giucose-6-phosph.ate 1 -dehydrogenase. In certain embodiments, the microorganism farther comprises a deletion of a second gene. In certain embodiments, the second gene is phosphofru.ctokinase-1 . In certain embodiments, the microorganism further comprises a deletion of a third gene. In certain embodiments, the third gene i s D-allulose-6-pliosphate 3 -epimerase. In certain embodiments, the microorganism further comprises a deletion of a fourth gene, In certain embodiments, the fourth gene is mannose-6-phosphate isomerase. In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is E. -colt. In certain embodiments, the bacterium is A snhri / A. In certain embodiments, the bacterium is L. lact'is.

[0142] In certain embodiments, the present disclosure provides a recombinant microorganism comprising an increased production of D-tagatose as compared to a naturally occurring microorganism. In certain embodiments, the recombinant microorganism comprises an exogenous epimerase, an exogenous nuclease, a sgRNA, and a deletion of four (4) genes. In certain embodiments, the exogenous epimerase is tagatose- 1 ,6-bisphosphate aldolase subunit GatZ and / or ketose biphosphate aldolase subunit KbaZ). In certain embodiments, the recombinant microorganism further comprises an exogenous phosphatase. In certain embodiments, the exogenous phosphatase is hexitol phosphatase A (HxpA), In certain embodiments, the four deleted genes are c In certain embodiments, the sgRNA targetsIn certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is E. coli. In certain embodiments, the bacterium is B, subtiiis. In certain embodiments, the bacterium is

[0143] In certain embodiments, the present disclosure provides a microorganism comprising a recombinant polynucleotide, wherein the microorganism comprises an increased production of tagatose as compared to a naturally occurring microorganism. In certain embodiments, the recombinant polynucleotide comprises a nucleotide sequence encoding an exogenous epimerase, and a nucleotide sequence encoding a nuclease. In certain embodiments, the exogenous epimerase is tagatose- 1 ,6-bisphosphate aldolase subunit GatZ and / or ketose biphosphate aldolase subunit KbaZ In certain embodiments, the recombinant polynucleotide further comprises a nucleotide sequence encoding an exogenous phosphatase. In certain embodiments, the exogenous phosphatase is hexitol phosphatase A (HxpA). In certain embodiments, the microorganism ..further comprises a deletion of a first gene. In certain embodiments, the first gene is cry / icertain embodiments, the microorganism further comprises a deletion of a second gene. In certain embodiments, the second gene is pfkA. In certain embodiments, the microorganism further comprises a deletion of a third gene. In certain embodiments, the third gene is alsE, In certain embodiments, the microorganism further comprises a deletion of a fourth gene. In certain embodiments, the fourth gene is matiA. In certain embodiments, the microorganism further comprises a sgRNA. In certain embodiments, the sgRNA targets p / MJ. In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is £ co / Z In certain embodiments, the bacterium is B. subtiiis. In certain embodiments, the bacterium is L.

[0144] In certain embodiments, the present disclosure provides a recombinant microorganism comprising an increased production of D-tagatose as compared to a naturally occurring microorganism. In certain embodiments, the recombinant microorganism comprises an exogenous epimerase. In certain embodiments, the exogenous epimerase is a tagatose- 1, 6-bisphosphate aldolase subunit GatZ and / or ketose biphosphate aldolase subunit KbaZ. In certain embodiments, the recombinant microorganism comprises an exogenous phosphatase. In certain embodiments, the exogenous phosphatase is hexitol phosphatase A (HxpA). In certain embodiments, the recombinant microorganism further comprises a mutation of at least one gene selected from glucose-6-phosphate I -dehydrogenase (cW), phosphofructokinase-lphosphoffuctokinase-2 (pfiB), pyruvate kinase ( / MF), D-alhdose-6-phosphate 3 -epimerase (aZs-£)4marmose-6-phosphate isomerase (wd), phosphoglucomutase ( / / gM, D-tagatose- 1 ,6- bisphosphate aldolase subunit (grrtl'), D-tagatose- 1 ,6-bisphosphate aldolase subunit (kbaY), or a combination thereof. In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is E. coll.

[0145] In certain embodiments, the present disclosure provides a recombinant microorganism comprising an increased production of D-tagatose as compared to a naturally occurring microorganism. In certain embodiments, the recombinant microorganism comprises an exogenous epimerase. In certain embodiments, the exogenous epimerase is a tagatose- 1 ,6- bisphosphate aldolase subunit GatZ and / or ketose biphosphate aldolase subunit KbaZ. In certain embodiments, the recombinant microorganism comprises an exogenous phosphatase. In certain embodiments, the exogenous phosphatase is hexitol phosphatase A (HxpA). In certain embodiments, the recombinant mi croorganism further comprises a mutation of at least one gene selected from. glucose~6~phosphate 1 -dehydrogenase (cty / ), phosphofructokinase-l (p / F-1), phospho fructokinase- 2 ( / / .O), pyruvate kinase ( / MZA, D-allulose-6-phosphate 3 -epimerase (MF). nia.nnose~6~phosp.hate isomerase (manA), phosphoghicomutase (pgm), D-tagatose- 1 ,6- bisphosphate aldolase subunit (grr / l), D-tagatose- 1 ,6-bisphosphate aldolase subunit (Fbuh), or a combination thereof In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is

[0146] In certain embodiments, the present disclosure provides a recombinant microorganism comprising an increased production of D-tagatose as compared to a naturally occurring microorganism. In certain embodiments, the recombinant microorganism comprises an exogenous epimerase. In certain embodiments, the exogenous epimerase is a tagatose -1 ,6- bisphosphate aldolase subunit GatZ and / or ketose biphosphate aldolase subunit KbaZ. In certain embodiments, the recombinant microorganism comprises an exogenous phosphatase, In certain embodiments, the exogenous phosphatase is hexitol phosphatase A (HxpA). In certain embodiments, the recombinant microorganism further comprises a mutation of at least one gene selected from glucose-6-phosphate I -dehydrogenase phosphofructokinase-l ( / z / fct),phosphofructokinase-2 pyruvate kinase D-allulose-6-phosphate 3 -epimerase(MF), mannose-6-phosphate isomerase phosphoglucomutase (pgm), D-tagatose- 1, 6-bisphosphate aldolase subunit D-tagatose- 1,6-bisphosphate aldolase subunit ( or acombination thereof. In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is L lactLs.X Methods for IfyodueiKg and Generating Microorganisms Tagatase

[0147] The present disclosure also provides methods for preparing and / or generating any of the microorganisms disclosed herein. Many recombinant techniques commonly known in the art may be used, to introduce one or more recombinant polynucleotides of the present disclosure into a microorganism, including without limitation protoplast fusion, transfection, transformation, conjugation, and transduction. These techniques include conventional molecular biology techniques fog., recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of die art. Additional information on these techniques can be found in Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al, 1989); Oligonucleotide Synthesis (Gait, ed., 1984); Animal Cell Culture (Freshney, ed., 1987); Gene Transfer Vectors for Mammalian Cells (Miller & Calos, eds., 1987); Current Protocols in Molecular Biology (Ausubel et al., eds,, 1987); PCR: The Polyniemse Chain Reaction, (Mullis et al., eds., 1994); and Current Protocols in Immunology (Coligan et ai., eds., 1991 ).3,1. Recontbinanf polynucleotides

[0148] In certain embodiments, the recombinant polynucleotides disclosed herein can be stably integrated into a microorganism chromosome. In certain embodiments, the recombinant polynucleotides disclosed herein are stably integrated into a microorganism chromosome using homologous recombination, transposition-based chromosomal integration, reconibmase-niediated cassette exchange (R.MCE; e g,, using a Cre-lox system), or an integrating plasmid fogy, a yeast integrating plasmid), A variety of integration techniques suitable for a range of microor gan isms are known in the art (see, e.g,, Griffiths, AJ.F., Miller, J.H., Suzuki, D.T. et al. An Introduction to Genetic Analysis, 7thed, New York; W.H. Freeman; 2000), In certain embodiments, the recombinant polynucleotides disclosed herein are maintained in a recombinant microorganism of the present disclosure on an extra-chromosomal plasmid fe.g., an expression plasmid or vector). A variety of extra-chromosomal plasmids suitable for a range of microorganisms are known in the art, including without limitation replicating plasmids (e.g., yeast replicating plasmids that include an autonomously replicating sequence, ARS), centromere plasmids (e.g, yeast centromere plasmids that include an autonomously replicating sequence, CEN), episomal plasmids (e.g., 2- p.m plasmids), and / or artificial chromosomes (e.g., yeast artificial chromosomes, YACs. or bacterial artificial chromosomes. BACs).

[0149] In certain embodiments. the present disclosure provides vectors including the nucleotide sequences disclosed herein. As used herein, the term “vector” refers to a polynucleotide construct designed to introduce nucleic acids into one or more microorganisms. Vectors can include, but without any limitation, cloning vectors, expression vectors, shuttle vectors, plasmids, and cassettes. As used herein, the term “plasmid” refers to a circular double-stranded DNA construct used as a cloning and / or expression vector. In certain embodiments, plasmids can be extrachromosomai self-replicating genetic elements (e.g,, episomal plasmids) when introduced into a microorganism. In certain embodiments, plasmids can integrate into a microorganism chromosome. In certain embodiments, vectors can direct the expression of coding regions to which they are operatively linked, e.«., “expression vectors.’’' These expression vectors allow the expression of exogenous polynucleotides and / or polypeptides in microorganisms. In certain embodiments, the vectors allow the integration of one or more polynucleotides into the genome of a microorganism.

[0150] In certain embodiments, a vector disclosed herein includes a promoter. In certain embodiments, the vector is a bacterial or prokaryotic expression vector. In certain embodiments, the vector is a yeast, or fungal cell expression vector.

[0151] In certain embodiments, a vector discloses herein comprises nucleotide sequences in a single operon,3.L2. Promoters

[0152] In certain non-limiting embodiments, the recombinant polynucleotides disclosed herein include a control sequence, an enhancer, or a promoter. For example, but without any limitation, a nucleotide sequence encoding the grr / Z gene and / or kxp£i gene can be operably linked to a control sequence, enhancer, or promoter.

[0153] As used herein, the term “promoter” refers to any nucleotide sequence that regulates the initiation of transcription for a particular coding sequence under its control. Biologically, promoters are not transcribed but coordinate the assembly of components that initiate the transcription of other nucleotide sequences. In addition, promoters can limit this assembly and subsequent transcription to specific prerequisite conditions. For example, but without any limitation, a promoter can allow transcription in response to one or more environmental, temporal, or developmental stimuli. Bacterial and fungal cells possess a multitude of proteins tha t sense external or internal conditions and initiate signaling cascades ending in the binding of proteins to specific promoters and subsequent initiation of transcription of nucleic acid( s) under the control of the promoters. In certain embodiments, the promoter is endogenous. In certain embodiments, the promoter is exogenous. In certain embodiments, the promoter is artificially designed for expression in a particular species.

[0154] In certain embodiments.. the promoter .is a constitutive promoter. A constitutive promoter is a promoter that drives the expression of a nucleotide sequence continuously and without interruption in response to internal or external stimuli. Constitutive promoters are commonly used in recombinant engineering to ensure the continuous expression of a desired nucleotide sequence. Constitutive promoters result in a robust amount of nucleic acid expression, and, as such, are used in many recombin an t engineering applications to achie ve a high level of recomb inant protein and enzymatic activity. Non-limiting examples of constitutive promoters encompassed by the present disclosure include £, co / z promotersand ft from wiB, and the lambda phage promoter ft. (Liang, S.T. et al. JMn 292(1): 19- 37 (1999)). In some embodiments,the promoter is active in the stationary phase of the microorganism. Exemplary stationary phase promoters can be found in, e,g,, Slumada, ei af,, JOU&VJI OF BACTERIOLOGY, Nov. 2004, p. 7112-7122; Pletnev al tft ACTA NATURAE | VOL. 7 Xs 4 (27) 2015.

[0155] In certain embodiments, the promoter is an inducible promoter. An inducible promoter is a promoter that drives the expression of a nucleotide sequence in response to a stimulus. An inducible promoter drives sustained expression upon exposure to a specific stimulus (e.g., IPTG). In certain embodiments, an inducible promoter dri ves a graded level of expression correlated with the amount of stimulus. Non-limiting examples of stimuli for inducible promoters include heat shock, exogenous compounds or a lack thereof ft", a sugar, metal, drug, or phosphate), salts or osmotic shock, oxygen, and biological stimuli ft.g, a growth factor or pheromone). Non4irniting examples of inducible promoters include the E. coil promoters P^c, P^), / U ftv, / ’BAD, and

[0156] In certain embodiments, the recombinant polynucleotide can include multiple promoters. In certain embodiments, the multiple promoters can be the same, For example, but without any limitation, the recombinant polynucleotide can include a nucleotide sequence encoding the gciiZ gene operably linked to a first promoter and a nucleotide sequence encoding the hpxfi gene operably linked to a second promoter, wherein the first and second promoter is the same. In certain embodiments, the multiple promoters can be different. For example, but without any limitation, the recombinant polynucleotide can include a nucleotide sequence encoding thegene operably linked to a first promoter and. a nucleotide sequence encoding thegene operably linked to a second promoter, wherein the first and second promoter are different. In certain embodiments, the promoter is a ftjacot promoter. In certain embodiments, the Pu^-oi promoter comprises the nucleotide sequence set forth in SEQ ID NO: 42. In certain embodiments, the ftwi promoter consists of the nucleotide sequence set forth in SEQ ID NO: 42. Thepromoter is a hybrid regulators region including the promoter ft, of phage lambda with the CIbinding sites replaced with lacO1. The hybrid design allows for a strong promotion that can be repressed by LacI, the Lac inhibitor (i.e.;repressor) or induced by IPTG.

[0157] In certain embodiments, the promoter is a A promoter. In certain embodiments, thepromoter comprises the nucleotide sequence set forth in SEQ ID NO: 43. In certain embodiments, the Puaroi promoter consists of the nucleotide sequence set forth in SEQ ID NO: 43.

[0158] In certain embodiments, the promoter is a Pn promoter. In certain embodiments, the Pn promoter comprises the nucleotide sequence set forth tn SEQ ID NO: 44, In certain embodiments, the promoter consists of the nucleotide sequence set forth in SEQ ID NO: 44.

[0159] In certain embodiments, the promoter is a Plet promoter. In certain embodiments, the Ptet promoter comprises the nucleotide sequence set forth in SEQ ID NO: 45, In certain embodiments, the promoter consists of the nucleotide sequence set forth in SEQ ID NO: 45.

[0160] In certain embodiments, the promoter is apromoter. In certain embodiments, the promoter comprises the nucleotide sequence set forth in SEQ ID NO: 46, In certain embodiments, the 7^® promoter consists of the nucleotide sequence set forth in SEQ ID NO: 46.promoter nucleotide sequence:AATT 1 GT G AGC G G AT AAG AA 1 T GA Al T QJ T G PWJQ GG AT AMIJ\AU AT AC i GAG c AC P\ I GAG c AGG A CGGACTGACCGAATTCATTAAAGAGGAGAAAAGATATACC ( SEQ ID DO : Q 2 )

[0162] Pr.tes n promoter nucleotide sequence: t ccctatcag tgatagagattgagatccctac cag tgatagagatac tgagcacatcagcagga cqcactgaocgaat.tcattaaagaggagaaaggtace ( SEQ I D NO : 4 3 )

[0163] PT7 promoter nucleotide sequence: t a a t a eg a o t c a a t a t ag g gg a a 11 g t g a g eg g a t a a c a a 11 c c c c t c t a g a a a t a a 1111 g 11 taa ctttaagaaggagatatacc (SEQ ID NO : 44 )

[0164] Pict promoter nucleotide sequence: gttgacactGtatcgttgatagagltatt ttaccaetccetateagtgatagagaaaagaatt: aaaagatataaagaggagaaaggatct (SEQ ID NO : 4 5 )

[0165] PgsdB promoter nucleotide sequence:

[0166] In certain embodiments, the promoter is a stationary phase promoter. As used herein, the term “stationary' phase promoter” refers to a promoter upstream of a gene that is transcribed dur ing the stationary phase of a microorganism growth. The life cycle of an E, t-cPi culture includes 5distinct phases; lag, logarithmic, stationary, death, and long-term stationary phase. The lag phase occurs when cells are inoculated into media and adjust their metabolic processes according to their new environment. The cells will then rapidly grow and divide, entering the logarithmic phase. It is at this time that enzymes related to central carbon metabolism are most important, and the transcription of corresponding genes will be npregulated. Once the cells sense environmental stressors such as scarcity of media nutrients, their growth and division slows, and the culture enters the stationary phase. The use of a stationary phase promoter prevents the production pathway from competing with central carbon metabolism for carbon flux during the fogarithmic phase of growth, a time when cells need, carbon to rigorously grow and. divide.

[0167] In certain embodiments, the stationary phase promoter is P^g. In certain embodiments, the promoter comprises the nucleotide sequence set forth in SEQ ID NO: 46 or SEQ I D NO:53. In certain embodiments, thepromoter consists of the nucleotide sequence set forth in SEQ ID NO: 53.

[0168] In certain embodiments, the stationary phase promoter ishi certain embodiments, the promoter comprises the nucleotide sequence set forth in SEQ ID NO: 54. In certain embodiments, thepromoter consists of the nucleotide sequence set forth in SEQ ID NO:54.

[0169] In certain embodiments, the stationary phase promoter is PIn certain embodiments, the promoter comprises the nucleotide sequence sei forth in SEQ ID NO: 55. In certain embodiments, thepromoter consists of the nucleotide sequence set forth in SEQ ID NO: 55.

[0170] In certain embodiments, the stationary phase promoter isIn certain embodiments, the Pdpspromoter comprises the nucleotide sequence set forth in SEQ ID NO: 56, In certain embodiments, the Pdpspromoter consists of the nucleotide sequence set forth in SEQ ID NO: 56,AC CTGTTTAAAATATGTTCAGCAACC CLATCTTGATGGC GACCTCC CTCC GGGATGA'i TTCAAIAACATATTCTGTGTTGGCATATGAAATTTTGAGGATTACCCTACACTTATAGGAGTTACCTTAC AGGGGTTCCTTCAATTTGTGTTGATTTACGCGAGATAACGCTCGAATTCATTAAAGAGGAGAAA GGTACCATG ( SEQ ID NO : 54 )■it.. JAC- 1 I.. AZ"k‘ .7 / "4. kj A A 1 kj k.- k-Tk-z J. z^V— k^ kj A kJ k.- A f_7 J. kj A 7't kJ jk^ j'k _L 7 A j'kz^\7't kj j'k'jjyGATTCCAGGCATCATTGAGGGATTGAACCTCG3.7.3, Generic Markers

[0171] In certain embodiments, the presently disclosed recombinant polynucleotides include genetic markers. These genetic markers allow the selection of microorganisms that have one or more desired polynucleotides (e.g., recombinant polynucleotides). In certain embodiments, the genetic marker is an antibiotic resistance marker selected from the group consisting of Aprarnyciti resistance. Ampicillin resistance, Kanamyc-in resistance, Spectinomycin resistance, Tetracyclin resistance, Neomycin resistance. Chloramphenicol resistance, Gentamycin resistance, Erythromycin resistance, Carbenicillin resistance. Actinomycin D resistance. Neomycin resistance, Polymyxin resistance, Zeocin resistance, and Streptomycin resistance. In certain embodiments, the genetic marker includes a coding sequence of an antibiotic resistance protein (e.g., a beta-lactamase for certain Ampicillin resistance markers) and a promoter or enhancer element that drives the expression of the coding sequence in a microorganism of the present disclosure. In certain embodiments, a microorganism of the present disclosure is grown under conditions in which an antibiotic resistance marker is expressed and confers resistance to the microorganism, thereby selected for the microorganism with successful integration of the marker. In certain embodiments, the genetic marker is an auxotrophic marker. In certain embodiments, the auxotrophic marker is a gene involved in vitamin, amino acid, fatty acid synthesis, or carbohydrate metabolism. In certain embodiments, the auxotrophic marker is a gene for synthesizing amino acid. In certain embodiments, the auxotrophic marker is a gene for synthesizing glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, methionine, asparagine, glutamine, lysine, arginine, histidine, aspartate or glutamate. In certain embodiments, the auxotrophic marker is a gene for synthesizing adenosine, biotin, thiamine, leucine, glucose, lactose, or maltose. In certain embodiments, a microorganism of the present disclosure is grown under conditions in which an auxotrophic resistance marker is expressed in an environment or medium lacking the corresponding nutrient, and confers growth to the microorganism (lacking an endogenous ability to produce the nutrient), thereby selected for the microorganism with success ful integrati on of the marker.

[0172] In certain embodiments, the present disclosure also provides methods to introduce a deletion of any of the genes or enzymes disclosed herein. These deletions can be generated by any suitable gene-editing methods. In certain embodiments, the deletion is generated by a method comprising homologous recombination, a Zinc finger nuclease, a meganuclease, a Transcription activator-like effector nuclease (TALEN), a Clustered regularly-interspaced short palindromic repeats (CRISPR) system, or a combination thereof.

[0173] In certain embodiments, the deletion is generated by a CRISPR system. Clustered regularly-interspaced short palindromic repeats (CRISPR) system is a genome-editing tool discovered in prokaryotic cells. When utilized for genome editing, the system includes Cas9 (a protein able to modi fy DNA utilizing crRNA as its guide), CRISPR RNA (crRNA, which contains the RNA used by Cas9 to guide it to the correct section of host DNA along with a region that binds to tracrRNA (generally in a hairpin loop form) forming an active complex with Cas91, transactivating crRNA (tracrRNA, binds to crRNA and forms an active complex with Cas9), and an optional section of DNA repair template (DNA that guides the cellular repair process allowing insertion of a specific DNA sequence). Multiple crRNA’s and the tracrRNA can be packaged together to form a single-guide RNA (sgR.NA). This sgRNA can be joined together with the Cas9 gene and made into a plasmid in order to be transfected into cells. In certain embodiments, the CRISPR system comprises base editors. In certain embodiments, the CRISPR system comprises transposasesZrecombinases. In certain embodiments, the CRISPR system comprises prime editors. In certain embodiments, the CRISPR system comprises an epigenetic modulator. In certain embodiments, the CRISPR system comprises a CRISPRoff system. Additional details on the CRISPR systems of the present disclosure can be found in Anzalone et al., Ato / re bfotec / inofoqu 38.7 (2020)'. 824-844 and in Nunez et al, Cell 184 ,9 (2021); 2503-2519, and Jiang et al., Appl Environ Microbiol 2015 Apr;81(7).'2506-I4, the contents of each of which are incorporated by reference in their entireties.

[0174] In certain embodiments, the deletion is generated by a zinc- finger nuclease. A zinc-finger nuclease (ZFN) is an artificial restriction enzyme, which is generated by combining a zinc finger DN A-binding domain with a DNA-cleavage domain. A zinc finger domain can be engineered to target specific DNA sequences and allows a zinc-finger nuclease to target desired sequences within genomes. The DNA-binding domains of individual ZFNs typically contain a plurality of individual zinc finger repeats and can each recognize a plurality of base pairs. The most common method to generate a new zinc-finger domain is to combine smaller zinc-finger “modules” of known specificity. The most common cleavage domain in ZFNs is the non-specific cleavage domain from the Type Ils restriction endonuclease Fokl.

[0175] In certain embodiments, the deletion is generated by a TALEN system. Transcription activator- like effector nucleases (TALEN) are restriction enzymes that can be engineered to cut specific sequences of DNA. TALEN system operates on almost the same principle as ZFNs. They are generated by combining a transcription activator-like effectors DNA-binding domain with a DNA cleavage domain. Transcription activator-like effectors (TALEs) are composed of 33-34 amino acid repeating motifs with two variable positions that have a strong recognition for specific nucleotides. By assembling arrays of these TALEs, the TALE DNA-binding domain can be engineered to bind desired DNA sequence, and thereby guide the nuclease to cut at specific locations in genome.

[0176] In certain embodiments, the deletion is generated by a meganuclease. A meganiiclease is an endodeoxyribonuclease that recognizes a double-stranded DNA site of approx. 12 to approx. 40 base pairs that occur only once in a genome. Meganucleases are some of the most specific naturally occurring restriction enzymes. Meganucleases are also defined as molecular DNA scissors since they can replace, eliminate or modify sequences in a highly targeted way. Protein engineering allows the modification of their recognition sequence and the targeted sequence.

[0177] In certain embodiments, the present disclosure also provides methods to reduce the expression of any of the genes or enzy mes disclosed herein. In certain embodiments, the reduced expression of genes and enzymes disclosed herein comprises using oligonucleotides that have complementary sequences to the mRNA of the genes disclosed herein (e.g., zn / i man J, a / sE, / tfkd , pftB, etc.). Non-limiting examples of these oligonucleotides include small interference RNA (siRNA), short hairpin RNA. (shRNA), and microRNA (niiRNA). In certain embodiments, these oligonucleotides can be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to at least a portion of a zu / mRNA sequence. In certain embodiments, these oligonucleotides can be identical to at least a portion of a CM / mRNA sequence. In certain embodiments, these oligonucleotides can be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to at least a portion of a p / M mRNA sequence. In certain embodiments, these oligonucleotides can be identical to at least a portion of a pfkA mRN A sequence. In certain embodiments, these oligonucleotides can be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to at least a portion of a pfkB mRNA sequence. In certain embodiments, these oligonucleotides can be identical to at least a portion of a pflcB mRNA sequence. In certain embodiments, these oligonucleotides can be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about96%. at least about 97%, at least about 98%, at least about 99% identical to at least a portion of an alsE mRNA sequence. In certain embodiments, these oligonucleotides can be identical to at least a portion of an alsE mRNA sequence. In certain embodiments, these oligonucleotides can be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to at least a portion of a mRN A sequence. In certain embodiments, these oligonucleotides can be identical to at least a portion of a manJ mRNA sequence. In certain embodiments, antisense nucleic acid, shRNA, miR'NA, or siRNA molecules can include DNA or atypical or uon-naturally occurring residues, for example, but not limited to, phosphorothioate residues.

[0178] In some embodiments, reduction of expression of genes and enzymes disclosed herein can comprise use of CRISPR, which can mutate the coding sequence or promoter, to lower or remove expression of the gene product, or CRISFRi can be targeted to the genes disclosed herein, thereby reducing expression of one of more of the genes. See, e.g., Arroya-Olarte, e / al., Microorganisms, 2021 Apr; 9(4); 844. Zhang ei al. , Front. Microbiol. (31 March 202'1),

[0179] In certain embodiments, the reduced expression of genes and enzymes disclosed herein comprises using a CRIS PR system. In certain embodiments, the CRISPR system includes a Cas9. In certain embodiments, Cas9 comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 85. In certain embodiments, Cas9 comprises the amino acid sequence set forth in SEQ ID NO: 85. In certain embodiments, Cas9 consists of the amino acid sequence set forth in SEQ ID NO: 85. SEQ ID NO: 85 is provided below:

[0180] In certain embodiments, the reduced expression of genes and enzymes disclosed herein comprises using a CRISPRi system. CRISPRi systems silence genes at the transcriptional level and can have fewer sequence-specific off-target effects than RNAi. In certain embodiments, the CRISPRi system includes a catalytically dead Cas9 (d€as9). dCas9 is a programmable transcription factor that can be targeted to promoters through sgRN As, where it can function as repressor. In certain embodiments, dCas9 comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 57. In certain embodiments, dCas9 comprises the amino acid sequence set forth in SEQ ID NO: 57. In certain embodiments, dCas9 consists of the amino acid sequence set forth in SEQ ID NO: 57. SEQ ID NO: 57 is provided below:KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRMLAS AGELQKGDELALPSKYVNFLYLASHYEKLKGSPEDDEQKQLFVEQHKHYLDE 11EQ1 SEFSKRV TLADANLDKVLSAYNf;HRDf;PIREQAENT THI;RTLPRLGAPAGFKYFDTTEDRKRYTSTKEVLEATLIHQS ITGLYETRI DLSQLGGD [ SEQ YD I'-ii > : GY s

[0181] In certain embodiments, the nuclease (e.g., Cas9 ordCas9) is regulated by a promoter (e.g., described in Section 3.1 .2). In certain embodiments, the promoter is an inducible promoter. In certain embodiments, the promoter is a stationary phase promoter. In certain embodiments, tbe CRISPiVCRISPRl system includes a small guide RNAs (sgRNA). In certain embodiments, the sgRNA of the CRISPR / CRISPRi system targets a gene encoding an enzyme of a competing pathway. For example, but without any limitation, the sgRNA can target the zwf gene, the pgm gene. the / r / M gene, the / ?$$ gene, the ManA gene, or the AlsE gene. In certain embodiments.the sgRNA can target any portion of a gene. For example, but wi thout any limi tation, the sgRNA can target a promoter, an operator, or a sequence encoding a protein,

[0182] In certain embodiments, the CRISPR / CRISPRi system includes a nuclease (e.g., Cas9 or dCas9) and a sgRNA. In certain embodiments, the nuclease (e.g., Cas9 or d.Cas9) is regulated by an inducible promoter. In certain embodiments, the inducible promoter is 7Aet. In certain embodiments, the sgRN A targets a pfldB gene. In certain embodiments, the sgRNA targets a promoter of thegene.3,5. rraw / bmfffcw and Gene Editing

[0183] In certain embodiments, the present disclosure provides the use of transformation of the plasmids and vectors disclosed herein. Vectors and plasmids disclosed herein can be transformed into cells through any known system in the art. For example, but without any limitation, the presently disclosed microorganisms can be transformed by particle bombardment, chemical transformation, Agrobaeterium transformation, nano-spike transformation, electroporation, and virus tr ansformati on ,

[0184] In certain embodiments, the vectors of the present disclosure may be introduced into the microorganisms using a variety of techniques, including transformation, transfection, transduction, viral infection, gene guns, or Ti-mediated gene transfer. Non-limiting examples of these methods include calcium phosphate transfection, DEAE-Dextran mediated transfection, lipofection, and electroporation (see, e.g., Davis, L., Dibner, M., Battey, .1., 1986 “Basic Methods in Molecular Biology”; Gietz et al.. Nucleic Acids Res. 27:69-74 (1992); Ito et al,, J. Bacterol. 153: 163-168 (1983); and Becker and Guarente, Methods in Enzymology 194: 182-187 (1991 )). In certain embodiments, transformed microorganisms are referred to as recombinant microorganisms.

[0185] In certain embodiments, the present disclosure provides methods for introducing exogenous proteins (e.g., nuclease), RNA (e,g., gRNA), and DNA (e.g., a recombinant polynucleotide disclosed herein) into the microorganism. Various methods for achieving this have been described previously including direct transfection of protein and nucleotide sequence or DN A transformation followed by intracellular expression of RNA and protein (see, e.g., Dicarlo, .1. E. et al. “Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems.” Nucleic Acids Res (2013). doi: 10,1093 / narigktl 35; Ren, Z. J., Baumann, R. G. & Black, L. W. “Cloning of linear DNAs in vivo by overexpressed T4 DNA ligase: construction of a T4 phage hoc gene display vector.” Gene 195, 303-311 (1997); Lin, S.tStaahl, B. T., Alia, R. K, & Doudna, .1. A. “Enhanced homology-directed human genome engineering by controlled timing ofCRISPR / Cas9 delivery.” Elite 3, e04766 (2014)).3,4. Recombination Systems

[0186] In certain embodiments. the present disclosure also provides homologous recombination systems for editing (e.g,, insertion, deletion) in a microorganism. In certain embodiments, the homologous recombination system can be native to the host cell or introduced to the cell host. For example, but without any limitation, genes for the homologous recombination system can be introduced on a plasmid, introduced on a linear DNA fragment, introduced as and translated from RNA or set of RNAs, or introduced as a protein or set of proteins. In certain embodiments, the methods inchide a recombinant polynucleotide disclosed herein. In certain embodiments, the polynucleotide includes sequence homologous (e.g., left and right homology arms) to a region in a nucleic acid (e.g., genome, plasmid, etc.) such that the left and right homology arms are separated by a designed genetic edit (e.g., promoter, insertion, substitution, SNP, terminator, degron, a sequence for a tag, sequence for a degradation signal or deletion). In certain embodiments, the recombinant polynucleotide includes a genetic marker, a counter selectable genetic marker (e.g., SacB or PheS), and an origin of replication (e.g., R6K ).

[0187] In certain embodiments, the recombinant polynucleotide including the homology arms and sequence for genetic editing is introduced into the microorganism using any of the methods disclosed herein (e.g., transformation via electroporation, conjugation, etc.). In certain embodiments, following transformation, the resulting transformants can be plated on a medium to select for transformants expressing the selectable genetic markers. The recombination of a plasmid comprising homology arms with a targeted locus in a nucleic acid (e.g., genome, plasmid, etc.) can occur at one of the two homology sites targeted by the homology arms present on the plasmid and that flank the designed genetic edit. In certain embodiments, the resulting transformants grow as colonies on the selective medium and can be selected and plated on a second type of selective medium (e, g. , counter-selectable medium). In certain embodiments, the second type of selective medium allows the selection of cells that comprise the desired genetic editing.

[0188] In certain embodiments, the methods disclosed herein include using proteins from one or more recombination systems. Said recombination systems can be endogenous to the microorganism or can be exogenous. In certain embodiments, the proteins from one or more recombination systems can be introduced as nucleic acids (e.g., as a plasmid, linear DNA or RNA, or integron) and be integrated into the genome of the host cell or be stably expressed from an cxtrachromosomal element. In certain embodiments, the proteins from one or more recombination systems can be introduced as RN A and be translated by the host cell. In certain embodiments, the proteins from one or more recombination systems can be introduced as proteins into the host. cell. Non- limiting examples of recombination systems include lambda red recombination system, RecET recombination system, Red / ET recombination system, any homologs, orthologs, orparalogs of proteins from a lambda red recombination system, RecET recombination system, Red / ET recombination system, lambda red-mediated recombination system, or any combination thereof. Details on the recombination systems from the RecET recombination system can be any of those as described in Zhang Y., Buchholz F.sMuyrers J.P.P. and Stewart A.F. “A new logic for DN A engineering using recombination in E. coEE Nature Genetics 20 (1998 ) 123-128; Muyrers, J.P.P., Zhang, Y., Testa, G., Stewart, A.F. “Rapid modification of bacterial artificial chromosomes by ET-recombination.” Nucleic Acids Res. 27 (1999) 1555- 1557; Zhang Y„ Muyrers J.P.P., Testa G. and Stewart A.F. “DNA cloning by homologous recombination in E. coEE Nature Biotechnology 18 (2000) 1314-1317 and Muyrers IP et al., “Techniques: Recombinogenic engineering— new options for cloning and manipulating DNA” Trends Biochem Sei. 2001 May;26(5 ):325-31 , which are herein incorporated by reference in their entirety.< Methods for Producing If ~ Tagatose

[0189] The present disclosure also provides methods for producing D-tagatose. Cell-free methods (e,g,, in vitro synthesis) utilize thermodynamically unfavorable enzymatic reactions. In certain embodiments, the presently disclosed methods for producing tagatose include culturing microorganisms (e.g., one disclosed in Section 2) and purifying D-tagatose.4.1. CeE CuEnre

[0190] The present disclosure provides methods of culturing microorganisms disclosed herein. As used herein, “culturing” a cell refers to introducing an appropriate culture medium, under appropriate conditions, to promote the growth of a cell. In certain embodiments, culturing is performed using a liquid or solid growth medium. In certain embodiments, culturing occurs under aerobic or anaerobic conditions based on the requirements of the microorganism and desired metabolic state of the same. In certain embodiments, culturing includes specific conditions such as temperature, pressure, light, pH, and ceil density.

[0191] In certain embodiments, the methods for producing methods of producing tagatose include a culture medium for culturing the recombinant bacteria. “Culture medium,” as used herein, refers to any composition or broth that supports the growth of the microorganism disclosed herein. A culture media can be liquid or solid. In certain embodiments, the culture media include nutrients, salts, buffers, elements, and other compounds that support the growth and viability of cells. Additionally, culture media can include sources of nitrogen, carbon, amino acids, carbohydrates, trace elements, vitamins, and minerals. In certain embodiments, the culture media include a complex extract (e.g., yeast extract). In certain embodiments, the culture medium is enriched in order to support rapid growth. In certain embodiments, the culture medium is modified in order to support slower growth. In certain embodiments, the culture medium includes an agent that can inhibit the growth of or kill contaminating organisms (e.g., an antibiotic). In certain embodiments,the culture medium includes an agent that can activate an inducible promoter or enzyme (e.g., IPTG). Non-limiting examples of culture media encompassed by the present disclosure include M9 medium. Lysogeny Broth (LB)tTerrific Broth (TB)„ and YT broth. In certain embodiments, the culture medium comprises a substrate that is converted by the recombinant microorganisms to tagatose.

[0192] In certain embodiments, the substrate is a sugar (e.g., glucose or fructose) that can be phosphorylated by the bacteria via a kinase (e.g., hexokinase) and converted into fructose-6- phosphate. In certain embodiments, the substrate is glucose. In certain embodiments, glucose can derive from cellulose, C5 sugars, hemicellulose, and / or xylose, to certain embodiments, the substrate is a constituent of the culture medium, to certain embodiments, the substrate is supplemented with the culture medium. In certain embodiments, the substrate is continuously present in the culture medium. In certain embodiments, the substrate is supplemented daring the growth phase. In certain embodiments, the substrate is supplemented during the stationary phase.4,2. Pnrificalk»? qf Tagatose

[0193] In certain embodiments, the methods of the present disclosure further comprise purifying tagatose produced by a microorganism of the present disclosure, e.g., from cell culture or cell culture medium. A variety of methods known in the art may be used to purify a product from a microorganism or microorganism culture. In certain embodiments, one or more products may be purified continuously, e.g., from a continuous culture. In certain embodiments, one or more products may be purified separately from fermentation, e.g., from a batch or fed-batch culture. One skilled in the art will appreciate that the specific purification tnethod(s) used may depend upon, inter alia, the microorganism, culture conditions, and / or particular product! s),

[0194] In certain embodiments, purifying D-tagatose comprises separating or filtering the microorganisms from a cell culture medium, separating the D-tagatose from the culture medium fe.g., by chromatography), concentration of water (e.g., by evaporation), and lyophilization of the D-tagatose.5. Food Frw / w c A

[0195] The present disclosure also provides delivery systems methods for use in food products including the D-tagatose prepared and / or generated by any of the microorganisms disclosed herein.

[0196] The term “food product,” as used herein, includes any food product, for example, those set forth in 21 CFR. 101.12. Non-limiting examples of such food products include fi'ozen desserts, baked goods, fillings, nutritional drinks, beverages, salad dressing or similar dressing, sauces, icings, puddings and custards, batters, and the like. Various baked goods are disclosed in U.S. Patent No. 6,536,599, the disclosure of which is herein incorporated by reference in its entirety.Non-limiting examples of bakery goods include cookies, cakes. rolls, pastries, pie dough, brownies, breads, bagels, and the like. The D-tagatose prepared and / or generated by any of the microorganisms disclosed herein are also suitable as a component in frozen foods.

[0197] In certain embodiments, the food product is prepared by admixing the D-tagatose in an ingestible vehicle, together with any optional ingredients, to form a uniform mixture. The final compositions are readily prepared using standard methods and apparatus generally known by those skilled in the corresponding arts, such as confectionary arts. The apparatus useful per the presently disclosed subject matter comprises mixing apparatus well known in the art, and therefore the selection of the specific apparatus will be apparent to the artisan.

[0198] As used herein “admixing,” for example, “admixing D-tagatose with a food product,” refers to the process where the flavor compositipn is mixed with or added to the completed product or mixed with some or all of the components of the product during product formation or some combination of these steps. When used in the context of admixing the term “product”' refers to the product or any of its components. This admixing step can incl ude a process selected from the step of adding D-tagatose to the product, spraying D-tagatose on the product, coating D-tagatose on the product, suspending the product in D-tagatose, painting D-tagatose on the product, pasting D-tagatose on the product, encapsulating the product with D-tagatose, mixing D-tagatose with the product and any combination thereof. The D-tagatose can be a liquid, dry powder, spray, paste, suspension and any combination thereof

[0199] In certain embodiments, the present application relates to the modified edible food products produced by the methods disclosed herein. In certain embodiments, the food products can be produced by processes for producing comestible products well known to those of ordinary skill in the art,

[0200] In certain embodiments, the D-tagatose prepared and / or generated by any of the microorganisms disclosed herein can be dissolved in or dispersed in one of many known comestible acceptable liquids, solids, or other carriers, such as water at neutral, acidic, or basic pH, fruit or vegetable juices, vinegar, marinades, beer, wine, natural water / fat emulsions such as milk or condensed milk, whey or whey products, edible oils and shortenings, fatty acids, certain low molecular weight oligomers of propylene glycol, glyceryl esters of fatty acids, and dispersions or emulsions of such hydrophobic substances in aqueous media, salts such as sodium chloride, vegetable flours, solvents such as ethanol, solid edible diluents such as vegetable powders or flours, and the like, and then combined with precursors of the comestible or medicinal products, or applied directly to the comestible or medicinal products.

[0201] Those of ordinary skill in the art of preparing and selling food products are well aware of a large variety of classes, subclasses, and species of the comestibl e compositions, and util ize well-known and recognized terms of art to refer to those comestible compositions while endeavoring to prepare and sell various of those comestible compositions. Such a list of terms of art is enumerated below, and it is specifically contemplated hereby that the D-tagatose prepared and / or generated by any of the microorganisms disclosed herein can be used to modify or enhance the taste of the following list edible compositions, either singly or in all reasonable combinations or mixtures thereof.

[0202] In certain embodiments, the food products to which the D-tagatose prepared and / or generated by any of the microorganisms disclosed herein are admixed with comprise, by way of example, the wet soup category, the dehydrated and culinary food category, the beverage category, the frozen food category, the snack food category, and seasonings or seasoning blends, described herein.

[0203] In certain embodiments, the D-tagatose prepared and / or generated by any of the microorganisms disclosed herein are admixed with one or more confectioneries, chocolate confectionery, tablets, countlines, bagged selfmies / softltnes, boxed assortments, standard boxed assortments, twist wrapped miniatures, seasonal chocolate, chocolate with toys, allsorts, other chocolate confectionery, mints, standard, mints, power mints, boiled sweets, pastilles, gums, jellies and chews, toffees, caramels and nougat, medicated confectionery, lollipops, liquorice, other sugar confectionery, gum, chewing gum, sugarised gum, sugar-free gum, functional gum, bubble gum, bread, packaged / industrial bread, unpackaged'artisanal bread, pastries, cakes, packaged / industrial cakes, unpackaged / artisanal cakes, cookies, chocolate coated biscuits, sandwich biscuits, filled biscuits, savory biscuits and crackers, bread substitutes, breakfast cereals, rte cereals, family breakfast cereals, flakes, muesli, other rte cereals, children's breakfast cereals, hot cereals, ice cream, impulse ice cream, single portion dairy ice cream, single portion water ice cream, multi-pack daily ice cream, multi-pack water ice cream, take-home ice cream, take-home dairy ice cream, ice cream desserts, bulk ice cream, take-home water ice cream, frozen yoghurt, artisanal ice cream, dairy products, milk, fresh / pasteurized milk, full fat ffesh / pasteurized milk, semi skimmed fresh / pasteurized milk, long-llfe / uht milk, foil fat long Hfe / uht milk, semi skimmed long life / ubt milk, fat-free long Hfe / uht milk, goat milk, condensed / evaporated milk, plain condensed / evaporated milk, flavored, functional and other condensed milk, flavored milk drinks, dairy only flavored milk drinks, flavored milk drinks with fruit juice, soy milk, sour milk drinks, fermented dairy drinks, coffee whiteners, powder milk, flavored powder milk drinks, cream, cheese, processed cheese, spreadable processed cheese, unspreadable processed cheese, unprocessed cheese, spreadable unprocessed cheese, hard cheese, packaged hard cheese, unpackaged hard cheese, yoghurt, plain / natural yoghurt, flavored yoghurt, fruited yoghurt, probiotic yoghurt, drinking yoghurt, regular drinking yoghurt, probiotic drinking yoghurt, chilledand shelf-stable desserts, dairy-based desserts, soy-based desserts, chilled snacks, frontage frais and quark, plain fromage frais and quark, flavored fromage frais and quark, savory fromage frais and quark, sweet and savory snacks, fruit snacks, chips / crisps, extruded snacks, tortilla / corn chips,popcorn, pretzels, nuts, other sweet and savory snacks, snack bars, granola bars, breakfast bars, energy bars, fruit bars, other snack bars, meal replacement products, slimming products, convalescence drinks, ready meals, canned ready meals, frozen ready meals, dried ready meals, chilled ready meals, dinner mixes, frozen pizza, chilled pizza, soup, canned soup, dehydrated soup, instant soup, chilled soup, uht soup, frozen soup, pasta, canned pasta, dried pasta, chilled / fresh pasta, noodles, plain noodles, instant noodles, cups / bowl instant noodles, pouch instant noodles, chilled noodles, snack noodles, canned food, canned meat and meat products, canned flsh / seafbod, canned vegetables, canned tomatoes, canned beans, canned fruit, canned ready meals, canned soup, canned pasta, other canned foods, frozen food, frozen processed red meat, frozen processed poultry, frozen processed flsh / seafood, frozen processed vegetables, frozen meat substitutes, frozen potatoes, oven baked potato chips, other oven baked potato products, non-oven frozen potatoes, frozen bakery products, frozen desserts, frozen ready meals, frozen pizza, frozen soup, frozen noodles, other frozen food, dried food, dessert mixes, dried ready meals, dehydrated soup, instant soup, dried pasta, plain noodles, instant noodles, cups / bowl instant noodles, pouch instant noodles, chilled food, chilled processed meats, chilled fish / seafood products, chilled processed fish, chilled coated fish, chilled smoked fish, chilled lunch kit, chilled ready meals, chilled pizza, chilled soup, chilled / fresh pasta, chilled noodles, oils and fats, olive oil, vegetable and Seed oil, cooking fats, butter, margarine, spreadable oils and fats, functional spreadable oils and fats, sauces, dressings and condiments, tomato pastes and purees, bouillon / stock cubes, stock cubes, gravy granules, liquid stocks and fonds, herbs and spices, fermented sauces, soy based sauces, pasta sauces, wet sauces, dry sauces / powder mixes, ketchup, mayonnaise, regular mayonnaise, mustard, salad dressings, regular salad dressings, low fat salad dressings, vinaigrettes, dips, pickled products, other sauces, dressings and condiments, baby food, milk formula, standard milk formula, follow-on milk formula, toddler milk formula, hypoallergenic milk formula, prepared baby food, dried baby food, other baby food, spreads, jams and preserves, honey, chocolate spreads, nut-based spreads, and yeast-based spreads.

[0204] fa certain embodiments, the D-tagatose prepared and / or generated by any of the microorganisms disclosed herein can be used in low-calorie gum formulations and can also be used in sugar chewing gum. Various specifics of chewing gum compositions are disclosed in U .S. Patent No. 6,899,911, the disclosure of which is incorporated herein by reference In its entirety. The chewing gum composition of the presently disclosed subject matter follows the generalpattern outlined below. In general, a chewing gum composition typically contains a chewable gum base portion that is essentially free of water and is water-insoluble, a water-soluble bulk portion, and flavors that are typically 'water- insoluble. The water-soluble portion dissipates with a portion of the flavor over a period of time during chewing. The gum base portion is retained in the mouth throughout the chew. The insoluble gum base generally comprises elastomers, elastomer solvents, plasticizers, waxes, emulsifiers, and inorganic fillers. Plastic polymers, such as polyvinyl acetate, which behave somewhat as plasticizers, are also often included. Other plastic polymers that can be used include polyvinyl laureate, polyvinyl alcohol, and polyvinylpyrrolidone. Elastomers can include polyisobutylene, butyl rubber, (isobutylene-isoprene copolymer), and styrene butadiene rubber, as well as natural latexes such as chicle. Elastomer solvents are often resins such as terpene resins. Plasticizers, sometimes called softeners, are typically fats and oils, including tallow, hydrogenated and partially hydrogenated vegetable oils, and cocoa butter. Commonly employed waxes include paraffin, microcrystalline, and natural waxes such as beeswax and carnauba. Microcrystalline waxes, especially those with a high degree of crystallinity, can be considered bodying agents or textural modifiers.

[0205] In certain embodiments, the insoluble gum base constitutes between about 5% to about 95% by weight of the gum. More preferably the insoluble gum base comprises between 10% and 50% by weight of the gum and most preferably about 20% to 35% by weight of the gum. The gum base typically also includes a filler component. The filler component can be calcium carbonate, magnesium carbonate, talc, dicalcium phosphate, or the like. The filler can constitute between about 5% and about 60% by weight of the gum base. Preferably the filler comprises about 5% to 50% by weight of the gum base.

[0206] Gum bases typically also contain softeners including glycerol monostearate and glycerol triacetate. Gum bases can also contain optional ingredients such as antioxidants, colors, and emulsifiers. The presently disclosed subject matter contemplates employing any commercially acceptable gum base.

[0207] The water-soluble portion of tire chewing gum can further comprise softeners, sweeteners, flavors, physiological cooling agents, and combinations thereof The sweeteners often fulfill the role of bulking agents in the gum. The bulking agents typically comprise about 5% to about 95% of the gum composition.

[0208] Softeners are added to the chewing gum in order to optimize die chewability and mouth feel of the gum. Softeners, also known in the art as plasticizers or plasticizing agents, generally constitute between about 0.5% to about 15% of the chewing gum. Softeners contemplated by the presently disclosed subject matter include glycerin, lecithin, and combinations thereof Further,aqueous sweetener solutions such as those containing sorbitol, hydrogenated starch hydrolysate, com syrup, and combinations thereof can be used as softeners and binding agents in gum.

[0209] As mentioned above, the D-tagatose prepared and / or generated by any of the microorganisms disclosed herein can be used in low-calorie gum formulations. However, formulations containing sugar are also within the scope of the invention. Sugar sweeteners generally include saccharide-containing components commonly known in the chewing gum art which comprise, but are not limited to, sucrose, dextrose, maltose, dextrin, dried invert sugar, fructose, galactose, corn syrup solids and the like, alone or in any combination. The D-tagatose prepared and / or generated by any of the microorganisms disclosed herein ean also be used in combination with sugarless sweeteners. Generally, sugarless sweeteners inc lude components with sweetening characteristics but which are devoid of the commonly known sugars and comprise, but are not limited to, sugar alcohols such as sorbitol, hydrogenated isomaltulose, mannitol, xylitol, lactitol, erythritol, hydrogenated starch hydrolysate, maltitol and the like alone or in any combination.

[0210] Depending on the particular sweetness release profile and shelf stability needed, coated or uncoated high-intensity sweeteners can be used in the chewing gum composition, or can be used in a coating applied to centers made from those gum compositions. High-intensity sweeteners, preferably aspartame, can be used at levels from about 0.01% to about 3.0%. Encapsulated aspartame is a high-intensity sweetener with improved stability and release characteristics, as compared to free aspartame. Free aspartame can also be added, and a combination of some free and encapsulated aspartame is preferred when aspartame is used. Other high-intensity sweeteners that can be used in the gum center are saccharin, Thaumatin, alitame, saccharin salts, sucralose, Stevia, and acesulfame K. Overall, the chewing gum composition will preferably comprise about 0.5% to about 90% sweetening agents. Most typically the sweetening agents will comprise at. least one bulk sweetener and at least one high-intensity sweetener. Optional ingredients such as colors, emulsifiers, and pharmaceutical agents can also be added as separate components of the chewing gum composition, or added as part of the gum base,

[0211] Aqueous syrups, such as corn syrup and hydrogenated corn syrup can be used, particularly if their moisture content is reduced. This can preferably be done by co-evaporating the aqueous syrup with a plasticizer, such as glycerin or propylene glycol, to a moisture content of less than10%. Preferred compositions include hydrogenated starch hydrolysate solids and glycerin. Such syrups and their methods of prepara tion are discussed in detail in U.S, Patent No, 4.671,967.

[0212] Methods of manufacturing chewing gum according to the presently disclosed subject matter include the sequential addition of the various chewing gum ingredients to any commercially available mixer known in the art. After the ingredients have been thoroughly mixed,the gum is discharged from the mixer and shaped into the desired form such as by rolling into sheets and cutting into sticks, extruding into chunks, or casting into pellets. Generally, the ingredients are mixed by first melting the gum base and adding it to the running mixer . The base can also be melted in the mixer itself. Color or emulsifiers can also be added at this time, along with syrup and a portion of the bulking agent. Farther portions of the bulking agent can then be added to the mixer. Flavor systems are typically added with the final portion of the bulking agent. If the flavor system is coated or otherwise modified, when incorporated into a delivery system to modify its release rate, it will preferably be added after the final portion of the bulking agent has been added. The entire mixing procedure typically takes from five to twenty minutes, but. longer mixing times can sometimes be required. Those skilled in the art will recognize that many variations of the above-described procedures can be followed.

[0213] If formed into pellets or balls, the chewing gum composition can be coated. The coating is initially present as a liquid syrup which contains from about 30% to about 80% or 85% sugars or sugar alcohols, and from about 15% or 20% to about 70% of a solvent such as water. In general, the coating process is carried out in conventional panning equipment. Gum center tablets to be coated are placed into the panning equipment to form a moving mass.

[0214] The material or syrup that will eventually form the coating is applied or distributed over the gum center tablets. The D-tagatose can be added before, during, and after applying the syrup to the gum centers. Once the coating has dried to form a hard surface, additional syrup additions can be made to produce a plurality of coatings or multiple layers of coating. The D-tagatose can be added to any or none of the coatings and / or layers.

[0215] In the panning procedure, syrup is added to the gum center tablets at a temperature range of from about 100°F to about 240°F. Preferably, the syrup temperature is from about UO^F to about 200"F. Most preferably, the syrup temperature should be kept constant throughout the process in order to prevent the polyol in the syrup from crystallizing, The syrup can be mixed with, sprayed upon, poured over, or added to the gum center tablets in any way known to those skilled in the art.

[0216] In certain embodiments, a soft coating is formed by adding a powder coating after a liquid coating. The powder coating can include natural carbohydrate gum 'hydrolysates, maltodextrin, gelatin, cellulose derivatives, starches, modified starches,, sugars, sugar alcohols, natural carbohydrate gums, and fillers like talc and calcium carbonate.

[0217] Each component of the coating on the gum center can be applied in a single layer or a plurality of layers. In general, a plurality of layers is obtained by applying single coats, allowing the layers to dry, and then repeating the process. The amount of solids added by each coating step depends chiefly on the concentration of the coating syrup. Any number of coats can be applied tothe gum center tablet Preferably, no more than about 75 coats are applied to the gum center. More preferably, less than about 60 coats are applied and most preferably, about 30 to about 60 coats are applied. In any event, the presently disclosed subject matter contemplates applying an amount of syrup sufficient to yield a coated chewing gum product containing about 10% to about 65% coating. Preferably, the final product will contain from about 20% to about 50% coating.

[0218] Those skilled in the art will recognize that in order to obtain a plurality of coated layers, a plurality of premeasured aliquots of coating syrup can be applied to the gum center. It is contemplated, however, that the volume of aliquots of syrup applied to the gum. center can vary throughout the coating procedure.

[0219] Once a coating of syrup is applied to the gum center, the syrup is dried in an inert medium . A preferred drying medium comprises air. Preferably, forced drying air contacts the wet syrup coating in a temperature range of from about 70°F to about 1 10°F. More preferably, the drying air is in the temperature range of from about 80aF to about 100%’. The invention also contemplates that the drying air possesses a relative humidity of less than about 15 percent. Preferably, the relative humidity of the drying air is less than about 8 %.

[0220] The drying air can be passed over and admixed with the syrup coated gum centers in any way commonly known in the art. Preferably, the drying air is blown over and around the syrup coated gum center at a flow rate, for large scale operations, of about 2800 cubic feet per minute. If lower quantities of material are being processed, or if smaller equipment is used, lower flow rates would be used. If a flavor is applied after a syrup coating has been dried, the presently disclosed subject matter contemplates drying the flavor with or without the use of a drying medium.

[0221] The amount of D-tagatose employed herein is normally a matter of preference subject to such factors as the type of final chewing gum composition, the individual flavor, the gum base employed, and the strength of flavor desired. Thus, the amount of D-tagatose can be varied in order to obtain the result desired in the final product and such variations are within the capabilities of those skilled in the art without the need for undue experimentation. In gum compositions, the D-tagatose prepared and / or generated by any of the microorganisms disclosed herein is generally present in amounts from about 0.02% to about 5%, and preferably from about. 0. 1 % to about 2%, and more preferably, from about O,8% to about 1 .8%, by weight of the chewing gum composition.5.2. confectionery

[0222] Another important aspect of the presently disclosed subject matter includes a confectionery composition incorporating the D-tagatose prepared and / or generated by any of the microorganisms disclosed herein and a method for preparing the confectionery' compositions. The preparation of confectionery formulations is well-known in the art. Confectionery items havebeen classified as either “hard” confectionery or “soft” confectionery. The D~tagatose prepared and / or generated by any of the microorganisms disclosed herein can be incorporated into the confections by admixing the compositions of the presently disclosed subject matter into the conventional hard and soft confections.

[0223] Hard confectionery can be processed and formulated by conventional means. In general, hard confectionery has a base composed of a mixture of sugar and other carbohydrate bulking agents kept in an amorphous or glassy condition. The hard confectionery can also be sugarless. The hard confectionery can also be low-calorie. This form is considered a solid syrup of sugars generally having from about 0,5% to about 1 ,5% moisture. Such materials normally contain up to about 92% sugar, up to about 55% corn syrup, and from about. 0. 1 % to about 5% water, by weight of the final composition. The syrup component is generally prepared from sucrose and corn syrups but can include other materials. In certain embodiments, the syrup component includes the D- tagatose prepared and / or generated by any of the microorganisms disclosed herein. Further ingredients such as flavorings, sweetening agents, acidulants, colorants, and so forth can also be added.

[0224] Such confectionery can be routinely prepared by conventional methods, including but not limited to methods involving fire cookers, vacuum cookers, and scraped-surface cookers also referred to as high-speed atmospheric cookers. The apparatus useful in accordance with the presently disclosed subject matter comprises cooking and mixing apparatus well known in the confectionery manufacturing arts, and therefore the selection of the specific apparatus will be apparent to the artisan.

[0225] Fire cookers involve the traditional method of making a candy base. In this method, the desired quantity of carbohydrate bulking agent is dissolved in water by heating the agent in a kettle until the bulking agent dissolves. Additional bulking agents can then be added and cooked until a final temperature of 145’'' C to 156° C is achieved. The batch is then cooled and worked as a plastic-like mass to incorporate additives such as flavoring agents, colorants, and the like.

[0226] A high-speed atmospheric cooker uses a heat-exchanger surface, which involves spreading a film of candy on a heat exchange surface, the candy is heated to 165° C to 170° C within a few seconds. The candy is then rapidly cooled to 100° C to 120c' C and worked as a plastic-like mass enabling incorporation of the additives, such as flavoring agents, colorants, and the like. In vacuum cookers, the carbohydrate bulking agent is boiled to 125° C to 132° C, vacuum is applied, and additional water is boiled off without extra heating. When cooking is complete, the mass is a semi-solid and has a plastic-like consistency. At this point, flavoring agents, colorants, and other additives are admixed in the mass by routine mechanical mixing operations.

[0227] The optimum mixing required to uniformly mix the flavoring agent, colorants, and other additives during conventional manufacturing of hard confectionery is determined by the time needed to obtain a uniform distribution of the materials. Generally , mixing times of from 2 to 10 minutes have been found to be acceptable.

[0228] Once the candy mass has been properly tempered, it can be cut into workable portions or formed into desired shapes. A variety of forming techniques can be utilized depending upon the shape and size of the final product desired A general discussion of the composition and preparation of hard confections can be found mH. A. Lieberman, 'Pharmaceutical Dosage Forms: Tablets, Volume I (1989), Marcel Dekker, Inc., New York, N.Y. at pages 419 to 582, which disclosure is incorporated herein by reference.

[0229] Compressed tablet confections contain particular materials and are formed into structures under pressure. These confections generally contain sugars in amounts up to about 95%, by weight of the composition, and typical tablet excipients such as binders and lubricants as well as flavoring agents, colorants, and so forth. These confections can also be sugarless.

[0230] Simi lar to hard confectionery, soft confectionery can be utilized in the embodiments of the disclosed subject matter. The preparation of soft confections, such as nougat, involves conventional methods, such as the combination of two primary components, namely (1) a high boiling syrup such as com syrup, or the like, and (2) a relatively light textured frappe, generally prepared from egg albumin, gum arable, gelatin, vegetable proteins, such as soy-derived compounds, sugarless milk-derived compounds such as milk proteins, and mixtures thereof. The frappe is generally relatively light, and can, for example, range in density from about 0,5 to about 0.7 grams / cc.

[0231] The high boiling syrup, or “bob syrup” of the soft confectionery, is relatively viscous, has a higher density than the frappe component, and frequently contains a substantial amount of carbohydrate bulking agent. Conventionally, the final nougat composition is prepared by the addition of the “bob syrup” to the frappe under agitation, to form the basic nougat mixture. Further ingredients such as flavoring, additional carbohydrate bulking agents, colorants, preservatives, medicaments, mixtures thereof and the like can be added thereafter also under agitation. Soft confectioneries can also be prepared sugarless, A general discussion of the composition and preparation of nougat confections can be found in B. W. Minifie, Chocolate, Cocoa and Confectionery: Science and Technology, 2nd edition, AVI Publishing Co., Inc., Westport, Conn. (1983), at pages 576-580, which, disclosure is incorporated herein by reference.

[0232] In general, the frappe component is prepared first and thereafter the syrup component is slowly added under agitation at a temperature of at least about 65° C, and preferably at least about 100°C. The mixture of components is continued to be mixed to form a uniform mixture, afterwhich the mixture is cooled to a temperature below 80° C, at which point, the flavor can be added. The mixture is further mixed for an additional period until it is ready to be removed and formed into suitabl e confectionery shapes,

[0233] In accordance with the present disclosure, amounts of the D-tagatose prepared and / or generated by any of the microorganisms disclosed herein can be admixed into the hard and soft confections. The exact amount of D-tagatose employed is normally a matter of preference subject to such factors as the particular type of confection being prepared, the type of bulking agent or carrier employed, the type of flavor employed, and the intensity of breath freshening perception desired. Thus, the amount of D-tagatose can be varied in order to obtain the result desired in the final product and such variations are within the capabilities of those skilled in the art withou t the need for undue experimentation. In general, the amount of D-tagatose normally present in a hard or soft confection will be from about 0,001% to about 20%, preferably from about 0.01 % to about 15%, more preferably from about 0.01% to about 10%, and more preferably from about 0.01 % to about 5%, and more preferably 0.01 % to about 0,5% by weight of the confection.

[0234] The presently disclosed subject matter extends to methods for making the improved confections. The D-tagatose prepared and / or generated by any of the microorganisms disclosed herein can be incorporated into an otherwise conventional hard or soft confection composition using standard techniques and equipment known to those skilled in the art. The apparatus useful in accordance with the presently disclosed subject matter comprises mixing and heating apparatus well known in the confectionery manufacturing arts, and therefore the selection of the specific apparatus will be apparent to the artisan.

[0235] In such a method, a composition is made by admixing the D-tagatose into the confectionery composi tion along with the other ingredients of the final desired composi tion. Other ingredients will usually be incorporated into the composition as dictated by the nature of the desired composition as well known by those having ordinary skill in the art. The ultimate confectionery compositions are readily prepared using methods generally known in the food technology and pharmaceutical arts. Thereafter the confectionery mixture can be formed into desirable confectionery shapes.

[0236] The D-tagatose prepared and / or generated by any of the microorganisms disclosed herein can be formulated with conventional ingredients that offer a variety of text ures to suit particular applications. Such ingredients can be in the form of hard and soft confections, tablets, toffee, nougat, chewy candy, chewing gum and so forth, center filled candies, both sugar and sugariess. The acceptable ingredients can be selected from a wide range of materials. Without being limited thereto, such materials include diluents, binders and adhesives, lubricants, disintegrants, bulkingagents, humectants, buffers, and adsorbents. The preparation of such confections and chewing gum products is well known, 5, 3, Oocofe / ey and Fdlings

[0237] The presently disclosed subject mater is also used with and / or in chocolate products, chocolate-flavored confections, and chocolate flavored compositions. Chocolates also include those containing crumb solids or solids fully or partially made by a crumb process. Various chocolates are disclosed, for example, in U.S. Patent Nos. 7,968,140 and 8,263,168, the disclosures of which are incorporated herein by reference in their entireties, A general discussion of the composition and preparation of chocolate confections can be found in B. W. Minifie, Chocolate, Cocoa and Confectionery: Science and Technology. 2nd edition, AVI PLiblishing Co., Inc,, Westport, Conn. (1982), which disclosure is incorporated herein by reference,

[0238] The term “chocolate” as used herein refers to a solid or semi-plastic food and is intended to refer to all chocolate or chocolate-like compositions containing a fat-based component phase or fat-like composition. The term is intended to include standardized or nonstandardized compositions conforming to the U.S. Standards of identity (SOI), CODEX Alimentarius and / or other international standards and compositions not conforming to the U.S. Standards of Identity or other international standards. The term includes dark chocolate, baking chocolate, sweet chocolate, bittersweet or semisweet chocolate, milk chocolate, buttermilk chocolate, skim milk chocolate, mixed dairy product chocolate, white chocolate, sweet cocoa and vegetable fat coating, sweet chocolate and vegetable fat coating, milk chocolate and vegetable fat coating, vegetable fat based coating, pastels including white chocolate or coating made with cocoa butter or vegetable fat or a combination of these, nutritionally modified chocolate-like compositions (chocolates or coatings made with reduced calorie ingredients) and low fat chocolates, aerated chocolates, compound coatings, non-standardized chocolates and chocolate- like compositions, unless specifical ly i dent ified otherwi se ,

[0239] Nonstandardized chocolates result when, for example, the nutritive carbohydrate sweetener is replaced partially or completely; or when the cocoa butter, cocoa butter alternative, cocoa butter equivalent, cocoa butter extender, cocoa butter replacer, cocoa butter substitute or milkfat are replaced partially or completely; or when components that have flavors that imitate milk, butter or chocolate are added or other additions or deletions in formula are made outside the FDA standards of identify of chocolate or combinations thereof. Chocolate-like compositions are those fat-based compositions that can be used as substitutes for chocolate in applications such as panning, molding, or enrobing; for example, carob.

[0240] In the United States, chocolate is subject to a standard of identity established by the U. S, Food and Drug Administration (FDA) under the Federal Food, Drug and Cosmetic Act.Definitions and standards for the various types of chocolate are well established in the U.S. Nonstandardized chocolates are those chocolates which have compositions that fall outside the specified ranges of the standardized chocolates .

[0241] In certain embodiments., the chocolate can contain D-tagatose prepared and / or generated by any of the microorganisms disclosed herein. Additionally, the chocolate can contain a sugar syrup / solids. invert sugar, hydrolyzed lactose, maple sugar, brown sugar, molasses, honey, sugar substitute and the like. Nutritive carbohydrate sweeteners with varying degrees of sweetness intensity can be any of those typically used in the art and include, but are not limited to, sucrose, e.g., from cane or beet, dextrose, fructose, lactose, maltose, glucose syrup solids, corn syrup solids, invert sugar, hydrolyzed lactose, honey, maple sugar, brown sugar, molasses and the like. Sugar substitutes can partially replace the nutritive carbohydrate sweetener. High potency sweeteners include aspartame, cyclamates, saccharin, acesulfame-K, neohesperidin dihydrochalcone. sucralose, alitame, stevia sweeteners, glycyrrhizin, thauniatin and the like and mixtures thereof. The preferred high potency sweeteners are aspartame, cyclamates, saccharin, and acesulfame-K, Examples of sugar alcohols can be any of those typically used in the art and include sorbitol, mannitol, xylitol, maltitol, isomalt, lactitol and the like,

[0242] The chocolates can also contain bulking agents. The term “bulking agents” as defined herein can be any of those typically used in the art and include polydextrose, cellulose and its derivatives, maltodextrin, gum arable, and the like.

[0243] The chocolate products can contain emulsifiers. Examples of safe and suitable emulsifiers can be any of those typically used in the art and include lecithin deri ved from vegetable sources such as soybean, safflower, com, etc., fractionated lecithins enriched in either phosphatidyl choline or phosphatidyl ethanolamine, or both, mono- and digylcerides, diacetyl tartaric acid esters of mono- and diglycerides (also referred to as DATEM), monosodium phosphate derivatives of mono- and diglycerides of edible fats or oils, sorbhan monostearate, hydroxylated lecithin, lactylaied fatty acid esters of glycerol and propylene glycol, polyglycerol esters of fatty acids, propylene glycol mono- and di-esters of fats and fatty acids, or emulsifiers that can become approved for the US FDA-defined soft candy category. In addition, other emulsifiers that can be used include polyglycerol polyricinoleate (PGPR), ammonium salts of phosphatidic acid, (e.g., YN) sucrose esters., oat extract, etc., any emulsifier found to be suitable in chocolate, or similar fat / solid system or any blend,

[0244] The term “chocolate-flavored confection” refers to food products, excluding “chocolate’’, having a chocolate fiavor / aroma and comprising a cocoa fraction. These products are stable at ambient temperatures for extended periods of time (e,g., greater than 1 week) and are characterizedas microbiologically shelf-stable at I8~30':C under .normal atmospheric conditions. Examples include chocolate-flavored hard candies, chewables, chewing gums, etc.

[0245] The term “chocolate- flavored compositions” refers to chocolate-flavored compositions, excluding “chocolate”, containing a cocoa fraction and having a chocolate flavor / aroma. Examples include chocolate-flavored cake mixes, ice creams, syrups, baking goods, etc. The term includes chocolate-flavored compositions (e.g., cakes, nougats, puddings, etc,), as well as compositions not having a chocolate flavor (e.g., caramels, etc,).

[0246] In certain embodiments, the D-tagatnse. prepared and / or generated by any of the microorganisms disclosed herein is incorporated into savory goods. In certain embodiments, a savory good is a food product that has savory flavors including, for example, but not limited to, spicy flavor, pepper flavor, dairy flavor, vegetable flavor, tomato flavor, dill flavor, meat flavor, poultry flavor, chicken flavor and reaction flavors that are added or generated during heating of a food product.

[0247] In certain embodiments, the D-tagatose prepared and / or generated by any of the microorganisms disclosed herein is incorporated into a wet soup category food product, which comprises wet / liquid soups regardless of concentration or container, including frozen soups. In certain embodiments, the soup food product .means a food prepared from meat, poultry, fish, vegetables, grains, fruit, and / or other ingredients, cooked in a liquid which may include visible pieces of some or all of these ingredients. It may be clear (as a broth) or thick (as a chowder), smooth., pureed or chunky, ready-to-serve, semi-condensed or condensed and may be served hot or cold, as a first course or as the main course of a meal or as a between meal snack (sipped like a beverage). Soup may be used as an ingredient for preparing other meal components and may range flora broths (consomme) to sauces (cream or cheese-based soups).

[0248] In certain embodiments, the D-tagatose prepared and / or generated by any of the microorganisms disclosed herein is incorporated into a dehydrated and culinary food category of food products, which comprises (i) cooking aid products such as: powders, granules, pastes, concentrated liquid products, including concentrated bouillon, bouillon and bouillon like products in pressed cubes, tablets or powder or granulated form, which are sold separately as a finished product or as an ingredient within a product, sauces and recipe mixes (regardless of technology); (ii) meal solutions products such as: dehydrated and freeze dried soups, including dehydrated soup mixes, dehydrated instant soups, dehydrated ready-to-cook soups, dehydrated or ambient preparations of ready-made dishes, meals and single serve entrees including pasta, potato and rice dishes; and (ill) meal embellishment products such as; condiments, marinades, salad dressings, salad toppings, dips, breading, batter mixes, shelf stable spreads, barbecue sauces, liquid recipemixes, concentrates, sauces or sauce mixes., including recipe mixes for salad, sold as a finished produc t or as an ingredient within a product, whether dehydrated, liquid or frozen.

[0249] In certain embodiments, the D-tagatose prepared and / or generated by any of the microorganisms disclosed herein is incorporated into a meat food product. In certain embodiments, meat food products Include food products made by processing the edible remains of any dead animal. Including birds, fish, crustaceans, shellfish, and mammals. Meat food products include, without limitation, for example, prepared beef, lamb, pork, poultry, or seafood products. Examples of such meat food products include, for example, bologna, frankfurters, sausage, luncheon, deli slices, loaves, bacon, meatballs, fish sticks, chicken fingers, and ground meats, e.g.. meatloaf meatballs, and hamburgers. A meat food product may be combined with a simulated meat food product. Simulated meat food products include, without limitation, for example, a meat alternative, meat analog, soy burger, soy bologna, soy frankfurter, soy sausage, soy luncheon loaves, soy bacon, and soy meatbail. A simulated meat food product may be combined with a meat food product.

[0250] In certain embodiments, the D-tagatose prepared and / or generated by any of the microorganisms disclosed herein is incorporated into a snack food category food product. In certain embodiments, snack food products include any food that can be a light informal meal including, but not limited to sweet and savory snacks and snack bars. Examples of snack food include, but are not limited to fruit snacks, ehips / crisps, extruded snacks, iortilh / corn chips, popcorn, pretzels, nuts, and other sweet and savory snacks. Examples of snack bars include, but are not limited to granola / muesli bars, breakfast bars, energy bars, fruit bars, and other snack bars.

[0251] In certain embodiments, the D-tagatose prepared and / or generated by any of the microorganisms disclosed herein is incorporated into frozen food products, which cornprises chilled or frozen food products, for example, but not limited to, ice cream, impulse ice cream, single portion dairy ice cream, single portion water ice cream, multi-pack dairy ice cream, multipack water ice cream, take-home ice cream, take-home dairy ice cream, ice cream desserts, bulk ice cream, take-home water ice cream, frozen yogurt, artisanal ice cream, frozen ready meals, frozen pizza, chilled pizza, frozen soup, frozen pasta, frozen processed red meat, frozen processed poultry, frozen processed fish / seafood, frozen processed vegetables, frozen meat substitutes, frozen potatoes, frozen bakery products and frozen desserts.

[0252] The D-tagatose prepared and / or generated by any of the microorganisms disclosed herein can also be in the form of a pharmaceutical . One non-limi ting example of a pharmaceutical form is a suspension. Pharmaceutical suspensions can be prepared by conventional compounding methods. Suspensions can contain adjunct materials employed in formulating the suspensions ofthe art. The suspensions of the presently disclosed subject matter can comprise preservatives, buffers, suspending agents, antifoaming agents, sweetening agents, flavoring agents, coloring or decoloring agents, solubilizers, and combinations thereof.

[0253] Flavoring agents such as those flavors well known to the skilled artisan, such as natural and artificial flavors and mints, such as peppermint, menthol, citrus flavors such as orange and lemon, artificial vanilla, cinnamon, and various fruit flavors, both individual and mixed and the like can be utilized, in amounts from about 0,01% to about 5%, and more preferably 0.01% to about 0.5% by weight of the suspension.

[0254] The pharmaceutical suspensions of the presently disclosed subject matter can be prepared as follows: (i) admix the thickener with water heated from about 40f>C to about 95° C. preferably from about 40° C to about 70° C, to form a dispersion if the thickener is not water-soluble or a solution if the thickener is water solub le; (ii) admix the D-lagatose prepared and / or generated by any of the microorganisms disclosed herein with water to form a solution; (iii) admix, if desired, a flavoring agent with the thickener-water admixture to form a uniform thickener-flavoring agent;(iv) combine the sweetener solution with the thickener-flavoring agent and mix until uniform; and(v) admix the optional adjunct materials such as coloring agents, flavoring agents, decolorants, solubil izers, ant i-foaming agen ts, buffers and additional water w ith the mixtur e of step (i v) to form the suspension,

[0255] TheD-tagatose prepared and / or generated by any of the microorganisms disclosed herein can also be in chewable form. To achieve acceptable stability and quality as well as good taste and mouth feel in a chewable formulation several considerations are important. These considerations include the amount of active substance per tablet, the flavoring agent employed, the degree of compressibility of the tablet, and additional properties of the composition. Chewable pharmaceutical candy is prepared by procedures similar to those used to make soft confectionery', A general discussion of the lozenge and chewable tablet forms of confectionery can be found in H. A. Lieberman and L, Lachman, Pharmaceutical Dosage Forms: Tablets Volume I, Marcel Dekker, InC, New York, N.Y. ( 1989) at pages 367 to 418, which disclosure is incorporated herein by reference. In a typical procedure, a boiled sugar-corn syrup blend is formed to which is added a frappe mixture, The boiled sugar-corn syrup blend can be prepared from sugar and corn syrup blended in parts by weight ratio of about 90; 10 to about 10’90. The sugar-corn syrup blend is heated to temperatures above about 120° C to remove water and to form a molten mass. The frappe is generally prepared from gelatin, egg albumin,, milk proteins such as casein, and vegetable proteins such as soy protein, and the like, which are added to a gelatin solution and rapidly mixed at ambient temperature to form an aerated sponge-like mass. The frappe is then added to the molten candy mass and mixed until homogeneous at temperatures between about 65° C and about120° C, The D-tagatose prepared and / or generated by any of the microorganisms disclosed her ein can then be added to the homogeneous mixture as the temperature is lowered to about 65° C>95° C whereupon additional ingredients can then be added such as flavoring agents and coloring agents. The formulation is further cooled and formed into pieces of desired dimensions.

[0256] In other pharmaceutical embodiments, the flavoring agent is incorporated into an ingestible topical vehicle which can be in the form of a mouthwash, rinse, ingestible spray, suspension, dental gel, and the like. Typical non-toxic ingestible vehicles known in the pharmaceutical arts can be used in the presently disclosed subject matter. The preferred ingestible vehicles are water, ethanol, and water-ethanol mixtures. The water-ethanol mixtures are generally employed in a weight ratio from about 1 : 1 to about 20:1, preferably from about 3: 1 to about 20: 1 , and most preferably from about 3:1 to about 10: 1, respectively. The pH value of the ingestible vehicle is generally from about 4 to about 7, and preferably from about 5 to about 6.5. An ingestible topical vehicle having a pH value below about 4 is generally irritating to the ingestible cavity and an ingestible vehicle having a pH value greater than about 7 generally results in an unpleasant mouth feel.

[0257] The ingestible topical flavoring agents can also contain conventional additives normally employed in those products. Conventional additives include a fluorine -providing compound, a sweetening agent, a flavoring agent, a coloring agent, a humectant, a buffer, and an emulsifier, providing the addi tives do not in terfere wi th the flavoring properties of the composition. The coloring agents and humectants, and the amounts of these additives to be employed, set out above, can be used in the ingestible topical composition. The flavoring agents (flavors, flavorants) that can be used include those flavors known to the skilled artisan, such as natural and artificial flavors. Suitable flavoring agents include mints, such as peppermint, citrus flavors such as orange and lemon, artificial vanilla, cinnamon, various fruit flavors, both individual and mixed, and the like. The amount of flavoring agent employed in the ingestible topical composition is normally a matter of preference subject to such factors as the type of final ingestible composition, the individual flavor employed, and the strength of flavor desired. Thus, the amount of flavoring can be varied in order to obtain the result desired in the final product and such variations are within the capabilities of those skilled in the art without the need for undue experimentation. The flavoring agents, when used, are generally utilized in amounts that can, for example, range in amounts from about 0.05% to about 6%, by weight of the ingestible topical composition.5.5. Pel Food Prodticis

[0258] The D-tagatose prepared and / or generated by any of the microorganisms disclosed herein can be used in a wide variety of pet food products.

[0259] As used herein, the terms “pet food" or “pet food product” refer to a product or composition that is intended for consumption by a companion animal, such as cats. dogs, guinea pigs, rabbits, birds and horses. For example, but not by way of limitation, the companion anima! can be a “domestic” dog, e.g. , Can is htpusJamiJiaris. A “pet food” or “pet food product” includes any food, feed, snack, food, supplement, liquid, beverage, treat, toy (chewable and / or consumable toys), meal substitute or meal replacement.

[0260] In certain embodiments, the D-tagatose prepared and / or generated by any of the microorganisms disclosed herein is directly added to a pet food product. In certain embodiments, the D-tagatose prepared and / or generated by any of the microorganisms disclosed herein can be added prior to, during or after formulation processing or packaging of the pet. food product.

[0261] Non-limiting examples of suitable pet food products include wet food products, dry food products, moist food products, pet food supplements (e.g., vitamins ), pet beverage products, snack and treats and pet food categories described herein.

[0262] In certain embodiments, the pet food product is a dry food product. A dry or low moisturecontaining nutritionally-complete pet food product can comprise less than about 15% moisture. In certain embodiments, the pet food product is a wet food product. A wet or high .moisturecontaining nutritionally-complete pet food product can comprise greater than about 50% moisture. In certain embodiments, the pet food product is a nutritionally complete moist food product, A moist, e.g... semi-moist or semi-dry or soft dry' or soft moist or intermediate or medium moisture containing nutritioually-complete pet food product comprises from about 15% to about 50% moisture.

[0263] hi certain embodiments, the pet food product is a pet food snack product. Non-limiting examples of pet food snack products include snack bars, pet chews, crunchy treats, cereal bars, snacks, biscuits and sweet products.

[0264] In certain embodiments, the D-tagatose can be incorporated into a delivery system for use in edible compositions. In certain embodiments, the composition will comprise another flavor or taste modifier such as a salty, umami, bitter, astringent and / or savory tastant. Delivery systems can be liquid or solid, aqueous or non-aqueous. Delivery systems are generally adapted to suit the needs of the flavor composition and / or the edible composition into which the D-tagatose will be incorporated.

[0265] The D-tagatose can be employed in liquid form, dried form, and / or solid form. When used in dried form, suitable drying means such as spray drying can be used. Alternatively, D-tagatose can be encapsulated or absorbed onto water soluble materials, including but not limited to materials such as cellulose, starch, sugar, maltodextrin, gum arabic and so forth. The actualtechniques for preparing such dried forms are well-known in the art, and can be applied to the presently disclosed subject matter.

[0266] The D-tagatose can be used in many distinct physical forms well known in the art to provide an initial burst of taste,, flavor and / or texture; and / or a prolonged sensation of taste, flavor and / or texture. Without being limited thereto, such physical forms include free forms, such as spray dried, powdered, and beaded forms, and encapsulated forms, and mixtures thereof.

[0267] In certain embodiments, D-tagatose is encapsulated. Encapsulating materials and / or techniques can be selected to improve the stability of the D-tagatose and / or food product. In certain embodiments, the encapsulating materials and / or techniques are selected to modify the release profile of D-tagatose.

[0268] Suitable encapsulating materials can include, but are not limited to, hydrocolloids such as alginates, pectins, agars, guar gums, celluloses, and the like, proteins, polyvinyl acetate, polyethylene, crosslinked polyvinyl pyrrolidone, polymethylmethacrylate, polylactidacid, pcdyhydroxyalkanoates, eihylcelluiose, polyvinyl acetatephthalate, polyethylene glycol esters, methacrylicacid-co-methyl-methacrylate, ethylene- vinylacetate (EVA) copolymer, and the like, and combinations thereof. Suitable encapsulating techniques can include, but are not limited to, spray coating, spray drying, spray chilling, absorption, adsorption, inclusion complexing (e.g.. creating a flavor / cyclodextrin complex), coacervation, fluidized bed coating, or other process can be used to encapsulate an ingredient with an encapsulating material.

[0269] Encapsulated delivery systems for flavoring agents or sweetening agents (e.g... D-tagatose) contain a hydrophobic matrix of fat or wax surrounding a sweetening agent or flavoring agent core. The fats can be selected from any number of conventional materials such as fatty acids, glycerides or poly glycerol esters, sorbitol esters, and mixtures thereof. Examples of fatty acids include but are not limited to hydrogenated and partial ly hydrogenated vegetable oils such as palm oil, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, soybean oil, cottonseed oil, sunflower oil, safflower oil, and mixtures thereof. Examples of glycerides include but are not limited to monoglycerides, di glycerides, and triglycerides,[027(1] Waxes useful can be chosen from the group consisting of natural and synthetic waxes, and mixtures thereof. Non-limiting examples include paraffin wax, petrolatum, carbowax, microcrysialline wax, beeswax, carnauba wax, candelilla wax, lanolin, bayberry wax, sugarcane wax, spermaceti wax, rice bran wax, and mixtures thereof

[0271] The fats and waxes can be use individually or in combination in amounts vafying from about 10 to about 70%, and alternatively in amounts from about 30 to about 60%, by weight of the encapsulated system. When used in combination, the fat and wax are preferably present in a ratio from about 70: 10 to 85:15, respectively.

[0272] Typical encapsulated compositions, flavoring agent or sweetening agent delivery systems are disclosed in U.S. Patent Nos. 4,597,970 and 4,722.345, the disclosures of which are incorporated herein by reference in their entireties.

[0273] Liquid delivery systems can include, but are not limited to, systems with a dispersion of D-tagatose, such as in carbohydrate syrups and / or emulsions. Liquid delivery systems can also include extracts where D-tagatose is solubilized in a solvent. Solid delivery systems can be created by spray drying, spray coating, spray chilling, fluidized bed drying, absorption, adsorption, coacervation, complexation, or any other standard technique. In some embodiments, the delivery system can be selected to be compatible with or to function in the edible composition. In some embodiments, the delivery system will include an oleaginous material such as a fat or oil. In some embodiments, the delivery system will include a confectionery fat such as cocoa butter, a cocoa butter replacer, a cocoa butter substitute, or a cocoa butter equivalent.

[0274] When used in dried form, suitable drying means such as spray drying may be used. Alternatively, D-tagatose may be adsorbed or absorbed onto substrates such as water soluble materials, such as cellulose, starch, sugar, maltodextrin, gum arable and so forth or may be encapsulated. The actual techniques for preparing such dried forms are well known in the art.EXAMPLES

[0275] The presently disclosed subject matter can be better understood by reference to the following. The below examples are exemplary only and should in no way be taken as limiting.Example ynfhesis pf D-Eagatose from Gfaense

[0276] E coll is naturally capable of producing trace amounts of D-tagatose. In the present example, D-tagatose production is improved by overexpressing key genes, removing competing pathway genes, and optimizing the production conditions.

[0277] Initially , it was tested if E. call possesses enzymes capable of producing tagatose. Cultures were grown on M9P media (M9 minimum media with 5 g / L yeast extract) with 10 g / L gl ucose at 30SC, w ith samples of the supernat ant taken at 24 hours after inoculation, and then analyzed using HPLC. Gene knockouts (KOs) were constructed in AL360I (Table 1). Double KO strain, AL4240, with A / ? / M and Azw / produced tagatose (Figure 2). The gene zwy encodes for the enzyme glucose~6-phosphate dehydrogenase (Zwf) (EC 1,1.1.363), which converts G6P to 6- phospho-D-glucono-I ,5-lactone as the first committed step in the pentose phosphate pathway (PPP). pJkA encodes for phosphofructokinase A (EC 2.7. LI 1), which converts F6P to fructose- 1,6-bisphosphate (F16BP) as part of the fust committed step of glycolysis. The double KO strain containing ApJkA and Azuy" (AL4240) produced 0.4 g / L of tagatose, indicating that E. colipossesses enzymes capable of producing D-tagatose ( Figure 2). Further deletion of competing pathways resulted in increased tagatose production.

[0278] Table 1. Stein listIMteminmg r agatose pmductMn enzvmex

[0279] Next, it was hypothesized that fructose-6-phosphate (F6P) is an intermediate of D- tagatose in E. coll. The production pathway starts with the native assimilation of glucose into £. cvfi via the phosphotransferase system (PTS) or GalP / GIk, which converts glucose to ghicose~6~ phosphate (G6P). G6P is then isomerized to F6P via glucose-6-phospTiate isomerase (Gpi) (EC 5,3.1 .9). F6P could be converted to tagatose-6-phosphate by an epimerase. A phosphatase could dephosphorylate tagatosefo-phosphate to free tagatose, after which it would be excreted from the cell, A tagatose production system using phosphorylation and dephosphorylation steps as system driving forces should be more efficient than the pathway currently used in industrial production of tagatose.

[0280] By literature search and genome mining, one epimerase and seven phosphatase candidates were identified. Tagatose- 1 ,6-bisohosphate aldolase subunit (GatZ) has shown activity towards D-tagatose. Seven phosphatases (HxpA, HxpB. YbiV, YidA, YigL, YihX and YqaB) have shown promiscuity towards a variety of hexoses.

[0281] The gatZ gene was deleted in AL4330 (ApffiA Azwf AmapA AafcE Apgtn), creating AL4386, AL43S6 did not produce detectable amounts of D-tagatose (Figure 3), while the expression of gatZ from an expression plasmid in AL4386 recovered D-tagatose production (Figure 3), The results indicate that GatZ is responsible for the conversion of F6P to tagatose-6- phosphate.

[0282] Seven E. colt phosphatases (HxpA, HxpB. YbiV?YidA, YigL, YihX, YqaB), which have promiscuous activity towards various hexoses, were tested for D-tagatose production in AL4330. Seven plasmids, pAL2490, pAL249l, pAL292;.pAL2493, pAL2494, pAL2495, pAL2496;were constructed to additionally express kxpA, fepB. y&E, >M4, jvgZ., yihX oralong with ga / Z under ail inducible promoter, Ppapp (Table 2). Cells were cultured in M9P media with 10 g / L glucose at 30 *C and induced with 1 mM IPTG. The strain with pAL2490 (hxpA) produced most D-tagatose (Figure 4),

[0283] 'fable 2. Plasmid list

[0284] Table 3Example 2 - dteflvaflng the b-tasatffse tmuitictifm caiMtdtv af Eseftericfew eelt

[0285] Growing awareness regarding the health implication of eating habits has spurred a significant demand for healthier food options (Alsubhi et al., 2023). In particular, there has been increased scrutiny regarding the role of dietary sugars in noncotnmunicable diseases (Blither,2019; Lustig etal., 2012; Prinz. 2019 ). In response to this public health problem, the food industry is adopting artificial sweeteners as a means to lower sugar caloric content without compromising consumer satisfaction, evident in the projected growth of the sugar substitute market to reach 20.6 billion USD by 2025 ('“Sugar Substitutes Market Size and Forecast” 2017).

[0286] The current sweetener market is predominantly comprised of non-sugar substitutes, despite recent guidelines released by the World Health Organization advising against using nonsugar sweeteners for weight loss or preventing non-communicable diseases (Rizas et al., 2023). Rare sugars, characterized by their slight chemical structure variations compared to common sugars like glucose, are increasingly sought after as alternative sweeteners (Ahmed et al., 2022). D~Tagatose, a rare sugar and C4 epimer of fructose, has received Generally Recognized as Safe status and is 92% as sweet as sucrose (Xu et al., 2014). D-Tagatose is marketed as a low-caloric sweetener, although there remains a debate regarding its calorific value, which ranges from 1.5 kcal / g to 3 kcal / g (Ahmed et al, 2022). D-Tagatose has high solubility (58% (wAv) at 2 PC] and lower viscosity than sucrose which makes it an ideal sugar substitute in beverages (Oh, 2007), Moreover, its favorable flavor profile and desirable browning effects further enhance its suitability for use as a sugar substitute (Oh, 2007), D-Tagatose is reported to aid in weight loss and exhibits additional beneficial effects such as anti-plaque, non-cariogenic, anti-halitosis, pre-biotic and anti- biofilm properties (Oh, 2007).

[0287] D-Tagatose is found in minute concentration in sterilized powdered milk, hot cocoa, cheese, yogurts, and other dairy products (Roy et al., 2018). However, the extraction of D-tagatose from natural sources is not economically feasible due to complex purification steps and low yield (Oh, 2007), Synthetic methods for D-tagatose production also suffer from similar disadvantages along with the additional use of acids, bases, and catalysts (Roy et al., 2018). A potential solution to this problem is the 'Izumormg' strategy which makes use of enzy mes and serves as a framework for the interconversion of aldohexoses , ketohexoses* and hexitols (Granstrdm et al., 2004). In this regard, two such enzymes have been extensively investigated (Figure 5A): 1) L-arabinose isomerase (EC 5,3. 1,4), which interconverts D-galactose and D-tagatose, and 2) galactitol 2- dehydrogenase (EC 1.1.1.16), which interconverts galactitol and D-Tagatose. However, these methods encounter challenges such as the requirement for purified enzymes, co- factors like NAD*, low thermal stability or a lack of thermodynamic driving force, resulting in low yields and high cost of production (Dai and Jin, 2024; Noor et al., 2012). To address these challenges, recent research encapsulated L-arabinose isomerase in Lactobacillus plantarum, achieving high yields of D-tagatose. However, this method relied on high-cost D-galactose as the starting material, which is needed to be separated from D-tagatose post-production, further elevating costs (Baumgartner et al., 2015). Alternatively, microbial production was applied to convert lactose to D-tagatose inSaccharomyces cerevisiae, but this method utilizes only D-galactose from lactose and D-glucose is used to maintain ceil growth, thus the yield was low (Liu et al,, 2019). Consequently, the final media contained galactose, galaciitol, and D-tagatose. resulting in higher separation and purification costs (Liu et al,, 2019).

[0288] While D-fructose holds potential as a starting material for D-tagatose production, a notable gap exists in nature, the absence of a C4 epimerase capable of directly converting D-fructose to D-tagatose (Jeon et al., 2023; Lee et al., 2017b; Shin etai., 2020). A C4-epimerizati on activity of the tagatrronate-fmcturonate epimerase from Thermotoga petrophila was identified (Shin et al,, 2020). Recently, a phosphorylation and dephosphorylation strategy has surfaced as a promising route for rare sugar production from glucose, presenting an economically advantageous feedstock (Dai et al., 2022; Taylor et al., 2023 ). This pathway involves a pivotal C4 epimerization reaction, transforming D-fiuctose-6-phosphate (F6P) into D-iagatose-6-phosphate (T6P). Subsequent dephosphorylation of T6P provides substantial thermodynamic driving force, facilitating efficient D-tagatose production. Utilizing this pathway, whole-cell biocatalysts have been developed, yielding 3.38 g / L of D-tagatose from 10 g / L of maltodextrin (Dai et al., 2022). However, glucose and fructose persist in the solution after production due to inefficient conversion (Dai et al,, 2022),

[0289] in the present disclosure, it was discovered that E, coll has all of the required enzymes to convert D-glucose to D-tagatose (Figure 5B). D-glucose is converted to F6P via glycolysis and the epimerization of F6P to T6P is accomplished by native enzymes: GatZ, a putative tagato&e- 1,6-bisphosphate aldolase 2 chaperone (Nobelmann and Lengeler, 1995), or KbaZ, a putative tagatose-l ,6-bisphosphate aldolase 1 chaperone (Brinkkbtter et al., 2000), Although these enzymes are annotated as aldolase chaperones (Brinkkbtter et at, 2002), this example demonstrates the C4 epimerization activity of these enzymes in E, coli, Moreover, the dephosphorylation of TSP can be achieved by hexitol-phosphatase A (HxpA), Production of D- tagatose from D-glucose in L. colt was achieved only with the native genes of E. coli. D-tagatose production was further improved by additionally expressing the pathway genes and removing competing pathways, including the pentose phosphate pathway, glycogen biosynthesis, glycolysis, the D-psicose production pathway, and the D-mannose degradation pathway.Natural Pda ga fore product ion cupability of£. coh

[0290] The natural ability of E. cull to produce D-tagatose from D-glucose was examined, The production strain, derived from MG 1655 (Table 4). did not produce D-tagatose under production conditions ( Figure 6A). To accumulate, a critical intermediate. F6P, the pjk.4 gene encoding for phosphoffuctokinase A was deleted, but the ApfkA strain did not produce D-tagatose (Figure 6A), The su / gete encoding for glucose-6-phosphate dehydrogenase was deleted to further increase the F6P pool. The Δzwf strain (AL4240, Table 4) produced 0.21 g L-1D-tagatose in 24 h (Figure6A) indicating that E. coli naturally has all required enzymes to produce D-tagatose from D- glucose.

[0291] Table 4. Strains used in the present example.

[0292] While E. coli has been used to produce D-tagatose, these efforts largely relied on heterologous enzymes (Dai et al., 2022; Liu et al,, 2023; Zhang el al,, 2020). It was postulated that the natural D-tagatose pathway involves the conversion of D-glucose to FOP through glycolysis, followed by the action of an epimerase to convert F6P into T6P, Subsequently, dephosphorylation of TSP by a phosphatase y ields D-tagatose (Figure 5B).

[0293] Five endogenous enzymes were identified as candidates for possessing C4 epimerase activity to convert F6P into T6P. First, it has been shown that D~fiaxctose-l ,(i-bisphosphate aldolase encoded by . / bad exhibits C4 epimerase activity in vitro (Lee et at, 2017b, 2017a). Next, although GatZ is annotated as a putative tagatose-1 ,6-bisphosphate aldolase 2 chaperone, it was used to faci litate growthtumefaciens on galactitol, suggesting GatZ can convertT6P to F6P, which is the reverse reaction used in this example (Kohlmeier et al., 2019), Additionally, it was found KbaZ annotated as a tagatose-1 ,6-bisphosphate aldolase 2 chaperonehas high structural similarity with GatZ. Finally, the enzymes Gat'Y (tagatose-Cd-bisphosphate aldolase 2) and KbaY (tagatose-l,6-bisphosphate aldolase 1) have high structural similarity with FbaA (Brinkkbtter et al., 2002).

[0294] In order to elucidate the C4 epimerase involved in the production of D-tagatose, each candidate gene was deleted in AL4240 (d$fc< zfew£ Table 4). The / bad gene could not be deleted as it is essential (Goodall etal., 2018). Tagatose production was abolished only with the deletion of gufZ (Figure 6B)Ssuggesting GatZ converts F6P to T6P in £. coli. However, the expression of gatZ under the IPTG-inducible promoter f’UacOJ (Lutz and Bujard, 1997), from an expression plasmid in AL4424Jew / ' zlgatZ, Table 4) did not compensate D-tagatose production (Figure 6C). It has been demonstrated that the expression of gatZ enhances the activity of tagatose- 1 ,6-bisphosphate aldolases (Brinkkbtter et al,, 2002), suggesting a potential diversion of T6P away from D-tagatose production towards glycolysis. It was hypothesized that additional expression of T6P phosphatase is required to redirect carbon flux to D-tagatose production. Promiscuous phosphatases were selected (Kuznetsova et al„ 2006) and screenings were conducted by expressing hexitol phosphatase B (HxpB), sugar phosphatase YbiV, sugar phosphatase YidA. hexitol phosphatase A (HxpA), a-D-glucose-1 -phosphate phosphatase YihX, phosphosugar phosphatase YigL. or fructose- 1 -phosphate phosphatase YqaB along with GatZ in AL4424 (Apfk<4 Jru / 'JgorZ, Table 4). Co-expression of GatZ and hexitol phosphatase A (HxpA) restored D- tagafose production in AL4424 (Figure 4D).

[0295] The other C4 epimerase candidate genes may not be expressed under the. instant culture conditions. Therefore, each epimerase candidate was expressed along with hxpA .from an expression plasmid in AL4424 (Jp / ZA dciyf dgafZ, Table 4). The additional expression of kbaZ restored D-tagatose production, but that of / Az.4, hbaY, or ga / Fdid not (Figure 7 A). gatZ and kbaZ were used for further modifications,

[0296] The search for a C4-epimerase to facilitate the conversion of D- fructose to D-tagatose has been an important effort in the sugar industry (Shin et al., 2020). A C4~epimerization capability of the tagaturonafe-iructuronate epimerase from Thermotoga petrophifa was identified (Shin et al, 2020). However, this reaction is thermodynamically unfavorable, leading to a mixture of D~fructose and D-tagatose, An enzyme capable of converting F6P to T6P from Agrobacterium tumefaciens C58 was identified (Wichelecki et al., 2015). This finding enabled a phosphorylation- dephosphorylation pathway for D-tagatose production(Dai et al, 2022). Another C4 epimerase was identified in Sinorhizabium meliloti which showed structural similarity to GatZ of E. coll (Kohlmeier et al., 2019),

[0297] GatZ and KbaZ were identified to be similar in structure. Clustal Omega was used to align the structure of E. call GatZ and KbaZ with other published C4 epimerase enzymes. Structures of these epimerases were obtained from Alphatbld and analyzed for structural similarity.Modulating the expression of the IfAagatose jurorfatf hw pa / fewy ge / ?es

[0298] The native hxpA. gene has a. GTG start codon. In order to increase translation efficiency of fe / M, the start codon was changed to ATG ( / wyvl *)( Sussman et al, 1996). This modification improved D-tagatose titers to 0.48 g L4with the additional expression of g« / Z and 0.60 g L4with the additional, expression kabZ’ (Figure 7B). The PLlacOl promoter was replaced with the PgadB promoter which is a stationary phase promoter and - 100 times stronger than PLiacOf (Taylor et aL, 2023 ). The D-tagatose productivity with PgadB was similar to that PLlcicOl (Figure 7B).Removing competing pathways

[0299] AL4240 zfeu; / . Table 4) with production plasmid produced 0.8 g L4D-mannose and 1.1 g L4D-psicose along with D-tagatose (Figure 10). The mcmA gene encodes for mannose- 6-phosphate isomerase, which interconverts F6P and mannose-S-phosphate (Gao et al., 2005), diverting carbon flux towards D-mannose production. The aAE gene encodes D-allulose 6- phosphate 3-epimerasere, which intereonverts F6P and D-psicose-6-phosphate, is responsible for the production of D-psicose (Taylor et aL 2023). The deletion of mcmA hi AL4240 (ApfkA zfewfi Table 4) resulted in the creation of AL4290 (ApfkA AxwfAmartA, Table 4). This deletion improvedD-tagatose production to 0.6 g L-l and eliminated D-mannose production (Fig, 5). Similarly, the deletion of 'alsE in AL4240Table 1) resulted in AL4314 (ApfkA AzwfdalsE, Table4). This deletion improved D-tagatose production to 0.67 g L-1and eliminated D-psicose production (Figure 8). Both these genes were deleted in A L4240 (ApfkA dswf Table 4), generating AL4315 (ApfkA AzwfAalsE AmanA, Table 4), AL4315 (dp / E4 AzwfdalsE AmanA , Table 4) with the production plasmid produced 0.53 g L-1D-tagatose (Figure 8) and did not produce D-psicose. and D-mannose ,

[0300] E. colt can store excess glucose in the form of glycogen (Sekar et al, 2020). The pgm gene encoding phosphoglueomtrtase converts G6P to glucose- 1 -phosphate (Eydallin et aL, 2007). The pgm gene was deleted in AL4315 (ApfkA AzwfAalsE AmanA Table 4), generating AL4330 (ApfkA AzwfAalsE AmanA Apgm, Table 4). AL4330 with the production plasmid produced 0.82 g L4D- tagatose (Figure 8),

[0301] T6P can be converted to D-tagatose 1,6-bisphosphate by tagatose-6-phoshate kinase (PflcB) that is subsequently converted to glycerone phosphate and D-glyceraldehyde 3-phoshate aldolase (GatY and KbaY)(Brinkkdtter et aL. 2002). The pfkE gene cannot be deleted as the pfkA gene was deleted in the strains. It has been sho wn that overexpression of gatZ enhances tagatose- 1 ,6-bisphosphate aldolase activity in E. coli (Brinkkbtter et aL, 2002). Activated tagatose-1 ,6-bisphosphate aldolase activity would divert carbon flux away from D-tagatose production and towards glycolysis. The MaF gene was deleted in AL4330 (Ap / kA dzw / ‘ Ja / s£ AmanA Apgm, Table 1), generating AI..4493 (ApfkA AzwfAaEE AmanA Apgm AkbaY, Table 4). Similarly, gaiY was deleted in AL4330 (ApfkA Azwf AalsE AmanA Apgng Table 4), generating AL4533 (ApfkA AzwfAaEE AmanA Apgm AgafAg Table 4). Both gu / Fand Adv / Fwere deleted in AL4330 (Z) / ? / L,4 AzwfAahE AmanA Apgm, Table 4), generating AL4534 (.J / jfZri dzu / rirtZsA AmanA Apgm AgatY AkbaY, Table 4). However, these deletions did not improve D-tagatose production (Figure 8),D-taga^ose praA / fcban imAer high caknre Aenxby concfyians

[0302] Ensuring production performance at high cell densities is vital for obtaining a comprehensive understanding of production capabilities (Theisen and Liao, 2017)(Crater and Lievense, 2018). To address potential production constraints arising from glucose availability, AL4534 with pAL2606 (Pgmffi: gaiZ-hxpA*) or pAL2607 (PgaciB: kbaZ-hxpA*, Table 4 and 5) were cultured under high cell density conditions for 24 h. Both these strains consumed 40 g L-1glucose in 24 h (Figure 1 1 ). Based on this resul t, tagatase production at high cell density condition was tested with 40 g L4glucose feeding each day for two days. After the first 24 h, AL4534 with pAL2606 (gatZ) and pAL2607 (kbaZ) produced 3,2 g L4and 3,4 g L4D-tagatose respectively (Figure 9 A). These strains also formed D-fructose and D-mannitol as side products (Figures 9B and 9C). A bolus of glucose was added to each culture, bringing the media glucose concentration back up to 40 g L-1. After an additional 24 h culturing, AL4534 with p.AL2606 (gaiZ) and pAL2607 (kbaZ) produced 7.0 g L4and 8.7 g L4D-tagatose, respectively (Figure 9A). D-fructose and D-mannitol production, also increased (Figure 9a), This observation suggested the low C4 epimerase activity of GatZ and KbaZ causes the accutmilated F6P to redirect the carbon flux towards D-fructose and D-mannitol D-mannitol production is possible through the conversion of F6P to D-mannitol- 1 -phosphate (Mill P) via the enzyme mannitol- 1 -phosphate 5-dehydrogenase, encoded by the gene mEDr

[0303] HxpA shows substrate promiscuity towards F6P and Mil 1 P (Kuznetsova et al, 2006; Sevin et al,, 2017). To minimize the expression of hxpA, the present example reverted to the weaker PLlacOl promoter plasmid system, pAL2574 (PLlacOi: gaiZ-fixpA*) and pAL2575 (PLfac&f: kbaZAbxpA*, Table 5). AL4534 with pAL2574 (g« / Z) and pAL2575 (kbaZ) produced 4,5 g L4and 3.5 g L-1of D-tagatose, respectively, after 24 h (Figure 9 A). After a bolus o f g l ucose and an additional 24 h, these strains produced 8. 1 g L4and 7.6 g L4of D-tagatose, respectively. Additionally, 8.8 g L4and 7,8 g L4of D-fructose and 7.3 g L4and 7. 1 g L-1of D-mannitol were also produced (Figures 9B and 9C). Lowering the expression levels of hxpA resulted in similar D-tagatose titers but D-fructose and D-mannitol were still produced.

[0304] Table 5. Plasmids used in this example.Carbon starvation strategy to eliminate side product formation

[0305] Strain AL4534 showed an inability to assimilate D-tagatose tinder the production conditions. Additionally it has been shown that various E. coli strains cannot grow on D-tagatose (Joo et al., n,d.), although £'. coli is capable of redirecting phosphorylated D-tagatose towards glycolysis (Ha et al., 2022).

[0306] Carbon starvation would be an effective approach to reduce side products formation for D-tagatose production. Under D-glucose deprived conditions, the strain is unable to assimilate the produced D-tagatose but can use other byproducts as carbon sources, AL4534 with pAL2574(PlJacOJ. g&tZ), pAL2575 (P / JaeO / fkbaZ), pAL2606 (PgadB. ga / Z) and pAL2607 (PgadB, kbaZ, Table 5) were employed for D-tagatose production under high-density conditions, supplemented with daily additions of 15 g L-1glucose (Figures 9D-9.F). Within 24 h, the strains produced 1.5 g L4, L2 g L-1, 1.4 g L-1and 1.5 g L-1of D-tagatose, respectively (Figure 9D). Notably, undetectable levels of D-fructose were formed, and AL4534 with pAL2574 {PLkicOl, gaiZg pAL2575 (PLkicOL kbaZ) produced 1.2 g L-1and 2.1 g L-1of D-mannitoI, respectively (Figures 9E and 9F). Upon additional glucose supplementation, D-tagatose titers increased to 4.6 g L-1, 4.5 g L-1, 4.7 g L-1, and 43 g L-1respectively, after 48 hours (Figure 9D). AL4534 with pAL2574 (PLIacOl, gatZ), pAL2575 (PLfacOP kbaZ) utilized the produced D-mannltol. However, AL4534 with pAL2606 (PgciiiB, gaiZ) and pAL2607 (PgadB, kbaZ) produced no D- mannitol, with 2.0 g L-1and 0.7 g L-1of D-fructose persisting in the media after 48 hours (Figures 9E and 9F). Following another 15 g L-1glucose addition and 78 hours of cultivation, they produced 7.3 g L-1, 6.9 g I / \ 7.3 g L-1, and 73 g L-1of D-tagatose, respectively (Figures 9D). While AL4534 with pAL2574 (PLlacOl, gaiZ), pAL2575 (PlJacOl, kbaZ.) produced undetectable side products, AL4534 with pAL2606 (PgadB, gatZ) and pAL2607 (PgadB, kbaZ) produced 5.2 g L-1and 5.4 g Usof D-fructose, respectively (Figure 9E).

[0307] Herein, the present example reveals that £. co / i, with only the knockout of g / kA and zwf genes, possesses the inherent capacity to convert D-glucose into D-tagatose. Further, it was demonstrated that GatZ and KbaZ are capable of converting F6F to D-tagatose-6-phosphate. Leveraging the substrate promiscuity of native phosphatases increased D-tagatose production, band y eliminating competing pathways and overexpression of native E, coli genes, the present disclosure engineered a strain capable of D-tagatose production through a thermodynamically favorable biosynthetic pathway from easily accessible feedstocks. The presently disclosed strain’s inability to utilize D-tagatose and their efficiency in consuming all D-glucose and side products present in the media reduces downstream purification requirements. These results suggest that addition maintaining low glucose concentration and carbon starvation during production prevents side products formation. The presently disclosed final strain achieved conversion of 45 g LJD~ glucose to D-tagatose, reaching a titer of 7.3 g L-1a productivity of 0.1 g L-1h-1without formation of any maj or side products.

[0308] Reagents. All enzymes involved in the molecular cloning experiments were purchased from New England Biolabs (NEB). All synthetic oligonucleotides were synthesized by integrated DNA Technologies. Sanger Sequencing was provided by Genewiz. D-Mamiitol and D-Tagatosewere purchased from Tokyo Chemical Industry. D-Psicose and D-Mannose were purchased from Sigma- Aldrich. D-Glucose and D-Fructose were purchased from Fisher Scientific.

[0309] Strains and Plasmids. All strains and plasmids used in this study are listed in Table 4 and Table 5, respectively. Plasmids for D-tagatose production were constructed using sequence and ligation independent cloning (SLIC)(Li and Elledge, 2007). The constructed plasmids were verified via sequencing.

[0310] Genome modifications such as gene deletion and gene insertion were constructed using CRISPR-Cas9-mediated homologous recombination (Jiang et al, 2015). Linear DNA repair fragments for gene deletions and insertions were constructed by amplifying genomic or plasmidDNA via PCR. assembly. Plasmids encoding sgRNA for CRlSPR-Cas9-mediated homologous recombination were constructed using Q5 site-directed mutagenesis (New England Bio labs) using pTargetF plasmid (Addgene #62226) as a template. Ail genomic modifications were verified via sequencing. A guide to the CRISPR.-Cas9~mediated gene modifications used in this study is detailed in Table 6,

[0311] Table 6. Guide for CRlSPR-Cas9-mediate gene deletions and insertions

[0312] Cui taring conditions. Overnight cultures 'were grown at 37 °C in 3 ml. of Luria-Bertani (LB) media with appropriate antibiotics. Antibiotic concentrations were as follows: spectinomycin (50 pg mL-1), ampicillin (200 pg mL-1), kanamycin (50 gg mL-1), M9 minimal media consists of33.7 mM NajHPCu, 22 mM KlTPCfo 8.6 mM NaCI, 9.4 mM NH4CI, 2 mM MgSCfo 0.1 mM CaCh, AS trace metals mix (2.86 mg L’{HjBO?, 1.81 mg L-1MnCfoTlTO, 0.079 mg L-1CuSO45H2O, 49.4 pg L-1Co(NOs)2-6H2O), varying concentrations of glucose, and appropriate antibiotics. M9P media for the D-tagatose production consists of M9 minimal media supplemented wi th 5 g L-1of yeast extract and appropriate antibiotics. Inducer concen trations are as follows: isopropyl~P-D-1 -thiogalactopyranosidc (IPTG) (I mM), The optical density at 600 am (ODftoo) was measured, with a Synergy Hl hybrid plate reader (BioTek Instruments, Inc,).

[0313] D-Tagatose production. For regular ceil density production experiments, overnight cultures were inoculated at 1% into 3 mL of M9P media. Cells were grown at 37CC until the ODepe described, then induced with IPTG if necessary, and grown at 30 °C for 24 h. For high cell density production experiments in M9P media, overnight cultures were inoculated at 1% into 150 mL of M9P media. Cells were grown at 37 °C until OD«M> -4).4-0.6. Cultures were then induced with IPTG if necessary and grown for a further 30 min. Cultures were centrifuged at 2,200 g tor 5 min and resuspended in MOP media with IPTG if necessary to a target OD«>Q. Cultures were grown at 30 °C.

[0314] HPLC ana / ysA. Analysis of D-tagatose. D-glucose, D-tnannose, D-psicose. D-mannitol and D-fructose concentrations was performed using HPLC (Shimadzu) equipped with a refractive index detector (RID) 10 A and Rezex™ RCU-USP sugar alcohol column (Phenomenex). Mobile phase was comprised of 100% MilliQ water. Samples were run with an injection volume of 1 pL at a flow rate of 0.5 mL min’1for 7.5 min, with the column oven at 83QC and RI D cell temperature of 40 °C. To prepare samples for HPLC analysis, 300 pL of culture was centrifuged at 17,000 g for 5 min. Supernatants were applied to a 0.2 um PVDF hydrophilic membrane 96 well filter plate and centrifuged at 2,000 rpm for 2 min into a polystyrene 96 well,Peferencex

[0315] Ahmed, A., Khan, T.A., Ramdath, D.D., Kendall, C.W.C., Sievenpiper, J.L., 2022. Rare sugars and their health effects in humans: a systematic review and narrative synthesis of the evidence from human trials, NMT. Rev, 80, 255,

[0316] Alsubhi, M., Blake, M., Nguyen, T., Majmudar, 1., Moodie, M., Ananthapavan, J., 2023. Consumer willingness to pay for healthier food products: A systematic review. Oto. Rev. 24, e 13525.

[0317] Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, ¥., Baba, M., Datsenko, K.A., Tomita, M., Wanner, B.L., Mori, H., 2006, Construction of Escherichia coli K-12 in-frame, singlegene knockout mutants: the Keio collection. M> / . Srn. Rfo / . 2, 2006.0008.

[0318] Baumgartner, F., Sprenger, G.A.. Albermann. C., 2015. Galactose- Limited fed-batch cultivation of Escherichia coli for the production of lacto-N-tetraose. Enzyme Mrofe Technol. 75-76, 37-43,

[0319] Blither , M., 2019. Obesity: global epidemiology and pathogenesis. AU / > Rev. Endocrinol. 15, 288-298.

[0320] Brinkkotter, A., KldB, H„ Alpert, C.-A.. Lengeler, J.W., 2000. Pathways for the utilization of N-aeetyl-galactosamine and galactosamine in Escherichia colt MoZ. JWkroZuoZ. 37, 125-135.

[0321] Brinkkdtter, A., Shakeri-Garakani, A., Lengeler, J.W., 2002. Two class II D-tagatose- bisphosphate aldolases from enteric bacteria. Arch. Microbiol. 177, 410-419.

[0322] Crater, J.S., Lieveme, J.C.S2018. Scale-up of industrial microbial processes. FEMS Microbiol Leu. 365, fnyl38.

[0323] Dai, D., Jin, Y.-S., 2024. Rare sugar bioproduction: advantages as sweeteners, enzymatic innovation, and fermentative frontiers. Curr. Opin. Food Sci. 56, 101137.

[0324] Dai, Y,, Li, C., Zheng, L., Jiang, B,, Zhang, T., Chen, J., 2022. Enhanced biosynthesis of d-tagatose from maltodextrin through modular pathway engineering of recombinant Escherichia coli. Biachein. Eng. J. 178, 108303.

[0325] Eydallin. G„ Viale, A.M., Moran-Zorzano, M.T., Munoz, F.J., Montero. M., Baroja- Feruandez, E., Pozueta-Romero, J,, 2007. Genome-wide screening of genes affecting glycogen metabolism in Escherichia coll K-12. FEES Left.. 581, 2947-2953.

[0326] Gao, H., Yu, Y., Leary, J.A., 2005. Mechanism and Kinetics of Metalloenzyme .Phosphomannose Isomerase.' Measurement of Dissociation Constants and Effect of Zinc Binding Using ESI-FTICR Mass Spectrometry, Anal Chem. 77. 5596-5603.

[0327] Granstrdm, T.B., Takata, G., Tokuda. M., Izumori, K., 2004. Ixumoring: A novel and complete strategy for bioproduction of rare sugars. <Z Biosci. Bioeng. 97, 89-94.

[0328] Goodall, E.C.A,, Robinson, A., Johnston, I.G., Ja.bbari, S., Turner, K.A,, Cunningham, A.F., Lund, P.A., Cole, J.A., Henderson, l.R. 2018. The Essential Genome of Escherichia coli K-12. inBio 9.T0.1128 / mbio.02096-17.

[0329] Ha, J., Kim. D, Yeom, J., Kim, Y.. Yoo, S.M., Yoon, S.H., 2022. Identification of a gene cluster for D-tagatose utilization in Escherichia coli B2 phylogroup. iScience 25, 105655.

[0330] Jeon, E.J., Lee, Y.-M., Choi, E.J., Kim, S.-B., Jeong, KJ., 2023. Production of Tagatose by Whole-cell Bioconversion from Fructose Using Corynebacteriiim glutamic urn. Bioieclmol, Bioprocess Eng. 28, 419-427.

[0331] Jiang, ¥., Chen. B., Duan, C., Sun, B„ Y ang, J.. Yang, S., 2015. Multigene Editing in the Escherichia coll Genome via the CR1'SPR-Cas9 System, AppL Environ. Microbiol. 81, 2506- 2514.

[0332] Joo, Y., Sung, J.-Y„ Shin, Park S.J., Kim, K.S., Park, K.D., Kim, S.-B., Lee, D.~ W., n.d. A Retro-Aldol Reaction Prompted the Evolvability of a Phosphotransferase System for the Utilization of a Rare Sugar. Microbiol. Spectr. 11, e03660-22.

[0333] Kohlmeier, M.G., White, C.E., Fowler, IE., Finan, T.M., Oresnik, LI, 2019. Galactitol catabolism in Sinorhizobium meltioti is dependent on a chromosomally encoded sorbitol dehydrogenase and a pSymB-encoded operon necessary for tagatose catabolism. MV Genet. Genomics 23)4, 739-755.

[0334] Kuznetsova, E., Proudfoot, M„ Gonzalez, C.F., Brown. G., Omelchenko, M.V., Borozan, I, Carmel, L., Wolf, Y.I., Mori, H.. Savchenko, A.V., Arrowsmith, C.H., Koonin, E.V., Edwards, A.M Yakunin, A.F., 2006. Genome-wide Analysis of Substrate Specificities of the Escherichia coli Haloacid Dehalogenase-like Phosphatase Family *. J. Biol. Chem. 281, 36149-36161 ,

[0335] Lee, S.-H,, Hong, S.-H., An, J.-LL, Kim, K..-R., Kim, D.-E., Kang, L.-W., Oh, D.-K., 2017a. Structure-based prediction and identification of 4-epimerization activity of phosphate sugars in class II aldolases, Act Rep. 7, 1934.

[0336] Lee, S.-H., Hong. S.-H., Kim. K.-R., Oh, D.-K., 2017b. High-yield production of pure tagatose from fructose by a three-step enzymatic cascade reaction, R / otechnol. Leu. 39, 1141- 1148.

[0337] LI, M.Z., Elledge, S.J., 2007. Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat. Methods 4, 251-256.

[0338] Liu, J.-I, Zhang. G.-C,, Kwak, S., Oh, E.J., Yun, E.J., Chomvong, K., Cate, J.H.D., Jin, Y.-S., 2019. Overcoming the thermodynamic equilibrium of an isomerization reaction through oxidoreductive reactions for biotransformation. AW. Cornmun. 10, 1356,

[0339] Liu, W., Zhang, Z., Li, Y., Zhu, L„ Jiang, L,, 2023. Efficient production of d-tagatose via DNA scaffold mediated axidoreductases assembly in vivo from whey powder. Food Res. Ink 166, 112637.

[0340] Lustig, R.H., Schmidt, L.A., Brindis, C.D., 2012. The toxic truth about sugar. Nature 482, 27-29.

[0341] Lutz, R., Bujard, H., 1997. Independent and fight Regulation of Transcriptional Units in Escherichia coli Via the LacR / O, the TetR / O and AraCZl 1-12 Regulatory Elements. Nucleic Acids Res. 25, 1203 -1210.

[0342] Nobehnann, B., Lengeler, J.W., 1995. Sequence of the gat operon for galactitol utilization from a wild-type strain EC3132 of Escherichia coli. Biochini. liiophys. Ada BB.A - Gene S / 'rud. Lxpr. 1262, 69-72.

[0343] Noor, E., Bar-Even, A., Flamholz, A., Lubling, Y., David! D., Milo, R.., 2012. An integrated open framework for thermodynamics of reactions that combines accuracy and coverage. Bioinformaticx 28, 2037—2044.

[0344] Oh, D.-K., 2007. Tagatose: properties, applications, and biotechnological processes. Appt.MicrobfoL Bioieehno / . 76, 1-8.

[0345] Prinz, P., 2019. The role of dietary sugars in health: molecular composition or just calories? Zi!tr. J. Clin. Nutr. 73, 1216-1223 ,

[0346] Rizas, K.D., Sams, L.E., Massberg, S., 2023. Non-nutritioaal sweeteners and cardiovascular risk. Nat Met / . 29, 539-540.

[0347] Roy, S., Chikkerur, J., Roy, S.C., Dhali, A., Kofte, A.P., Sridhar, M., Samanta, A.K.., 2018. Tagatose as a Potential Nutraceutical: Production, Properties, Biological Roles, and Applications. J. Food ScL 83, 2699-2709,

[0348] Sekar, K., Linker, S.M., Nguyen. J., Griinhagen, A.. Stocker, R., Sauer, U., 2020. Bacterial Glycogen Provides Short-Term Benefits in Changing Environments. Appt Frtviixm. Mierobiol, 86, e00049-20.

[0349] Sevin, D.C., Fuhrer, T,, Zamboni, N,, Sauer, th, 2017. Nontargeted in vitro metabolomics for high-throughput identification of novel enzymes in Escherichia coli. AGz. MedrnN 14, 187 - 194,

[0350] Shin, K.-C., Lee, T.-E., Seo, M.-I, Kim, D.W., Kang, L.-W., Oh, D.-K., 2020. Development of Tagaturonate 3-Epimerase into Tagatose 4-Epimerase with a Biocataiytic Route from Fructose to Tagatose. ACS Cafcd. 10, 12212-12222.

[0351] Sugar Substitutes Market Size and Forecast [Latest] [WWW Document], 2017. .AfozTjezw / rZAfo / 'Zuzv. URL https', / / www.marketsandmarkets.com / Mai'ket-Reports / sugar- substitute-market-1134.html (accessed 3. 12.24).

[0352] Sussman, J.K., Simons, EX., Simons, R.W., 1996. Escherichia coli translation initiation factor 3 discriminates the initiation codon in vivo. Md. MicroMot 21 , 347-360.

[0353] Taylor, IE., Pahtr, D.S.K., Zhang, A., Gonzales, J.N., Arredondo, A., Coulther, T.A., Lechner, A.B.J., Rodriguez, E.P., Fiehn, O., Didzbalis, J., Siegel, LB., Atsumi, S., 2023. Awakening the natural capability of psicose production in Escherichia coli. Np / Set Food 7, 54.

[0354] Theisen, M., Liao, J.C., 2017. Industrial Biotechnology; Escherichia coli as a Host, in: Industrial Biotechnology. John Wiley & Sons, Ltd, pp. 149-181.

[0355] Wichelecki, D.J., Vetting, M.W., Chou, L., Al-ObaidisN,. Bouvier, J.T.SAlmo, S,C., Gerlt, J. A., 2015. ATP-binding Cassette (ABC) Transport System Solute-binding Protein-guided Identification of Novel d-Altritol and Galactitol Catabolic Pathways in Agrobacterium tumefaciens C58 *. <Z Biol. Chem. 290, 28963-28976.

[0356] Xu, Z., Li, S., Feng, X., Liang, J., Xu, IL, 2014. L-Arabinose isomerase and its use for biotechnological production of rare sugars. 4PP*- MicroPro / . BiofechttoL 98, 8869-8878.

[0357] Y'oueda, H., Tantillo, D..L, Atsumi, S , 2014. Biological Production of 2-Butanone in Escherichia coli. ChemSuxOwm 1, 92-95.

[0358] Zhang, G., Zabed, H.M., Yun, I, Yuan, J., Zhang, Y., Wang, Y.?Qi, X., 2020. Two-stage biosynthesis of D-tagatose from milk whey powder by an engineered Escherichia coli strain expressing L-arabinose isomerase from Lactobacillus plantarum. Bioresour. Tec / wol. 305, 123010.

[0359] Although the presently disclosed subject matter and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims, Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the presently disclosed subject mater, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized according to the presently disclosed subject matter. Accordingly, the appended claims are intended to include within their scope such, processes, machines, manufacture, compositions of matt er, means, methods, or steps.

[0360] Patents, patent applications, publications, product descriptions and protocols are cited throughout this application the disclosures of which are incorporated herein by reference in their entireties for all purposes.

Claims

WHAT IS CLAIMED IS:

1. A recombinant microorganism comprising an exogenous epimerase, wherein the recombinant microorganism produces an increased amount of D~tagatose as compared to a naturally occurring microorganism.

2. The recombinant microorganism of claim 1, wherein the epimerase is a tagatose-1,6- bisohosphate aldolase subunit GatZ or a ketose biphosphate aldolase subunit KbaZ.

3. The recombinant microorganism of clai m 1 or 2, wherein the epi merase is an £. coti. GatZ.

4. I he recombinant microorganism of claim 3, wherein the epimerase comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 58.

5. The recombinant microorganism of claim 3 or 4, wherein the epimerase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58.

6. The recombinant microorganism of claim 1 or 2. wherein the epimerase is an £. coH KbaZ.

7. The recombinan t microorganisms of claim 6, wherein the epimerase comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 60,8. The recombinant microorganism of claim 6 or 7, wherein the epimerase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 60,9. The recombinant microorganism of any one of claims 1-8, wherein the recombinant microorganism further comprises exogenous phosphatase.

10. The recombinant microorganism of claim 9, wherein the phosphatase is a hexitol phosphatase A (HxpA).11 . The recombinant microorganism of claims 9 or 10, wherein the phosphatase is an A*, co / z HxpA.

12. The recombinant microorganism of any one of claims 9-1 1 , wherein the phosphatase comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 62.

13. The recombinant microorganism of any one of claims 9-12, wherein the phosphatase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62.

14. The recombinant microorganism of any one of claims 1-13, wherein the recombinant microorganism further comprises exogenous galactose: Hx‘ symporter (GalP) and glucokinase (Glk).

15. The recombinant microorganism of claim 14, wherein the GalP is an £. coZz GalP and the Glk is £ m / z Glk.

16. The recoinbinani microorganism of claim 14 or 15, wherein the GalP comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO; 38 and the Glk comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 40.

17. The recombinant microorganism of any one of claims 14-16, wherein the GalP comprises or consists of the amino acid sequence set forth in SEQ ID NO: 38 and the Glk comprises or consists of the amino acid sequence set forth in SEQ I D NO: 40,18. The recombinant microorganism of any one of claims 1 - 17 further comprising a mutation of a gene encoding for an enzyme of the pentose phosphate pathway compared to a naturally occurring microorganism.

19. The recombinant microorganism of claim 18, wherein enzyme of the pentose phosphate pathway' is glucose-6-phosphate 1 -dehydrogenase (Zwf).

20. The recombinant microorgani sm of any' one of claims 1-19 further comprising a mutation of a gene encoding for an enzyme of the glycolysis compared to a naturally occurring microorganism.

21. The recombinant microorganism of claim 20, wherein the enzyme of the glycolysis is phosphofructokinase- 1 (PflcA), phospho fructokinase- 2 (PfkB), or pyruvate kinase (PykF).

22. The recombinan t microorganism of claim 20 or 21, wherein the enzyme of the glycolysis is phosphofructokinase- 1 (PfkA).

23. The recombinant microorganism of any one of claims 1 -22 further comprising a mutation of a gene encoding for an enzyme that converts fiisctose~6~phosphate to psicose-6- phosphate.

24. The recombinant microorganism of claim 23, wherein the enzyme that converts fructose- 6-phosphate to psicose-6-phosphate is D-aHulose-6-phosphate 3-epimerase (AlsE).

25. The recombinant microorganism of any one of claims 1-24 further comprising a mutation of a gene encoding for an enzyme of mannose biosynthesis pathway.

26. The recombinant microorganism of claim 25, wherein the enzyme of mannose biosynthesis pathway is mannose-6-phosphate isomerase (ManA).

27. The recombinan t microorganism of any one of claims 1-26 further comprising a mutation of a gene encoding for an enzyme of carbohy drate metabolism,28. The recombinant microorganism of claim 27, wherein the enzyme of carbohydrate metabolism is D-tagatose- 1,6-bisphosphate aldolase subunit (GatY),29. The recombinant microorganism of claim 27. wherein the enzyme of carbohydrate metabolism is D-tagatose- 1,6-bisphosphate aldolase subunit (KbaY).

30. The recombinant microorganism of any one of claims 1-29, further comprising a mutation of a gene encoding for an enzyme of glycogen biosynthesis selected from phosphoglucomutase (Pgm), UDP-glucose pyrophosphorylase, glycogen synthase, glycogen branching enzyme, glycogenin, or combinations thereof31. The recombinant microorganism of claim 30, wherein the enzyme of glycogen biosynthesis is phosphoglucomutase (Pgm),32. A microorganism comprising a recombinant polynucleotide encoding an epimerase, wherein expression of the epimerase results in an increased production of D-tagatose as compared to a microorganism lacking the recombinant polynucleotide.

33. The microorganism, of claim 32, wherein the epimerase is a tagatose-l fo-bisohosphate aldolase subunit GatZ or a ketose biphosphate aldolase subunit KbaZ,34. The microorganism of claim 32 or 33, wherein the epimerase is anco / / GatZ.

35. The recombinant microorganism of claim 32 or 33, wherein the epimerase is anco / / KbaZ.

36. The recombinant microorganism of any one of claims 32-35, wherein the recombinant microorganism further comprises exogenous phosphatase.

37. The recombinant microorganism of claim 36, wherein the phosphatase is a hexitol phosphatase A (HxpA).

38. The recombinant microorganism of claim 37, wherein the phosphatase is an £. co / z HxpA.

39. The microorganism of any one of claims 32-38 further comprising a mutation of at least one gene.

40. The microorganism of claim 39, wherein the at least one gene is selected from glucose-6- phosphate 1 -dehydrogenase (zuy), phosphofructokinase- 1 ( / yfod), phosphofructokinase-2 (pfkB), pyruvate kinase (p.yk / ;'), D-allulose-6-phosphate 3-epimerase (n / yh), mannose-6- phosphate isomerase (mauA), phosphoglucomutase (pgm), D-tagatose- 1 ,6-bisphosphate aldolase subunit (grtzF), D-tagatose- 1,6-bisphosphate aldolase subunit (kha I), or a combinati on thereof.

41. The microorganism of claim 39, wherein the at least one gene is giucose-6-phosphate 1~ dehydrogenase (zw / J, phosphofructokinase- 1 (p / M), D-allufose-6-phosphate 3 -epimerase (o / .v£), mannose~6-phosphate isomerase (man A), phosphoglucomutase (pgw), D-tagatose- 1,6-bisphosphate aldolase subunitD-tagatose-l,6-bisphosphate aldolase subunit (A'h«y), or a combination thereof.

42. The microorganism of any one of claims 18-31 and 39-41, wherein the mutation is a deletion.

43. The microorganism of any one of claims 18-31 and 39-41 , wherein the mutation reduces or eliminates expression or activity of the enzyme.

44. The microorganism of any one of claims 1-43, wherein the microorganism is Zi, cxzZr, BadZZm .vwZfoZts or / .aczococcw lactis.

45. A method for producing D-tagatose comprising culturing the microorganism of any one of claims 1-44 under conditions suitable for converting a substrate to D-tagatose.

46. The method of claim 45, wherein the substrate comprises D-glucose,47. A method of making a food product comprising D-tagatose comprising a) culturing the microorganism of any one of clai ms I -45 under conditions suitable for converting a substrate to D-tagatose; and b) admixing the tagatose with one or more foods to form a food comprising tagatose.

48. The method of claim 47, wherein the food product is a beverage, yogurt, ice cream. a baked good, or a nutritional bar.

Images