Microorganisms for the production of d-ribose

Abstract

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

Description

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

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 537,138, 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 lias 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 081906-1459717 253610PC SL.xml, and is 92,611 bytes in size.TECHNICAL FIELD

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

[0004] The market for rare sugars as a foodstuff, dietary supplement, and health aid is expanding. Among rare sugars, D-ribose has attracted particular attention. However, the current method of producing D-ribose is costly and inefficient, limiting its potential for wide-spread use. Thus, there is a need for producing D-ribose in an economic and efficient manner.SUMMARY

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

[0006] In some embodiments, the isomerase is a ribose 5-phosphate isomerase A (RpiA). In some embodiments, the isomerase is an E. call Rpi A. In some embodiments, the epimerase comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the epimerase comprises the amino acid sequence set forth in SEQ ID NO: 1 . In some embodiments, the epimerase consists of the amino acid sequence set forth in SEQ ID NO: 1 .

[0007] In some embodiments, the isomerase is a ribose 5-phosphate isomerase B (RpiB), In some embodiments, the isomerase is an E coli (RpiB). In some embodiments, the isomerase comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the epimerase comprises the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the epimerase consists of the amino acid sequence set forth in SEQ ID NO: 3.

[0008] In some embodiments, the phosphatase .is a sugar phosphatase YbiV (YbiV). In some embodiments, the phosphatase is an £, co / / YbiV. 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: 5, In some embodiments, the phosphatase comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the phosphatase consists of the amino acid sequence set forth m SEQ ID NO: 5.

[0009] In some embodiments, the phosphatase is a sugar phosphatase YidA (YidA). In some embodiments, the phosphatase is an E. coli YidA. 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: 7. In some embodiments, the phosphatase comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the phosphatase Consists of the amino acid sequence set forth in SEQ ID NO: 7.

[0010] In some embodiments, the recombinant microorganism further comprises at least one exogenous enzyme of the pentose phosphate pathway. In some embodiments the at least one exogenous enzyme of the pentose phosphate pathway comprises glucose-6-phosphate dehydrogenase (Zwf). In some embodiments, the Zwf comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 51. In some embodiments, the Zwf comprises the amino acid sequence set forth in SEQ ID NO: 51. In some embodiments, the at least one exogenous enzyme of the pentose phosphate pathway comprises phosphogluconate dehydrogenase (Gnd). In some embodiments, the Gnd comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 53. In some embodiments, the Gnd comprises the amino acid sequence set forth in SEQ ID NO: 53.

[0011] In some embodiments, the recombinant microorganism farther comprises exogenous galactoserH* symporter (GalP) and glucokinase (Glk). In some embodiments, the GalP is an E. co / i GalP and the Glk is E. (:<Ei 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: 36 and the Glk comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 38. In some embodiments, the GalP comprises the amino acid sequence set forth in SEQ ID NO: 36 and the Glk comprises the amino acid sequence set forth in SEQ ID NO; 38.

[0012] In some embodiments, the microorganism further comprises a mutation of a gene encoding for an enzyme of glycogen biosynthesis selected from the group consisting of phosphoglucomutase (Pgm), UDP-glucose pyrophosphoiylase, glycogen synthase, glycogen branching enzyme, and glycogenin. In some embodiments, the enzyme of glycogen biosynthesis is phosphoglucomutase ( Pgm).

[0013] In some embodiments, the recombinant microorganism farther comprises a mutation of a gene encoding for an enzyme of the carbohydrate biosynthesis pathway compared to a naturally occurring microorganism. In some embodiments, the enzyme of the carbohydrate biosynthesis pathway is gIucose-6-phosphate isomerase (Pgi).

[0014] In some embodiments, the recombinant microorganism further comprises a mutation of a gene encoding for an enzyme of the purine / pyrhnidine ribonucleosides degradation pathway compared to a naturally occurring microorganism. In some embodiments, the enzyme of the purine / pyrimidine ribonucleosides degradation pathway is phosphopentomutase (DeoB).

[0015] 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 transketolase I (TktA), In some embodiments, the enzyme of the pentose phosphate pathway is transketolase 2 (TktB).

[0016] In some embodiments, the recombinant microorganism further comprises a mutation of a gene encoding for an enzy me of glycolysis. In some embodiments, the enzyme of the glycolysis is phosphofructokinase- 1 (PfkA), In some embodiments, the recombinant microorganism further comprises a mutation of a gene encoding for an epimerase compared to a naturally occurring microorganism. In some embodiments, the epimerase is allulose-6-phosphate~3 -epimerase (AIsE). In some embodiments , the recombinant microorganism further comprising a mutation of a gene encoding for an enzyme of the gluconate degradation pathway compared to a naturally occurring microorganism. In some embodiments, the enzyme of the gluconate degradation pathway is phosphogluconate dehydratase (Edd)„ In some embodiments, the recombinant microorganism further comprises a mutation of at least one gene encoding for an enzyme of the ribose catabolism pathway. In some embodiments, at least one gene encoding for an enzyme of the ribose catabolism pathway is selected from D-ribose transporter ATP binding subunit (RbsA), D-ribose transporterperiplasmic binding protein (RbsB), D-ribose transporter membrane subunit (RbsC), D-ribose pyranase (RbsD), D-ribose kinase (RbsK), DNA-biuding transcriptional dual regulator (RbsR), small regulatory RNA (RbsZ), or combinations thereof.

[0017] In some embodiments, the exogenous isomerase and the exogenous phosphatase are expressed by a stationary phase promoter. In some embodiments, exogenous isomerase and the exogenous phosphatase are expressed by an inducible promoter.

[0018] In another aspect, the present disclosure relates to a microorganism comprising a recombinant polynucleotide encoding an isomerase and a phosphatase, wherein expression of the isomerase and the phosphatase results in an increased production of D-ribose as compared to a microorganism lacking the recombinant polynucleotide,

[0019] In some embodiments, the isomerase is a ribose 5-phosphate isomerase A (RpiA). In some embodiments, the isomerase is an £ a.> / z RpiA. In some embodiments, the isomerase comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 1 . In some embodiments, the isomerase comprises the amino acid sequence set forth in SEQ ID NO: I. In some embodiments, the isomerase consists of the amino add sequence set forth in SEQ ID NO: 1.

[0020] In some embodiments, the isomerase is a ribose 5-phosphate isomerase B (RpiB). In some embodiments, the isomerase is an E. coll RpiB. In some embodiments, the isomerase comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the isomerase comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the isomerase consists of the amino acid sequence set forth in SEQ ID NO: 2.

[0021] In some embodiments, the phosphatase is a sugar phosphatase YbiV (YbiV), In some embodiments, the phosphatase is anca / i YbiV. 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: 5, In some embodiments, the phosphatase comprises the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the phosphatase consists of the amino acid sequence set forth in SEQ ID NO: 5.

[0022] In some embodiments,, the phosphatase is a sugar phosphatase YidA (YidA). In some embodiments, the phosphatase is an E. coll YidA. In some enibodinients, the phosphatase comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the phosphatase comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the phosphatase consists of the amino acid sequence set forth in SEQ ID NO: 7.

[0023] In some embodiments, the recombinant microorganism further comprises at least one exogenous enzyme of the pentose phosphate pathway. In some embodiments the at least one exogenous enzyme of the pentose phosphate pathway comprises glucose-6-phospha.te dehydrogenase (Zwf). In some embodiments, the Zwf comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 51. In some embodiments, the Zwf comprises the amino acid sequence set forth in SEQ ID NO: 51. In some embodiments, the at least one exogenous enzyme of the pentose phosphate pathway comprises phosphogluconate dehydrogenase (Gnd). In some embodiments, the Gnd comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 53. In some embodiments, the Gnd comprises the amino acid sequence set forth in SEQ ID NO: 53.

[0024] In some embodiments, the microorganism further comprises a mutation of a gene encoding for an enzyme of glycogen biosynthesis selected from the group consisting of phosphoglucomutase (Pgm), UDP-glucose pyrophosphorylase, glycogen synthase, glycogen branching enzyme, and glycogenin. In some embodiments, the enzyme of glycogen biosynthesis is phosphoglucomutase (Pgm).

[0025] In some embodiments, the microorganism further comprises a mutation of a gene encoding for an enzyme of the carbohydrate biosynthesis pathway compared to a naturally occurring microorganism. In some embodiments, the enzyme of the carbohydrate biosynthesis pathway is glucose-6-phosphate isomerase (Pgi).

[0026] In some embodiments, the recombinant microorganism further comprises a mutation of a gene encoding for an enzyme of the purine / pyrimidine ribonucleosides degradation pathway compared to a naturally occurring microorganism. In some embodiments, the enzyme of the purine- •'pyrimidine ribonucleosides degradation pathway is phosphopentom utase (DeoB).

[0027] In some embodiments, the microorganism further comprises a mutation of a gene encoding tor an enzyme of the pentose phosphate pathway compared to a naturally occurring microorganism. In some embodiments, the enzyme of the pentose phosphate pathway is transketolase 1 (TktA). In some embodiments, the enzyme of the pentose phosphate pathway is transketolase 2 (TktB).

[0028] In some embodiments, the recombinant microorganism further comprises a mutation of a gene encoding for an enzyme of glycolysis. In some embodiments, the enzyme of the glycolysis is phosphoftuctpkinase-l (PfkA). In some embodiments, the recombinant microorganism further comprises a mutation of a gene encoding for an epimerase compared to a naturally occurring microorganism. In some embodiments, the epimerase is alluIose-6-phosphate-3-epimerase (AlsE). In some embodiments, the recombinant microorganism further comprising a mutation of a gene encoding for an enzyme of the gluconate degradation pathway compared to a naturally occurring microorgan ism. In some embodiments, the enzyme of the gluconate degradation pathway is phosphogiuconate dehydratase (Edd). In some embodiments, the recombinant microorganism further comprises a mutation of at least one gene encoding for an enzyme of the ribose catabolism pathway. In some embodiments, the at least one gene encoding for an enzyme of the ribose catabolism pathway selected from D-ribose transporter ATP binding subunit (RbsA), D-ribose transporter periplasmic binding protein (RbsB), D-ribose transporter membrane subunit (RbsC), D-ribose pyranase (RbsD), D-ribose kinase (RbsK), DNA-binding transcriptional dual regulator (RbsR), small regulatory RNA (RbsZ), or combinations thereof.

[0029] Ih some embodiments, the exogenous isomerase and the exogenous phosphatase are expressed by a stationary phase promoter. In some embodiments, exogenous isomerase and the exogenous phosphatase are expressed by an inducible promoter.

[0030] In a further aspect, the present disclosure relates to a microorganism comprising a recombinant polynucleotide encoding a ribose 5-phosphate isomerase A (RpiA) or ribose 5- phosphate isomerase B (RpiB) and a sugar phosphatase YbiV or YidA; a mutation of phosphoglucomutase (pg / »); a mutation of glucose-6-phosphate isomerase (pgi); a mutation of phosphopeutomutase (deoB); a mutation of transketolase I (tfori); a mutation of transketolase 2 ( / &$); and optionally a recombinant polynucleotide encoding GalP, Glk, or both. In some embodiments, the microorganism optionally comprises a .recombinant polwucleotide encoding Zwf, Gnd, or both. In some embodiments, the microorganism optionally comprises a mutation of allulose-6-phosphate 3 -epimerase (o&£). In some embodiments, the microorganism optionally comprises a mutation of phosphogluconate dehsdratase (edd) In some embodiment * the microorganism optionally comprises a mutation of phosphofructokinase-1 (p / kA), In. some embodiments, the microorganism optionally comprises a mutation of at least one gene encoding for an enzyme of the ribose catabolism pathway selected from D-ribose transporter ATP binding subunit (RbsA), D-ribose transporter perip lasmic binding protein (RbsB), D-ribose transporter membrane subunit (RbsC), D-ribose pyranase (RbsD), D-ribose kinase (RbsK). DNA-binding transcriptional dual regulator (RbsR), small regulatory RNA. (RbsZ), or combinations thereof.

[0031] Further, in one aspect, the present disclosure relates to a microorganism comprising: a recombinant polynucleotide encoding a ribose 5-phosphate isomerase A (RpiA) or ribose-5- phosphate isomerase B (RpiB); a recombinant polynucleotide encoding a sugar phosphatase YbiV (YbiV) or sugar phosphatase YidA (YidA); a mutation of phosphoglucomutase (pgm)', a mutation of glucose-6-phospliate isomerase (pgf); a mutation of phosphopentom utase (deoB); a mutation of transketolase 1 (W,4); a mutation of transketolase 2 (ikiB); and optionally a recombinant polynucleotide encoding GalP, Glk, or both. In some embodiments, the microorganism optionally comprises a recombinant polynucleotide encoding Zwf, Gnd, or both. In some embodiments, the microorganism optionally comprises a mutation of allulose-6-phosphate 3-epimerase (Mr£). In some embodiments, the microorganism optionally comprises a mutation of phosphogluconate dehydratase (edd). In some embodiments, the microorganism optionally comprises a mutation of phosphoftuctokinase-1 (pfkA). In some embodiments, the microorganism optionally comprises a mutation of at least one gene encoding for an enzyme of the ribose catabolism pathway selected from D-ribose transporter ATP binding subunit (RbsA), D-ribose transporter periplasmic binding protein (RbsB), D-ribose transporter membrane subunit (RbsC), D-ribose pyranase (RbsD), D-ribose kinase (RbsK), DNA-binding transcriptional dual regulator ( RbsR), small regulatory RNA (RbsZ), or combinations thereof.

[0032] 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 7f ooh, Bacilhis subtilis or Laciococcus lactis.

[0033] In one aspect, the present disclosure also relates to a method for producing D-ribose comprising culturing the microorganism disclosed herein under conditions suitable for converting a substrate to D-ribose, In some embodiments, the substrate comprises glucose.

[0034] Additionally, the present disclosure relates to methods of making a food product comprising D-ribose, the method comprising culturing the microorganism disclosed herein under conditions suitable for converting a substrate to D-ribose; and admixing the D-ribose with one or more foods to form a food comprising D-ribose. In some embodiments, the food product is a beverage, yogurt, ice cream, a baked good, or a nutritional bar.BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figures 1 A and IB show exemplary pathways disclosed herein for the production of D- ribose.

[0036] Figure 2 shows strategy for the biosynthesis of D-ribose. The proposed pathway for the biosynthetic production of D-ribose incoff, PTS, the phosphotransferase system; GaiP, galactoseTT' symporter; Glk, glucokinase; Zwf, NADP' -dependent glucose-6-phosphate dehydrogenase; Pgi, 6-phosphogIucolactonase; Gnd, 6-phosphogIuconate dehydrogenase, decarboxylating; RpiA, D-ribose~6-phosphate-isomerase A.

[0037] Figure 3 shows the effect of gene deletions on the D-ribose production. Cel ls were grown in M9P media with 10 g L-1glucose to OD600 ~0.4 at 37 °C, then grown at 30 °C for 24 h. Error bars indicate s.d. (n = 3 biological replicates).

[0038] Figure 4 shows screening for D-ribose-6~phosphate phosphatase in E. coli. Cells were grown in M9P media with 10 g L-1glucose at 37°C to OD600 ~0.4, then grown at 30 °C for 24 h. At OD600 ~0.4, 1 niM IPTG was added. Various sugar phosphatase genes with rpiA under were tested for D-ribose production (Table 3 ). Plasmids containing galP and gft under Puseoi and zwf and gnd underwere also added to the strain. (-) indicates that no pathway genes and / or no phosphatase genes were additionally expressed. Error bars indicate s.d. (n = 3 biological replicates).DETAILED DESCRIPTION

[0039] The market for rare sugars as a foodstuff, dietary supplement, and health aid is expanding. Among rare sugars, D-ribose has attracted particular attention. However, the current method of producing D-ribose is costly and inefficient, limiting its potential for wide-spread use.Significantly, the present disclosure addressed various obstacles in D-ribose production including thermodynamic barriers, limited yield, the requirement for purified enzymes and the addition of cofactors. Another significant finding disclosed herein was that 7i. eoli naturally possesses a thermodynamically favorable pathway for D-ribose production allowing the improvement of D- ribose production by increasing the expression of native genes and eliminating competing pathways without the introduction of heterologous genes. The present disclosed subject matter facilitates the industrial-scale production of D-ribose without the need for costly enzyme purification or difficult feedstock and product separation.

[0040] 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 D-ribose. 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 Ribose;3. Methods for Producing and Generating Microorganisms;4. Methods for Producing Ribose; and5. Food Products.7. Definitio

[0041] 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.

[0042] 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.”

[0043] The term “about” or “approximately'’ means within an acceptable error range for the partic ular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i ,e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.

[0044] 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.

[0045] 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 ftrngi. 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,

[0046] 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-sul&r bacteria, radioresistant micrococci, and ihermotoga and Thermosipho thermophiles.

[0047] 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,Zfonfe / eZZa, Escherichia, Salmonella, Shigella, Klebsiella, Proteus, HMo, Pseudomonas, bacteroides, Ace / obacter, Aerobaefer, Agrobacterium, Asotobacter, Spirilla, Serratla, Vibrio, IdHcobimn, Chlamydia, Rickettsia, Treponema, and Fysobacterium,

[0048] As used herein, the term “gram-positive bacteria” includes cocci, nonsporulating rods, and sporulatmg rods, The genera of gram-positive bacteria include, for example, and without any limitation, Actinomyces, bacillus, Clostridium. Cotynebacterium, Erysipelothrbc, Lactobacillus, Listeria, Mycobacterium, Myxococcus, Nocardia, Staphylococcus, Streptococcus, and Streptomyces.

[0049] As used herein, the term “recombinant microorganism” refers to a microorganism that contains one or more recombinant polynucleotides,

[0050] 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. Theterm “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.

[0051] 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 phosphodi ester bonds. The nucleic acid molecule can include deoxyribonucleotide bases or ribonucleotide bases and can be manufactured synthetically in wire or isolated from natural sources.

[0052] 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 na turally found in a given host, but in an unnatural (e.g., greater than or less than expected) amount, or additionally if the sequence of a polynucleotide comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, but without any limitation, a recombinant polynucleotide could have two or more sequences from unrelated polynucleotides or from endogenous nucleotides arranged to make a new polynucleotide. In certain embodiments, the present disclosure provides the introduction of a recombinant polynucleotide into a microorganism, wherein the polynucleotide encodes for a polypeptide that is not normally found in the microorganism. With reference to the microorganism's genome, then, the polynucleotide sequence that encodes the polypeptide is recombinant or heterologous.

[0053] A “gene,” as used herein, refers to a DNA region (including exons and introns) encoding a gene product, as well as all DNA. regions which regulate the production of the gene product, whether or not such regulatory sequences are ad jacent to coding and / or transcribed sequences. In certain non-limiting embodiments, a gene includes promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions.

[0054] The terms “polypeptide,” “peptide,” “amino acid sequence” and “protein,” used interchangeably herein, refer to a molecule formed from the linking of at least two amino acids, The link between one amino acid residue and the next is an amide bond and is sometimes referred to as a peptide bond, A polypeptide can be obtained by a suitable method known in the art, including isolation from natural sources, expression in a recombinant expression system, chemical synthesis, or enzymatic synthesis. The terms can apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.TO

[0055] 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, eg., 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, / .e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, and methionine methyl sulfonium. Such analogs can have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics mean chemical compounds that have a structure that is different from the general chemical structure of amino acid, but that function in a manner similar to a naturally occurring amino acid. Non-hnuting 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.

[0056] 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).

[0057] 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%,

[0058] As used herein, the term “increase,” “elevate” and “elevation” refers to a measurable augmentation of an end-point (e.g. , enzymatic activity, production of compound, expression of a protein) by at least about 10%, at. least about 50%, at least about 75%, or at least about 90%. In certain embodiments, the increase can be from about 10% to about 100%. In certain embodiments, the increase can be at least about 10-fold, about 100-fold, or about 1000-fold or more. In certain embodiments, the increase can be about 100-fold or more, about 1000-fold or more, or about 10,000- fold or more.

[0059] Techniques for determining nucleic acid and amino acid sequence identity are known in the art. Typically, such techniques include determining the nucleotide sequence of the mRN A for a gene and / or determining the amino acid sequence encoded thereby and comparing these sequences to a second nucleotide or amino acid sequence. Genomic sequences can also be determined and compared in this fashion. In general, identity refers to an exact uucleotide-to- nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptidesequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their percent identity. The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. Unless indicated otherwise, percent identity is determined for two sequences when compared and aligned for maximum correspondence over a comparison window or designated region as measured using a BLAST or BLAST 2,0 sequence comparison algorithms with default parameters. See. e.g., the NCBI web site at ncbi.nlm.nih.gov / BLAST. For example, BLASTN and BLAST? can be used using the following default parameters: genetic code-standard; fdtei-none; strand=both; cutoflMO; expect- 10; Matrix:::BI,()SUM62; Descriptions-150 sequences; sort by- HIGH SCORE; Databases-non<ednndant, GenBaftk+EMBL+DDBJ+PDBXrenBank CDS translations+Swiss protein'FSpupdate't'PIR. 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,2, Microorganisms Producing Ribose

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

[0062] D-ribose is simple sugar and carbohydrate. It is an aldopentose that has the same empirical formula as common monosaccharides such as glucose and fructose. Importantly, D-ribose is a 5- catbon sugar with the empirical formula CsHwQs, while glucose and fructose are 6 carbon sugars. D-ribose has the following- formula:

[0063] The present disclosure provides genetically engineered microorganisms that have increased production of ribose as compared to naturally-occurring microorganisms. Figures 1A~ 2 illustrate the biochemical pathways modulated in exemplary microorganisms of the present disclosure. In these exemplary microorganisms, glucose is imported and phosphorylated toglucose-6-phosphate (G6P) by the phosphotransferase system (PTS) or GalP / Glk.. G6P is then catalyzed by glncose-6-phosphate dehydrogenase (Zwf) to 6-phosphoghicono lactone (6PGL). 6PGL is then converted to D-Gluconate 6-phosphate by 6-phosphogluconoIactonase (Pgl). D- Gluconate 6-phosphate is then converted to D-Ribtilose 5-phosphate by 6-phosphogluconate dehydrogenase (Gnd). D-Ribulose 5-phosphate is then isomerized to D-Ribose 5-Pbosphate by ribose 5-phosphate isomerase A (RpiA) or ribose-5-phosphate isomerase B (RpiB), which is then dephosphorylated to free ribose by sugar phosphatase YbiV (YbiV) or sugar phosphatase YldA (YidA).

[0064] Finally; free ribose 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 glycogen biosynthesis, catalyzed by the enzyme phsphoghicomutase (Pgm); carbohydrate biosynthesis, catalyzed by glucose-6-phsphate isomerase (Pgi); the purine / pyrimidine ribonucleosides degradation pathway,, catalyzed by phosphopentomutase (DeoB); and the pentose phosphate pathway, catalyzed by transketolase I (TktA) and / or transketeolase 2 (TktB).rR ibosePro dictionEn

[0065] in certain embodiments, the presently disclosed microorganisms include overexpression of at least one gene encoding an enzyme cataly zing reactions for the production of D-ribose. In certain embodiments, the presently disclosed microorganisms include a recombinant polynucleotide encoding at least one enzyme catalyzing reactions for the production of D-ribose. In certain embodiments, the enzyme is an isomerase, for example an isomerase that converts D- ribulose 5-phosphate to D-ribose 5-phosphate, As used herein, the term “isomerase” refers to a class of enzymes that catalyze the inversion of asymmetric groups in a substrate with several centers of asymmetry. Non-limiting examples of isomerase include ribose 5-phosphate isomerase A (RpiA) and ribose-5-pbosphate isomerase B (RpiB),

[0066] In certain embodiments, the isomerase is a ribose-5-phosphate isomerase A (RpiA) (UniProt No, P0A7Z0). RpiA catalyzes the reversible conversion of ribose-5-phosphate to ribulose 5-phosphate. In certain embodiments. RpiA is an A. co2 RpiA. In certain embodiments, RpiA 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: I . hi certain embodiments, RpiA comprises the amino acid sequence set forth in SEQ ID NO; 1 . In certain embodiments, RpiA consists of the amino acid sequence set forth in SEQ ID NO: 1. SEQ ID NO: 1 is provided below:

[0067] in certain embodiments, the gene r / AA is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the nucleotide sequence set forth in SEQ ID NO: 2. In certain embodiments, rp / 'A comprises the nucleotide sequence set forth in SEQ ID NG: 2. In certain embodiments, r / AA consists of the nucleotide sequence set forth in SEQ ID NO: 2. SEQ ID NO: 2 is provided below:

[0068] In certain embodiments, the isomerase is a ribose 5-phosphate isomerase B (RpiB) (UniProt No. P37351 ). RpiB catalyzes the reversible conversion of ribpse-5 -phospha te to ribulose 5-phosphate. In certain embodiments, RpiB is an E. coli RpiB. In certain embodiments, 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; 3. In certain embodiments, RpiB comprises the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, RpiB consists of the amino acid sequence set forth in SEQ ID NO: 3. SEQ ID NO: 3 is provided below:

[0069] In certain embodiments, the gene r / AB 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: 4. In certain embodiments, rpiB comprises the nucleotide sequence set forth in SEQ ID NO: 4. hi certain embodiments, rplB consists of the nucleotide sequence set forth in SEQ ID NO: 4. SEQ ID NO: 4 is provided below:

[0070] In certain embodiments, the enzyme is a phosphatase, for example a phosphatase that dephosphorylates ribose 5-phosphate to free ribose. 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 pentose.

[0071] In certain embodiments, the phosphatase is a sugar phosphatase, e.g„ YbiV, G6425 (EcoCyc) P75792 (UniProt) and catalyzes the dephosphorylation of D-ribose 5-phosphate. In certain embodiments. YbiV is an £. coZz YbiV. In certain embodiments, YbiV comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO; 5. In certain embodiments, YbiV comprises the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, YbiV consists of the amino acid sequence set forth in SEQ ID NO: 5. SEQ ID NO: 5 is provided below:

[0072] In certain embodiments, the gene WF 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: 6. la certain embodiments, ybr F comprises the nucleotide sequence set forth in SEQ ID NO: 6. In certain embodiments, ybiF consists of the nucleotide sequence set forth in SEQ ID NO: 6, SEQ ID NO: 6 is provided below;

[0073] In certain embodiments, the phosphatase is a sugar phosphatase, e.g., YidA, P0A8Y5 (UniProt) and catalyzes the dephosphoiylatfon of D-ribose 5-phosphate. In certain embodiments, YidA is an Zi.'. co / i YidA. In certain einbodinients, YidA comprises an amino acid sequence that is at least about 80%, at least about 85%, at ieast 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: 7. In certain embodiments, YidA comprises the amino acid sequence set forth in SEQ ID NO: 7, In certain embodiments, YidA consists of the amino acid sequence set forth in SEQ ID NO: 7. SEQ ID NO: 7 is provided below:

[0074] In certain embodiments, the gene wrfd 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: 58, In certain embodiments, yidA comprises the nucleotide sequence set forth in SEQ ID NO: 58, In certain embodiments,consists of the nucleotide sequence set forth in SEQ ID NO: 58. SEQ ID NO: 58 is provided below:

[0075] In certain embodiments,, one or more transporter that transports glucose into the cell can be expressed (i.e,, overexpressed) in the cell, thereby increasing glucose in the cell. In some embodiments, one or both of galactose:Hrsymporter (GalP) and glucokinase (Glk) are expressedin the .microorganism. For example, in some embodiments. GalP transports glucose into the cell where it is phosphorylated to gIucose-6-phosphate by Glk and assimilated into central carbon metabolism.

[0076] In certain embodiments, the galactose:H5' symporter (GalP) is as described in EGU148 (EcoCyc) or P0AEP1 (LhiiProt). In certain embodiments, GalP is an E. colt 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: 36. In certain embodiments, GalP comprises the amino acid sequence set forth in SEQ ID NO: 36. In certain embodiments, GalP consists of the amino acid sequence set. forth in SEQ ID NO: 36. SEQ ID NO: 36 is provided below:

[0077] In certain embodiments, the gene g&LP is encoded by a nucleotide sequence that is at least about 80%, at least about 85%sat least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the nucleotide sequence set forth in SEQ ID NO: 37. In certain embodiments, gn / F comprises the nucleotide sequence set forth in SEQ ID NO: 37. In certain embodiments, galP consists of the nucleotide sequence set forth in SEQ ID NO: 37. SEQ ID NO: 37 is provided below:

[0078] In certain embodiments, the glucokinase (Glk) is as described in EG 12957 (EcoCyc) or P0A6V8 (UniProt). In certain embodiments, Gik. is an .£ call 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: 38. In certain embodiments, Glk comprises the amino acid sequence set forth in SEQ ID NO: 38. In certain embodiments, Glk consists of the amino acid sequence set forth in SEQ ID NO: 38. SEQ ID NO: 38 is provided below:

[0079] In certain embodiments, the gene glk is encoded by a nucleotide sequence that is at least about 80%. at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the nucleotide sequence set forth in SEQ ID NO: 39. In certain embodiments, glk comprises the nucleotide sequence set forth in SEQ ID NO: 39. In certain embodiments, glk consists of the nucleotide sequence set. forth in SEQ ID NO: 39. SEQ ID NO: 39 is provided below:

[0080] 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 conversion of ribulose 5-phosphate to ribose-5-phosphate. In another non-limiting example, the presently disclosed microorganism can include any enzyme that dephosphorylates ribose-5-phosphate to free ribose.

[0081] In certain embodiments, the presently disclosed microorganisms further include a recombinant polynucleotide encoding at least one enzyme of the pentose phosphate pathway. In certain embodiments, the at least one enzyme of the pentose phosphate pathway comprises gIucose-6-phosphate dehydrogenase (Zwf) (Entrez Gene ID: 946370; EcoCyc ID: EG1122I; UniProt ID: P0AC53). Zwf catalyzes the oxidation of glucose-6-phosphate to 6- phosphog I ucono lactone. In certain embodiments. Zwf is an E. coZz Zwf A representative nucleotide sequence of the gene zwf is set forth hi SEQ ID NO: 50 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 50. SEQ ID NO: 50 is provided below:

[0082] In certain embodiments, E. coU 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 aminoacid sequence set forth in SEQ ID NO: 51. In certain embodiments, E, coll Zwf comprises the amino acid sequence set forth in SEQ ID NO: 51. SEQ ID NO: 51 is provided below:

[0083] In certain embodiments, the at least one enzyme of the pentose phosphate pathway comprises phosphogluconate dehydrogenase (Gnd). In certain embodiments, the phosphogluconate dehydrogenase is as described in AAC75090 (EnsemblBaeteria) or P00350 (UiiiProt). Gnd catalyzes the decarboxylation of 6 -phosphogluconate to ribulose-5-phosphate, In certain embodiments, Gnd is an E. eoli Gnd. A representative nucleotide sequence of the gene gnd is set forth in SEQ ID NO: 52 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 52. SEQ ID NO; 52 is provided below;

[0084] In certain embodiments, E coll Gnd 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 aminoacid sequence set forth in SEQ ID NO: 53. In certain embodiments. £. coli Gad comprises the amino acid sequence set forth in SEQ ID NO: 53. SEQ ID NO: 53 is provided below:2.2. Competing Pathways

[0085] In certain embodiments, the presently disclosed microorganisms include a mutation of one or more gene encoding one or more enzymes regulating biochemical pathways that can reduce the production of D-ribose. 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 D-ribose. Physiologically, ceils 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 D-ribose production.

[0086] In certain embodiments, the presently disclosed microorganisms include a mutation of a gene encoding an enzyme of the glycogen biosynthesis. In certain embodiments, the presently disclosed microorganisms include a reduced expression of a gene encoding an enzyme of the glycogen biosynthesis. In certain embodiments, the enzyme of the glycogen biosynthesis is selected from the group consisting of phosphoglucomutase (Pgm), UDP-glucose pyrophosphorylase, glycogen synthase, glycogen branching enzyme, and glycogenin. In certain embodiments, the enzyme of the glycogen biosynthesis is phosphoglucomutase (Pgm) (Entrez Gene ID: 946370). Pgm (EC 5.4.2.2) is an enzyme that transfers a phosphate group on an a-D~ glucose monomer from the 1 to the 6 position in the forward direction or the 6 to the 1 position in the reverse direction. In certain embodiments, Pgm is an E. colt Pgm. A representative nucleotide sequence of gene pgm is set forth in SEQ ID NO: 9 or at least 90%. 95%, 95%, or 99% identical to SEQ ID NO: 9. SEQ ID NO: 9 is provided below:

[0087] In certain embodiments, £. call 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: 10. In certain embodiments, E. coll Pgm comprises the amino acid sequence set forth in SEQ ID NO: 10. SEQ ID NO: 10 is provided below;

[0088] In certain embodiments, Pgtn is a JBactlk®Pgm. A representative nucleotide sequence of gene pgm is set forth in SEQ ID NO: 59 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 59. SEQ I D NO: 59 is provided below:

[0089] In certain embodiments, Bacillus sublilis 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 ammo acid sequence set forth in SEQ ID NO: 60. In certain embodiments, Bacillus sublilis Pgm comprises the amino acid sequence set forth in SEQ ID NO: 60. SEQ ID NO: 60 is provided below:

[0091] In certain embodiment, facfowccus Zac / zs 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: 14. In certain embodiments, LacMcacctts Zaciitt Fgm comprises the amino acid sequence set forth in SEQ ID NO: 14. SEQ ID NO: 14 is provided below:

[0092] In certain embodiments, the presently disclosed microorganisms include a mutation of a gene encoding an enzyme of carbohydrate biosynthesis. In certain embodiments, the presently disclosed microorganisms include a reduced expression of a gene encoding an enzyme of the carbohydrate biosynthesis. In certain embodiments, the enzyme of the carbohydrate biosynthesis is glucose-6-phosphate isomerase (Pgi) ( Eiitrez Gene ID 948535), Pgi catalyzes the reversible isomerization of glucose-6-phosphate to fructose-6-phosphate. In certain embodiments, Pgi is an E, coli Pgi . A representa ti ve nucleotide sequence of gene j / gf is set forth in SEQ ID NO: 15. SEQ ID NO: 15 is provided below:

[0093] In certain embodiments, £ co / i Pgi comprises an amino acid sequence that is at least about 8G%Sat 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: 16. In certain embodiments, £ call Pgi comprises the amino acid sequence set forth in SEQ ID NO: 16, SEQ ID NO; 16 is provided below:

[0094] In certain embodiments, Pgi is a Bacillus sublilis pgi. A representative nucleotide sequence of a Bacillus subtilis gene pgi is set forth in SEQ ID NO: 17 or al least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 1.7. SEQ ID NO: 17 is provided below:

[0095] A representative amino acid sequence of / foctZ / zts SMMZIS Pgi is found as P80860 (Uniprot) or as set forth in SEQ ID NO: 18 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 18. SEQ ID NO: 18 is provided below:

[0096] In certain embodiments, Pgi is a Lactococcus lactis Pgi. A representative nucleotide sequence of a Z-ucfocoocw kwlis gene pgi 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:

[0097] A representative ainino acid sequence of Lactococcus lactis Pgi is found as P8118I (UniProt), or as set forth in SEQ ID NO: 20 or at least 90%. 95%, 95%, or 99% identical to SEQ ID NO.‘20, which is provided below.

[0098] In certai n embodiments, the enzyme of the purine / pyrimidine ribonucleosides degradati on pathway is phosphopentomutase (DeoB) (Entrez Gene ID 948910; EcoCyc ID; 10220; UniProt ID: P06A6K6). DeoB catalyzes the phosphyl group transfer between the C I and C5 carbon atoms of pentose. In certain embodiments, DeoB is an E, colt DeoB. A representative nucleotide sequence of gene deaB is set forth in SEQ ID NO: 21. SEQ ID NO: 21 is provided below:

[0099] In certain embodiments, coli DeoB 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 io the amino acid sequence set forth in SEQ ID NO; 22. In certain embodiments, E. coli DeoB comprises the amino acid sequence set forth in SEQ ID NO: 22. SEQ ID NO: 22 is provided below:

[0100] In certain embodiments, deo8 is a Bacillus subtilis deoB, A representative nucleotide sequence of a Bacillus subtilis gene deoB is set forth in SEQ ID NO: 23 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 23. SEQ ID NO: 23 is provided below:

[0101] A representative aniino acid sequence of Bacillus subtilis DeoB is found as P46353 (IJniprot) or as set forth in SEQ ID NO: 24 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO:24. SEQ ID NO: 24 is provided below:

[0102] In certain embodiments, DeoB is a ; l^c / ococcus herb deoB. A representative nucleotide sequence of a Laerococcus lacfis gene tfeoB is set forth in SEQ ID NO: 25 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 25. SEQ ID NO: 25 is provided below:

[0103] A .representative amino acid sequence Lactococcus lactis DeoB is found as Q9CH 12 (UniProt), or as set forth in SEQ ID NO: 26 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 26, which is provided below:

[0104] 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 inchide a reduced expression of a gene encoding an enzyme of the pentose phosphate pathway. In certain embodi ments, the enzyme of the pentose phosphate pathway is transketolase 1 (TkiA) and / or transketolase 2 (TktB). In certain embodiments, the enzyme of the pentose phosphate pathway is transketolase I (TktA) (Entrez Gene ID 947420; EcoCyc ID: EG11427; UniProt ID: P27302), Catalyzes the transfer of a two-carbon ketol group from a ketose donor to an aldose acceptor, via a covalent intermediate with the cofactor thiamine pyrophosphate. In certain embodiments, tktA is an E, coU tktA. A representative nucleotide sequence of gene tktA. is set forth in SEQ ID NO: 27. SEQ ID NO: 27 is provided below:

[0105] In certain embodiments, ZJ. COZZ TktA 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; 28. In certain embodiments, K co / Z TktA comprises the amino acid sequence set forth in SEQ ID NO: 28. SEQ ID NO: 28 is provided below:

[0106] In certain embodiments, zAv / l is a Bacillus subtilis (fad. A representative nucleotide sequence of a Bacillus subtilis gene tktA is set forth in SEQ ID NO; 29 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 29, SEQ ID NO: 29 is provided below:

[0107] A representative amino acid sequence of Bacillus subtilis TktA is found as P45694 (Uniprot) or as 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:

[0108] In certain embodiments, tklA is a Lactococcus lactis tklA. A representative nucleotide sequence of a Lactococcus lactis gene fktA is set forth in SEQ ID NO: 31 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 3 L SEQ ID NO: 31 is provided below:

[0109] A representative amino acid sequence of Lacfococcus lactis TktA is found as Q9F178 (UniProt), or as set forth in SEQ ID NO: 32 or at least 90%, 95%. 95%, or 99% identical to SEQ ID NO:32, which is provided below:

[0110] In certain embodiments, the enzyme of the pentose phosphate pathway is transketolase 2 (TktB) (Entrez. Gene ID 945865; EcoCyc ID: 12100: UniProt ID; P33570), Catalyzes the transfer of a two-carbon keto! group from a ketose donor to an aldose acceptor, via a covalent intermediate with the cofactor thiamine pyrophosphate.. In certain embodiments, TktB is an £. col'i TktB. A representative nucleotide sequence of gene tktB is set forth in SEQ ID NO: 33. SEQ ID NO: 33 is provided below:

[0111] In certain embodiments, E. coll TktB 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 leastabout 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: 34. In certain embodiments. E. coli TktB comprises the amino acid sequence set forth in SEQ ID NO: 34, SEQ ID NO: 34 is provided below;

[0112] In certain embodiments, the presently disclosed .microorganisms include a mutation of a gene encoding an epimerase. In certain embodiments; the epimerase is a D-allulose-6-phosphate 3-epimerase (AlsE) (UniProt No, P32719), AlsE catalyzes the reversible epimerization of D- aHulose-6-phosphate to D-h‘uctose-6-phospha.te. In certain embodiments, AlsE is ancol'i AlsE. In certain embodiments, the gene alsE is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the nucleotide sequence set forth in SEQ ID NO: 54. In certain embodiments, alaE comprises the nucleotide sequence set forth in SEQ ID NO: 54, SEQ ID NO: 54 is provided below.

[0113] In certain embodiments, AlsE comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 35. In certain embodiments, AlsE comprises the amino acid sequence set forth in SEQ ID NO: 35. SEQ ID NO; 35 is provided below:

[0114] In certain embodiments, the presently disclosed microorganisms include a mutation of a gene encoding an enzyme of glycolysis is pyruvate kinase. In certain embodiments, the enzyme of glycolysis is phosphofructokinase A (PfkA) (Enirez Gene ID: 948412). PfkA catalyzes the phosphorylation of D-fnictose-d-phosphate to fructose l,6~bisphosphate by ATP, the first committing step of glycolysis. In certain embodiments, PfkA is an E. colt PfkA. A representative nucleotide sequence of gene pjkA is set forth in SEQ ID NO: 55. SEQ ID NO: 55 is provided below:

[0115] In certain embodiments, E call. PfkA comprises an amino acid sequence that is at least about 80%. at least about 85%, at least about 90%. at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO; 56. In certain embodiments, E. coll PfkA comprises the amino acid sequence set forth in SEQ ID NO: 56. SEQ ID NO: 56 is provided below:

[0116] In certain embodiments, the presently disclosed microorganisms include a mutation of a gene encoding an enzyme of the gluconate degradation pathway. In certain embodiments, the enzyme of the gluconate degradation pathway is a phosphogluconate dehydratase (Edd) (EcoCyc No. EG10257; UniProt No. P0ADF6). Edd catalyzes the dehydration of D-gluconaie-6~ phosphate. In certain embodiments, Edd is an E. co / i Edd. Further details on the gene sequencesand amino acid sequences of phosphogl neonate dehydratase can be found in EcoCyc No. EG 10257.

[0117] In certain embodiments, the presently disclosed microorganisms include a mutation of a gene encoding at least one enzyme of the rtoOOZ operon, which is responsible for ribose catabolism. This operon includes the ribose transporter (RbsABC), D-ribose pyranase (RbsD), ribokinase (RbsK), and regulatory protein and sRNA RbsR and RbsZ, respectively. Inforniation regarding the gene sequences and amino acid sequences of enzyme of the rbsA&CDKllZ operon can be found in EcoCyc Nos: EG10814, EG10815, EG10816, EG 10817, EG10818, EG10819, and GO- 17077.

[0118] 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 glucok inase. In certain embodiments, the presently disclosed microorganisms do not include a deletion or a reduced expression of gIucose-6-phosphate isomerase.

[0119] In certain embodiments, the deletion of a gene comprises a non-frameshift deletion, a fiameshift deletion, or a combination thereof. In certain embodiments, die deletion of a gene can be achieved by an insertion (e.g., a non-frameshift insertion, a frameshift insertion, or a combination thereof). In certain embodiments, the deletion of a gene comprises a nonsense mutation.25. Cells

[0120] The present disclosure provides recombinant microorganisms. Any culturable microorganism is suitable for use in the compositions and methods described herein. In certain embodiments, the microorganism is a bacterium. In certain embodiments, the microorganism is selected from the group consisting of Aceiobacter aceti, Achromobacter, Acidiphilium. Acbiefobacien AetinOrnadura, Acti / Mpkines, Aeropynmpemix, Agrobacteriutn. Alcaligenes, Ananas comosus (M), Arthrobacter, Bacillus alcalophilus, Bacillus amyloliqutfaciens, BadOus hmiy, Bacillus czra / Zanv, j-fco / Zur c.kmsii, Bacillus lentus, Bacillus lickenifirmis, Bacillus mace / ans, Bacillus sietn'alhennophilus, Bacillus subtilis, Bifidobacterium, Breuibacillus bm-fr, Butkliolderia cepacia, Candida cylindracea, Carica papaya tly, Cellulosimicrobium. Cephalosporium, Chaetomium erraticum, Chaetomium gracile, Clostridium, Clostridium butyricum, Clostridium acetobutylicum, Clostridium thermacellum, Corynebacterium (ghtiamiatm), Catynebaclerium efficiens, Escherichia coli, Enierocacajs, Erwina chrysanthemi, Gliconobacter. (■lluconacetobacter, Haloarcula. Humicola msolens. Kifasatospora setae, Klebsiella, Klebsiella oxy f oca, Kocuria, Lacflactis, Lactobacilhis. Lactobacillus fermentum, Lactobacillus sake, Laclococcus, Lacfocaccus lactis. Leuconosloc, Methylocystis, Melhanolobus sickiae, Methanogenium organophihitn. Methanabacterium bryantii,Microbactemtm imperiale, Micrococcus lysodeikticus, Microbiriahis, Mifcor / avanictrs, MvwZxtczezwn, Myrotheclum, MzroEiezer, Mitrosomonas, Nocardia, Papaya coriea, Pediococcus. Fediococcits halophihis. Faraeoccus / KuUatrapkus, PrtipioriibacieriUfn, Pseudomoiuis. Pseudamomisfluorcscens, Pseudomonas denitrlfictms, Pyrococcus, Pyracoccusfi.iriosas, Pyrococcus horikashii, iffiteohfam, Rhisomucor miehei, Rh&omncor pasi / lus Tindi, RhEopus, Rhizopiis delemar, Rhisopus japonicas, Rhizopus niveus. Rhisopus orysae, Rhisopus oligosparus, Rhoaococcus, Sckroiinu libertina, SphingobacrcZerztzzzz mulfivorum, Sphuigahiim, Sphirrpmionas, Streptococcus. Streptococcus thermophilus Yd, Slreptomyces, Suvptomyccs griseus, Streplomyces lividans, Streplomyces tfiurmus. Streploniyccs ruhiginosus, Strepiomyces vMlaceoruber, Streploverticillium mobaraense, Teiragenococcus. Thermos. Tkiosphaera pantoiropha, Trametes, Vibrio aigiuoiyticus, Aanthomonas, Zyinomonas, and Z ' ymomonus inobilis. In certain embodiments, the microorganism is Escherichia coii IE. coii). In certain embodiments, the microorganism is Raciihis subiihs. In certain embodiments, the microorganism is ZacioeocczA' Zoezir.

[0121] In certain embodiments, the E. coli is selected from the group consisting of Enterotoxigenic E. colt (ETEC), Enteropathogentc E. coli (EPEC), Enteroinvasive E. coli (E1EC), Enteroheniorrhagic Zf coii (EHEC), Uropathogenic E. coli (UPEC), Verotoxin-producing E. coll, E cali 0157:H7, E. coil 0104:144, E coil 0121 , £ coii O104:H21 , E. eo / z KI, and E. coli NCI 0 L In certain embodiments, the E. coli is E. coii KI2. In certain embodiments, the E. coli is E. coiiB. In certain embodiments, the E. coli is E. coli C.

[0122] In certain embodimente, the E eo / z is derived from a strain selected from the group consisting of NCTC 12757, NCTC 12779, NCTC 12790, NCTC 12796, NCTC 12811, ATCC 11229, ATCC 25922, ATCC 8739, DSM 30083, BC 5849, BC 8265, BC 8267, BC 8268, BC 8270, BC 8271, BC 8272, BC 8273, BC 8276, BC 8277, BC 8278, BC 8279, BC 8312, BC 8317, BC 8319, BC 8320, BC 8321, BC 8322, BC 8326, BC 8327, BC 8331, BC 8335, BC 8338, BC 8341, BC 8344, BC 8345, BC 8346, BC 8347, BC 8348, BC 8863, and BC 8864.

[0123] In certain embodiments, the E. coii is derived from a strain selected from the group consisting of BC 4734 (O26:H11), BC 4735 (O157:H-), BC 4736 , BC 4737 (n.d.), BC 4738 (O157CH7), BC 4945 (O26:H-)}BC 4946 (Oi57:H7), BC 4947 (0111:H-), BC 4948 (0157:14), BC 4949 (05), BC 5579 (O157:H7), BC 5580 (O157:H7), BC 5582 (03 :H), BC 5643 (02:H5), BC 5644 (0128), BC 5645 (055:11-), BC 5646 (069:11-), BC 5647 (0101:119), BC 5648 (O103:H2), BC 5850 (O22:H8), BC 5851 (O55:H-), BC 5852 (O48:H21), BC 5853 (O26:H11), BC 5854 (0157:147), BC 5855 (0157:14-), BC 5856 (026:11-), BC 5857 (O103;H2)tBC 5858 (O26:H11), BC 7832, BC 7833 (O raw form:H-), BC 7834 (ONT:H-), BC 7835 (O103:H2), BC 7836 (057:14-), BC 7837 (ONT:H-), BC 7838, BC 7839 (O128:H2), BC 7840 (OI57:H-), BC 7841 (O23:H-), BC 7842 (O157:H-), BC 7843, BC 7844 (0157:14-), BC 7845 (0103 :H2), BC7846 (026:1111 ), BC 7847 (O145:H~), BC 7848 (O157:H-), BC 7849 (O156:H47), BC 7850, BC 7851 (0157:14-), BC 7852 (O157:H-), BC 7853 (05:H-), BC 7854 (O157:H7)SBC 7855 (0157:117), BC 7856 (026:11-), BC 7857, BC 7858, BC 7859 (ONT(H-), BC 7860 (012941-), BC 7861, BC 7862 (Ol03:H2), BC 7863, BC 7864 (0 raw fotm:H-), BC 7865, BC 7866 (O26:H- ), BC 7867 (0 raw form:H-), BC 7868, BC 7869 (ONT.'H-), BC 7870 (0113:H-), BC 7871 (0NT:H-), BC 7872 (ONT:H-), BC 7873, BC 7874 (O raw formiH-), BC 7875 (O157:H-), BC 7876 (Oll IrH-), BC 7877 (O146:H21), BC 7878 (O145;H-), BC 7879 (O22;H8), BC 7880 (O raw fomrH-), BC 7881 (O145:H-), BC 8275 (O157:H7), BC 8318 (O55:K-:H-), BC 8325 (O157:H7), BC 8332 (ONT), and BC 8333.

[0124] In certain embodiments, the E. call is derived from a strain selected from the group consisting of BC 8246 (O152:K-:H-), BC 8247 (O124;K(72):H3), BC 8248 (0124), BC 8249 (0112), BC 8250 (O136:K(78):H-)SBC 8251 (0124:11-), BC 8252 (O144:K-:H-), BC 8253 (O143:K:H-), BC 8254 (0143), BC 8255 (0112), BC 8256 (028a.e), BC 8257 (0124:11-), BC 8258 (0143), BC 8259 (OI67:K-:H5), BC 8260 (0128a, e.:H35), BC 8261 (0164), BC 8262 (O164:K-:H-), BC 8263 (0164), and BC 8264 (0124),

[0125] In certain embodiments, the E. col'i is derived from a strain selected from the group consisting of BC 5581 (O78:H11), BC 5583 (O2:K1), BC 8221 (0118), BC 8222 (O148:H-), BC 8223 (0111), BC 8224 (0110: H-), BC 8225 (0148), BC 8226 (0118), BC 8227 (O25;H42), BC 8229 (06), BC 8231 (0153:1145), BC 8232 (09), BC 8233 (0148), BC 8234 (0128), BC 8235 (0118), BC 8237 (Ol l i), BC 8238 (O110:H17), BC 8240 (0148), BC 8241 (O6H16), BC 8243 (0153), BC 8244 (015:11-), BC 8245 (020), BC 8269 (O125a.c:H-), BC 8313 (06:116), BC 8315 (0153:H-), BC 8329, BC 8334 (O118:H12), and BC 8339,

[0126] In certain embodiments, the £.is derived from a strain selected from the group consisting of BC 7567 (086), BC 7568 (0128), BC 7571 (0114), BO 7572 (0119), BC 7573 (0125), BC 7574 (0124), BC 7576 (0127a), BC 7577 (0126), BC 7578 (0142), BC 7579 (026), BC 7580 (OK26), BC 7581 (0142), BC 7582 (055), BC 7583 (0158), BC 7584 (0-), BC 7585 (0-), BC 7586 (0-), BC 8330, BC 8550 (026), BC 8551 (055), BC 8552 (0158), BC 8553 (026), BC 8554 (0158), BC 8555 (086), BC 8556 (0128), BC 8557 (OK26), BC 8558 (055), BC 8560 (0158), BC 8561 (0158), BC 8562 (0114), BC 8563 (086), BC 8564 (0128), BC 8565 (0158), BC 8566 (0158), BC 8567 (0158), BC 8568 (Ol l i ), 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 (01581 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 (Ol 14), BC 8594 (0114), BC 8595 (0125), BC 8596 (0158), BC 8597 (026), BC 8598 (026), BC 8599 (0158), BC 8605 (0158), BC 8606 (0158), BC 8607(0158), BC 8608 (0128), BC 8609 (055), BC 8610 (O114), 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).

[0127] In certain embodiments, tbeK sub / ilis is derived from Strain 168.

[0128] In certain embodiments, the / ., lacfis is derived from Strain A 12.

[0129] In certain embodiments, the microorganism is a fungal cell In certain embodiments, the fimgal cell is selected from the group consisting of Aspergillus, Aspergilhrs rudulans, Aspargillus n?ger, frspargi / te onse, Aspergillus melletrs, / ispergi / tos pulyendenfus, Aspergillus saiioi, Aspergillus so / ea, Aspergillus lerreus, Aspergillus pseudtuerreuy Aspergillus usamli, Candida rugose, Jssatchenkia orietrlaiis, Kluyveromyces, Kh / yx^eram^esfreigilis. .Kitywmrnyces laclis. Kluyvemmyces marxianas, Penicillhm, Penicillium camemberti, Periicillmm eitrmum, Penicillium ernersonii, Penicillium roquefvrti, Penicillum iilactimnn, Perricilluui multicolor, Elurdosporidium torul aides, Saccharornyces cerevixiae, Schizosaccharomyces pombe, Trichoderrna, Trichoderrna kmgibrachjalum, liichoderma reesei, I'richutErma viride, Tricl-ursporcm pemclllaiimi, Yorrawia lipofylica, eosdZygosaccharomyces rmixii.

[0130] In certain embodiments, the microorganism is a yeast cell. In certain embodiments, the yeast cell is Saccharomyces cerevisiae.2.4. Exemplary microorganisms

[0131] In certain embodiments, the present disclosure provides a recombinant microorganism comprising an increased production of D-ribose as compared to a naturally occurring microorganism. In certain embodiments, the recombinant microorganism comprises an exogenous isomerase and an exogenous phosphatase. In certain embodiments, the exogenous isomerase is a ribose 5 -phosphate isomerase A (RpiA) or ribose-5-phosphate isomerase B (RpiB). In certain embodiments, the exogenous phosphatase is sugar phosphatase YbiV (YbiV) or sugar phosphatase YidA (YidA), In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is E. coll.

[0132] In certain embodiments, the present disclosure provides a recombinant microorganism comprising an increased production of D-ribose as compared to a naturally occurring microorganism. In certain embodiments, the recombinant microorganism comprises an exogenous isomerase, an exogenous phosphatase, and a deletion of one, two, three, four or, five [5] genes. In certain embodiments, the exogenous isomerase is a ribose 5-phosphate isomerase A (RpiA) or ribose-5~phosphate isomerase B (RpiB). In certain embodiments, the exogenous phosphatase is sugar phosphatase YbiV (YbiV) or sugar phosphatase YidA (YidA).[01.33] In certain embodiments, the five deleted genes are pbosphogiucomutase ( / / gm), glucose- 6-phosphate isomerase (pg / ), phosphopentomutase (deoE), transketolase 1 (thfA), andtransketolase 2 ( / AzB). In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is A’, coZz.

[0134] In certain embodiments, the present disclosure provides a microorganism comprising a recombinant polynucleotide, wherein the microorganism comprises an increased production of D- ribose as compared to a naturally occurring microorganism. In certain embodiments, the recombinant polynucleotide comprises a nucleotide sequence encoding an exogenous isomerase and an exogenous phosphatase. In certain embodiments, the exogenous isomerase is a ribose 5- phosphate isomerase A (RpiA) or ribose-5-phosphate isomerase B (RpiB). In certain embodiments, the exogenous phosphatase is sugar phosphatase YbiV (YbiV) or sugar phosphatase YidA (YidA). In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is A. colt

[0135] In certain embodiments, the present disclosure provides a microorganism comprising a recombinant polynucleotide, wherein the microorganism comprises an increased production of D- ribose as compared to a naturally occurring microorganism. In certain embodiments, the recombinant polynucleotide comprises a nucleotide sequence encoding an exogenous isomerase and an exogenous phosphatase. In certain embodiments, the exogenous isomerase is a ribose 5- phosphate isomerase A (RpiA) or ribose-5-phosphate isomerase B (RpiB). In certain embodiments, the exogenous phosphatase is sugar phosphatase YbiV (YbiV) or sugar phosphatase YidA (YidA). In certain embodiments, the microorganism further comprises a deletion of a first gene. In certain embodiments, the first gene is phosphoglucomutase (pgm). In certain embodiments, the microorganism further comprises a deletion of a second gene. In certain embodiments, the second gene is gIucose-6-phosphate isomerase (pgz). In certain embodiments, the m icroorganism further comprises a deletion of a third gene. In certain embodiments, the third gene is phosphopentomutase (oWA), In certain embodiments, the microorganism further comprises a deletion of a fourth gene. In certain embodiments, the fourth gene is transketolase 1 (zAzft), In certain embodiments, the microorganism further comprises a deletion of a fifth gene, hi certain embodiments, the fifth gene is transketolase 2 (ikt8). In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is A. coft, In certain embodiments, the bacterium is A vubft / A. In certain embodiments, the bacterium is A. laclis.

[0136] In certain embodiments, the recombinant microorganism further comprises at least one exogenous enzyme of the pentose phosphate pathway. In certain embodiments, the at least one exogenous enzyme of the pentose phosphate pathway comprises glucose-6-phosphate dehydrogenase (Zwf). In certain embodiments, the at least one exogenous enzyme of the pentose phosphate pathway comprises phosphogluconate dehydrogenase (Gnd). In certain embodiments,the recombinant microorganism further comprises a mutation of a gene encoding for an enzyme of glycolysis. In certain embodiments, the enzyme of the glycolysis is phosphofructokinase- 1 (PfkA). In certain embodiments, the recombinant microorganism further comprises a mutation of a gene encoding for an epimerase compared to a naturally occurring microorganism. In certain embodiments, the epimerase is alIulose-6-phosphate-3-epimeraseIn certain embodiments, the recombinant mi croorgani sm further comprising a mutation of a gene encoding for an enzyme of the gluconate degradation pathway compared to a naturally occurring microorganism. In certain embodiments, the enzy me of the gluconate degr adat ion pathway is phosphogluconate dehy dratase (Edd). In certain embodiments, the recombinant microorganism further comprises a mutation of at least one gene encoding for art enzyme of the ribose catabolism pathway. In certain embodiments, the at least one gene encoding for an enzyme of the ribose catabol ism pathway is selected from D-ribose transporter ATP binding subunit (RbsA), D-ribose transporter periplasmic binding protein (RbsB), D-ribose transporter membrane subunit (RbsC), D-ribose pyranase (RbsD), D-ribose kinase (RbsK), DNA-binding transcriptional dual regulator (RbsR), small regulatory RNA (RbsZ), or combinations thereof.

[0137] In certain embodiments, the present disclosure provides a recombinant microorganism comprising an increased production of D-ribose as compared to a naturally occurring microorganism. In certain embodiments, the recombinant microorganism cornprises an exogenous isomerase, an exogenous phosphatase, an exogenous nuclease, a sgRNA, and a deletion of five (5) genes. In certain embodiments, the exogenous isomerase is a ribose 5- phosphate isomerase A (RpiA) or ribose-5-phosphate .isomerase B (RpiB). In certain, embodiments, the exogenous phosphatase is sugar phosphatase YbiV (YbiV) or sugar phosphatase YidA (YidA), In certain embodiments, the five deleted genes are pgm^pgi, deaB, ik / A, and tkiB. In certain embodiments, the sgRNA targets pgngpgg cfeo / I, nlxd, or tklft. In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is E. cap. In certain embodiments, the bacterium is B. stforihw. In certain embodiments, the bacterium is L. k / cik. In certain embodiments, the recombinant microorganism further comprises a recombinant polynucleotide encoding Zwf, Gnd, or both. In certain embodiments, the recombinant microorganism further comprises a mutation of alIulose-6-phosphate 3-epinierase (AlsE). In certain embodiments, the recombinant microorganism further comprises a mutation of phosphogluconate dehydratase (Edd). In certain embodiments, the recombinant microorganism further comprises a mutation of phosphofiructokinase-1 (PfkA). In certain embodiments, the recombinant microorganism further comprises a mutation of at least one gene encoding for an enzyme of the ribose catabolism pathway is selected from D-ribose transporter ATP binding subunit (RbsA), D-ribose transporter periplasmic binding protein (RbsB ), D-ribose transportermembrane subunit (RbsC), D-ribose pyranase (RbsD), D-ribose kinase (RbsK), DNA-binding transcriptional dual regulator (RbsR), small regulatory RNA (RbsZ), or combinations thereof.

[0138] In certain embodiments, the present disclosure provides a microorganism comprising a recombinant polynucleotide, wherein the microorganism comprises an increased production of D- ribose as compared to a naturally occurring microorganism. In certain embodiments, the recombinant polynucleotide comprises a nucleotide sequence encoding an exogenous isomerase, a nucleotide sequence encoding an exogenous phosphatase, and a nucleotide sequence encoding a nuclease. In certain embodiments, the exogenous isomerase is a ribose 5-phosphate isomerase A (RpiA) or ribose-5-phosphate isomerase B (RpiB). In certain embodiments, the exogenous phosphatase is sugar phosphatase YbiV (Yb.iV) or sugar phosphatase YidA (YidA). In certain embodiments, the microorganism further comprises a deletion of a first gene. In certain embodiments, the first gene is phosphoglucomutase (pgm). In certain embodiments, the microorganism further comprises a deletion of a second gene. In certai n embodiments, the second gene is glucose-6-phosphate isomerase (ngi), In certain embodiments, the microorganism further comprises a deletion of a third gene. In certain embodiments, the third gene is phosphopentomutase (rfeo / f). In certain embodiments, the microorganism further comprises a deletion of a fourth gene. In certain embodiments, the fourth gene is transketolase 1 ( / fed). In certain embodiments, the microorganism further comprises a deletion of a fifth gene. In certain embodiments, the fifth gene is transketolase 2 (tk / R). In certain embodiments, the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is £. coik In certain embodiments, the bacterium is ,B. sadii / is, In certain embodiments, the bacterium is L. factis. In certain embodiments, the recombinant microorganism further comprises a recombinant polynucleotide encoding Zwf, Gnd, or both. In certain embodiments, the recombinant microorganism further comprises a mutation of allulose-6-phosphate 3-epimerase (ahE). In certain embodiments^ the recombinant microofganisro further comprises a mutation of phosphogluconate dehydratase (trie / ). In certain embodiments, the recombinant microorganism further comprises a mutation of phosphofructokinase- 1 (pflcA), In certain embodiments, the recombinant microorganism further comprises a mutation of at least one gene encoding for an enzyme of the ribose catabolism pathway is selected from D-ribose transporter ATP binding subunit (RbsA), D-ribose transporter periplasmic binding protein (RbsB), D-ribose transporter membrane subunit (RbsC), D-ribose pyranase (RbsD), D-ribose kinase (RbsK), DNA-binding transcriptional dual regulator (RbsR), small regulatory RNA (RbsZ), or combinations thereof.J. MetJwds far Producing and Generating Microotganistns Pilose

[0139] 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 maybe used to introduce one or more .recombinant polynucleotides of the present disclosure into a microorganism, including without limitation protoplast fusion, transfection, transformation, conj ugation, and transduction. These techniques include con ventional molecular biology techniques (e.g,, recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Additional information on these techniques can be found in Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al, 1989); Oligonucleotide Synthesis (Gait, ed., 1984); Animal Cell Culture (Freshney, ed., 1987); Gene Transfer Vectors for Mammalian Cells (Miller & Calos, eds., 1987); Current Protocols in Molecular Biology (Ausubel et al, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al, eds,, 1994); and Current Protocols in Immunology (Coligan et aL, eds., 1991).3. / . Recombinant polynucleotides

[0140] In certain embodiments, the recombinant polynucleotides disclosed herein can be stably integrated into a microorganism chromosome. In certain embodiments, the recombinant polynucleotides disclosed herein are stably integrated into a microorganism chromosome using homologous recombination, transposition-based chromosomal integration, recombinase-mediated cassette exchange (RMCE; e.g.}using a. Cre-fox system), or an integrating plasmid (c.g., a yeast integrating plasmid). A variety of integration techniques suitable for a range of microorganisms are known in the art (see, e g., Griffiths, A.J.F., Miller, J.H., Suzuki, D.T. et al. An Introduction to Genetic Analysis. 7tned. New York: W.H. Freeman; 2000), hi certain embodiments, the recombinant polynucleotides disclosed herein are maintained in a recombinant microorganism of the present disclosure on an. extra-chromosomal plasmid (e.g., an expression plasmid or vector). A variety of extra -chromosomal plasmids suitable for a. range of microorganisms are known in the art, including without limitation replicating plasmids (e,g, yeast replicating plasmids that include an autonomously replicating sequence, ARS), centromere plasmids (e.g., yeast centromere plasmids that include an autonomously replicating sequence, GEN), episomal plasmids (b.g., 2- p.m plasmids), and / or artificial chromosomes A<g,, yeast artificial chromosomes, YACs, or bacterial artificial chromosomes. BACs).3. / . / Sectors

[0141] 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 DMA construct used as a. cloning and / or expression vector. In certain embodiments, plasmids can be extrachromosomal self-replicating genetic elements (e.g., episomal plasmids) when introducedinto a microorganism, In certain embodiments, plasmids can integrate into a microorganism chromosome. In certain embodiments, vectors can direct the expression of coding regions to which they are operatively linked, e.g. “expression vectors.” These expression vectors allow the expression of exogenous polynucleotides and / or polypeptides in microorganisms. In certain embodiments, the vectors allow the integration of one or more polynucleotides into the genome of a microorganism,

[0142] 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.

[0143] In certain embodiments, a vector discloses herein comprises nucleotide sequences in a single operon,3, L2. Promoters

[0144] 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 rpiA. or tp / B gene and / or ybiF or yid4 gene can be operably linked to a control sequence, enhancer, or promoter.

[0145] As used herein, the term “promoter ’ refers to any nucleotide sequence that regulates the initiation of transcription for a particular ceding sequence under its control. Biologically, promoters are not : transcribed but coordinate the assembly of components that initiate the transcription of other nucleotide sequences. In addition, promoters can limit this assembly and subsequent iranscription to specific prerequisite conditions. For example, but without any limitation, a promoter can allow transcription in response to one or more euvironmeutaL temporal, or developmental stimuli. Bacterial and fungal cells possess a multitude of proteins that sense external or internal condi tions and initiate signaling cascades ending in die 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,

[0146] to 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 io internal or external stimuli. Constitutive promoters are commonly used in recombinant engineering to ensure the continuous expression of a desired nucleotide sequence. Constitutive promoters result in a robust amount of nucleic acid expression, and, as such, are used in many recombinant engineering applications to ach ie ve a high level of recomb inant protein and enzymatic activity. Non-limiting examples of constitutive promoters encompassed by the present disclosure include E, colt promoters Pxpe, Ao, / Nxw, PRAAIC Pi and A from traB, and the lambdaphage promoter ft, (Liang, S.T. et al. ftWft. ftft 292( 1): 19-37 (1999)). In some embodiments, the promoter is active in the stationary phase of the microorganism. Exemplary stationary phase promoters can be found in, e.g., Shimada, et a / ., JOURNAL OF BACTERIOLOGY. Nov. 2004, p. 7112-7122; Pletnev at elsACTA NATURAE i VOL. 7 M 4 (27) 2015.

[0147] In certain embodiments, rhe 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). Ln certain embodiments, an ind uci ble promoter drives a graded level of expression correlated with the amount of stimulus. Non-limiting examples of stimuli for inducible promoters include heat shock, exogenous compounds or a lack thereof ft " , a sugar, metal, drug, or phosphate), salts or osmotic shock, oxygen, and biological stimuli fe.g., a growth factor or pheromone). Non-limiting examples of inducible promoters include the ft colt promotersftac, ft'?, ftvro, and

[0148] 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 rpiA or rpiB gene operably linked to a first promoter and a nucleotide sequence encoding the jftz'F or yidA gene operably linked to a second promoter, wherein the first and second promoter is the same. In certain embodiments, the multiple promoters can be different For example, but without any limitation, the recombinant polynucleotide can include a nucleotide sequence encoding the rpiA or rpiB gene operably linked to a first promoter and a nucleotide sequence encoding iheyft T or yidA gene operably linked to a second promoter, wherein the first and second promoter are different. In certain embodiments, the promoter is a ftjaroi promoter. In certain embodiments, the promoter comprises the nucleotide sequence set forth in SEQ ID NO: 40. In certain embodiments, the , / h.UcO ! promoter consists of the nucleotide sequence set forth in SEQ ID NO:40. The Puacor promoter is a hybrid regulatory region including the promoter ft. of phage lambda with the CI binding sites replaced with lacOr The hybrid design allows for a strong promotion that can be repressed by Lad, the Lac inhibitor (i.e., repressor) or induced by IPTG.

[0149] In certain embodiments, the promoter is a Ptwioi promoter. In certain embodiments, the ftwEtoi promoter comprises the nucleotide sequence set forth in SEQ ID NO: 41. In certain embodiments, the ft -rerot promoter consists of the nucleotide sequence set forth in SEQ ID NO:41.

[0150] In certain embodiments, the promoter is a ft? promoter. In certain embodiments, the ft? promoter comprises the nucleotide sequence set forth in SEQ ID NO: 42. In certain embodiments, the ft? promoter consists of the nucleotide sequence set forth in SEQ ID NO: 42.

[0151] In certain embodiments, the promoter is a Ptetpromoter. In certain embodiments, the promoter comprises the nucleotide sequence set forth in SEQ ID NO: 43. In certain embodiments, the A* promoter consists of the n ucleotide sequence set forth in SEQ ID NO: 43.

[0152] In certain embodiments, the promoter is apromoter. In certain embodiments, the promoter comprises the nucleotide sequence set forth in SEQ ID NO: 44. In certain embodiments, thepromoter consi sts of the nucleotide sequence set forth in SEQ ID NO: 44,

[0153] / h.ueoi promoter nucleotide sequence:

[0154] promoter nucleotide sequence:

[0155] An promoter nucleotide sequence:

[0156] Aet promoter nucleotide sequence:

[0157] Pga<iB promoter nucleotide sequence:

[0158] In certain embodiments, the promoter is a stationary phase promoter. As used herein, the term “stationary phase promoter” refers to a promoter upstream of a gene that is transcribed during the stationary phase of a microorganism growth. The life cycle of an E. coli culture includes 5 distinct phases: lag, logarithmic, stationary, death, and long-term stationary phase. The lag phase occurs when cells are inoculated into media and adjust their metabolic processes according to their new environment. The cells will then rapidly grow and divide, entering the logarithmic phase. It is at this time that enzymes related to central carbon metabolism are most important, and the transcription of corresponding genes will be upregalated. Once the cells sense, environmental stressors such as scarcity of media nutrients, their growth and division slows, and the culture enters the stationary phase. The use of a stationary phase promoter prevents the production pathway from competing with central carbon metabolism for carbon flux during the logarithmic phase of growth, a time when cells need carbon to rigorously grow and divide.

[0159] In certain embodiments, the stationary phase promoter is In certain embodiments, the Pg^g promoter comprises the nucleotide sequence set forth in SEQ ID NO: 44 or SEQ ID NO:45. In certain embodiments, the PgM-gi promoter consists of the nucleotide sequence set forth in SEQ ID NO: 45.

[0160] In certain embodiments, the stationary phase promoter is / Q7A.?. In certain embodiments, the PebpAi promoter comprises the nucleotide sequence set forth in SEQ ID NO: 46. In certain embodiments, the PcbpM promoter consists of the nucleotide sequence set forth in SEQ ID NO:46.

[0161] In certain embodiments, the stationary phase promoter is PWM. In certain embodiments, the promoter comprises the nucleotide sequence set forth in SEQ ID NO: 47. In certain embodiments, thepromoter consists of the nucleotide sequence set forth in SEQ ID NO: 47.

[0162] In certain embodiments, the stationary phase promoter is P^. In certain embodiments, the Pdps promoter comprises the nucleotide sequence set forth in SEQ ID NO: 48. In certain embodiments, the P^ promoter consists of the nucleotide sequence set forth in SEQ ID NO: 48.3. 13„ Genetic Markers

[0163] In certain embodiments, the presently disclosed recombinant polynucleotides include genetic markers. These genetic markers allow the selection of microorganisms that have one ormore desired polynucleotides (e.g., recombinant polynucleotides ). In certain embodiments, the genetic marker is an antibiotic resistance marker selected from the group consisting of Apramycin resistance. Ampicillin resistance, Kanamycin resistance. Spectiuomycin resistance, Tetracycline 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 fogy 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, hi certain embodiments, the auxotrophic marker is a gene for synthesizing adenosine, biotin, thiamine, leucine, glucose, lactose, or maltose. In certain embodiments, a microorganism of the present disclosure is grown under conditions in which an auxotrophic resistance marker is expressed in an environment or medium lacking the corresponding nutrient and confers growth to the microorganism (lacking an endogenous ability to produce the nutrient), thereby selected for the microorganism with successful integration of the marker.X2. De / ezfonx and Reduced Expression of Genes

[0164] 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 repeals (CRISPR.) system, or a combination thereof

[0165] 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 (aprotein able to .modify DNA utilizing crRNA as its guide), CRISPR RNA (crRNA, which contains the RNA used by Cas9 to guide it to the correct section of host DNA along with a region that binds to tracrRNA (generally in a hairpin loop form.) forming an active complex with C-as9), transactivating crRNA (tracrRNA, binds to crRNA and. forms an active complex with Cas9), and an optional section of DNA. repair template (DNA that guides the cellular repair process allowing insertion of a specific DNA sequence). .Multiple crRNA’s and. the tracrRNA can be packaged together to form a single-guide RNA (sgRNA). This sgRNA can be joined together with the Cas9 gene and made into a plasmid in order to be transfected into cells. In certain embodiments, the CRISPR system comprises base editors. In certain embodiments, the CRISPR system comprises transposasesZtecombinases. In certain embodiments, the CRISPR system comprises prime editors. In certain embodiments, ths 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.. Arifore824-844 and in Nunez et al,, Cell 184,9 (2021 ); 2503-2519, and Jiang et al.. Appt Environ MierobioL 2015 Apr;81(7):2506-14, the contents of each of which are incorporated by reference in their entireties.

[0166] In certain embodiments, the deletion is generated by a zinc- finger nuclease. A zine-finger nuclease (ZFN) is an artificial restriction enzyme, which is generated by combining a zinc finger DNA-binding domain with a DNA-cleavage domain. A zinc finger domain can be engineered to target specific 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 FokI,

[0167] 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.

[0168] hi certain embodiments, the deletion is generated by a meganuclease. A meganuc lease 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.

[0169] In certain embodiments, the present disclosure also provides methods to reduce the expression of any of the genes or enzymes disclosed herein. In certain embodiments, the reduced expression of genes and enzymes disclosed herein comprises using oligonucleotides that have complementary sequences to the mRNA of the genes disclosed herein (e.g., pgm.pgi, deuB, tkM, tkiB, etc.). Non-limiting examples of these oligonucleotides include small interference RNA (siRNA), short hairpin RNA (shRNA), and microRNA (miRNA). In certain embodiments, these oligonucleotides can be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to at least a portion of a pg® mRNA sequence. In certain embodiments, these oligonucleotides can be identical to at least a portion of a / tg / u 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 pgi mRNA sequence. In certain embodiments, these oligonucleotides can be identical to at least a portion of a pgz mRNA sequence. In certain embodiments, these oligonucleohdes 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% i dentical to at least a portion of a tfeoZi mRN A sequence. In certain embodiments, these oligonucleotides can be identical to at least a portion of a BeoB 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 a t least a portion of a tkfA mRNA sequence. In certain embodiments, these oligonucleotides can be identical to at least a portion of a tktA mRNA sequence. In certain embodiments, these oli gonucl eotides 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 iklB mRNA sequence. In certain embodiments, these oligonucleotides can be identical to at least a portion of a iktft mRNA sequence. In certain embodiments, antisense nucleic acid, shRNA, miRNA, or siRNA molecules can include DNA or atypical or non-naturally occurring residues, for example, but not limited to, phosphorothioate residues.

[0170] In some embodiments, reduction of expression of genes and enzymes disclosed herein can comprise use of CRISPR , which can mutate the coding sequence or promoter, to lower or remove expression of the gene product, or CRISPRi can be targeted to the genes disclosed herein, thereby reducing expression of one of more of the genes. See, e.g., Arroya-OIarte, ef a / ., A^craorganisms, 2021 Apr; 9(4): 844. Zhang et al , Frtmf. Microhm / . (31 March 2021).

[0171] In certain embodiments, the reduced expression of genes and enzymes disclosed herein comprises using a CRISPR system. In certain embodiments, the CRISPR system includes a Cas9. In certain embodiments, Cas9 comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 57. In certain embodiments, Cas9 comprises the amino acid sequence set forth in SEQ ID NO: 57. In certain embodiments, Cas9 consists of the amino acid sequence set forth in SEQ ID NO: 57. SEQ ID NO: 57 is provided below:

[0172] 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 inchides a catalytically dead Cas9 (dCas9). dCas9 is a programmable transcription factor that can be targeted to promoters through sgRNAs, where it can function as repressor. In certain embodiments, dCas9 comprises an amino acid sequence that is at least about5Q80%. 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: 49, In certain embodiments, dCa§9 comprises the amino acid sequence set forth in SEQ ID NO: 49, In certain embodiments, dCas9 consists of the amino add sequence set forth in SEQ ID NO: 49. SEQ ID NO: 49 is provided below:

[0173] In certain embodiments, the nuclease (e,g.. Cas9 or dCas9) is regulated by a promoter (e.g,. described in Section 3,1.2), In certain embodiments, the promoter is an inducible promoter. In certain embodiments, the promoter is a stationary phase promoter. In certain embodiments, the CRISPR / CRISPRi system includes a small guide RNAs (sgRNA), In certain embodiments, the sgRNA of the CRISPR / CRISPRi. system targets a gene encoding an enzyme of a competing pathway. For example, but without any limitation, the sgRNA can target the pgm gene, the pgi gene, the deoB gene, the tktA gene, or the fktB gene. In certain embodiments, the sgRNA can target any portion of a gene. For example, but without any limitation, the sgRNA can target a promoter, an operator, or a sequence encoding a protein,

[0174] In certain embodiments, the CRISPR / CRISPRi system includes a nuclease (e.g„ Cas9 or dCas9) and a sgRNA, In certain embodiments, the nuclease (e.g., Cas9 or dCas9) is regulated by an inducible promoter. In certain embodiments, the inducible promoter is / -%. Transformation and Gene e Ek dditing

[0175] 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 transformedinto 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 tansformanou, Agrobacterium transformation, nano-spike transformadon, electroporation, and virus transformation.

[0176] 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., Dibuer, M., Battey, I., 1986 ‘'Basic Methods in Molecular Biology”; Gietz ei at.. Nucleic Acids Res. 27:69-74 (1992); Ito et al., J. BacieroL 153:163-168 (1983); and Becker and Guareme, Methods in Enzymology 194:182-187 (1991)). In certain embodiments, transformed microorganisms are referred to as recombinant microorganisms.

[0177] 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. Ii. ei al. “Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems.” Nucleic Acids Res (2013). doi:10:1093 / nar / gkil35; Ren, Z. J., Baumann, R. G. & Black, L. W. “Cloning of linear DNAs in vivo by overexpressed T4 DNA ligase: construction of a T4 phage hoc gene display vector,” Gene 195, 303-31 1 (1997); Lin, S., Staahl, B. T., Alla, R. K. & Doudua, J. A. “Enhanced homology-directed human genome engineering by controlled timing of CRISPR / Cas9 delivery.” Elite 3, e04766 (2014)).3.4. Recombination Systems

[0178] 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 DN A fragment, introduced as and translated from RNA or set of R'NAs, or introduced as a protein or set of proteins. In certain embodiments, the methods include a recombinant polynucleotide disclosed herein. In certain embodiments, the polynucleotide includes sequence homologous (e.g. left and right homology arms) to a region in a nucleic acid (e.g., genome, plasmid, etc.) such that the left and right homology arms are separated by a designed genetic edit (e.g., promoter, insertion, substitution, SNR terminator,degron, a sequence for a tag, sequence for a degradation signa! 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 repli cat ion (e.g., R6K).

[0179] 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.

[0180] 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 RN A, or integron) and be integrated into the genome of the host cell or be stably expressed from an exfrachrotuosomal element. In certain embodiments, the proteins from one or more recombination systems can be introduced as RNA and be translated by the host ceil. In certain embodiments, the proteins from one or more recombination systems can be introduced as proteins into the host cell. Non-limiting examples of recombination systems include lambda red recombination system, RecET recombination system, Red / ET recombination system, any homologs, orthologs, or paralogs of proteins from a lambda red recombination system, RecET recombination system. Red / ET recombination system, lambda red-mediated recombination system, or any combination thereof. Details on the recombination systems from the RecET recombination system can be any of those as described in Zhang ¥., Buchholz F., Muyrers J.P.P. and Stewart A.F. “A new logic for DNA engineering using recombination In E. call ” 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, co / iT Nature Biotechnology 18 (2000) 1314-1317 and Muyrers JP et al., “Techniques: Recombinogenicengineering-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.4 Methods for producing ribose

[0181] The present disclosure also provides methods for producing D-ribose. Certain cell- free methods (e.g,, za vz / z-o synthesis) can have a predicted thermodynamically unfavorable reaction. While production of D-ribose can be energetically favorable, it is a significant costly process. In certain embodiments, the presently disclosed methods for producing D-ribose include culturing microorganisms (e.g,, one disclosed in Section 2) and purifying D-ribose,1 Cell

[0182] The present disclosure provides methods of culturing microorganisms disclosed herein. As used herein, “culturing” a cell refers to introducing an appropriate culture medium, under appropriate conditions, to promote the growth of a cell. In certain embodiments, culturing is performed using a liquid or solid growth medium. In certain embodiments, culturing occurs under aerobic or anaerobic conditions based on the requirements of the microorganism and desired metabolic state of the same. In certain embodiments, culturing includes specific conditions such as temperature, pressure, light, pH, and cell density.

[0183] In certain embodiments, the methods for producing methods of producing D-ri bose include a culture medium for culturing the recombinant bacteria, ^Culture medium,” as used herein, refers to any composition or broth that supports the growth of the microorganism disclosed herein. A culture media can be liquid or solid. In certain embodiments, the culture media include nutrients, salts, buffers., elements, and other compounds that support the growth and viability of cells. Additionally, culture media can include sources of nitrogen, carbon, amino acids, carbohydrates, trace elements, vitamins, and minerals. In certain embodiments, the culture media include a complex extract (e.g., yeast extract). In certain embodiments, the culture medium is enriched in order to support rapid growth. In certain embodiments, the culture medium is modified in order to support slower growth. In certain embodiments, the culture medium includes an agent that can inhibit the growth of or kill contaminating organisms (e.g., an antibiotic). In certain embodiments, the culture medium includes an agent that can activate an inducible promoter or enzyme (e.g., IPTG), Non-limiting examples of culture media encompassed by the present disclosure include M9 medium, Lysogeny Broth (LB), Terrific Broth (TB), and YT broth. In certain embodiments, the culture medium comprises a substrate that is converted by the recombinant microorganisms to D-ribose.

[0184] In certain embodiments, the substrate is a sugar (e.g., glucose) that can be phosphorylated by the bacteria via a kinase (e.g., hexokinase). In certain embodiments, the substrate is glucose. In certain embodiments, glucose can derive from cellulose, C» sugars, hemicellulose, and / orxylose. In certain embodiments, the substrate is a constituent of the culture medium. In certain embodiments, the substrate is supplemented with the culture medium. In certain embodiments, the substrate is continuously present in the culture medium, hi certain embodiments, the substrate is supplemented during the growth phase. In certain embodiments, the substrate is supplemented during the stationary phase, Purificatio nof

[0185] In certain embodiments, the methods of the present disclosure further comprise purifying r ibose produced by a microorganism of the present discl osure, e.g, , from cell culture or cell culture medium. A variety of methods known in the art may be used to purify a product from a microorganism or microorganism culture. In certain embodiments, one or more products may be purified continuously, e.g„ from a continuous culture. In certain embodiments, one or more products may be purified separately from fermentation, e.g, from a batch or fed-batch culture. One skilled in the art will appreciate that the specific purification method(s) used may depend upon, inter alia, the microorganism, cul ture conditions, and / or particular product(s),

[0186] In certain embodiments, purifying D-ribose comprises separating or filtering the microorganisms from a cell culture medium, separating the D-ribose from the culture medium (e.g., by chromatography), concentration of water fy.g,, by evaporation), and lyophilization of the D-ribose.5. Food Products'

[0187] The present disclosure also provides delivery' systems methods for use in food products including the D-ribose prepared and / or generated by any of the microorganisms disclosed herein.

[0188] The term “food product,” as used herein, includes any food product, for example, those set forth in 21 CFR 101.12. Non-limiting examples of such food products include frozen desserts, baked goods, fillings, nutritional drinks, beverages, salad dressing or similar dressing, sauces, icings, puddings and custards, batters, and the like. Various baked goods are disclosed in U.S, Patent No. 6,536,599, the disclosure of 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-ribose prepared and / or generated by any of the microorganisms disclosed herein are also suitable as a component in frozen foods,

[0189] In certain embodiments, the food product is prepared by admixing the D-ribose 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,

[0190] As used herein “admixing,” for example, “admixing D-ribose with a food product,” refers to the process where the flavor composition is mixed with or added to the completed product or mixed with some or all of the components of the product during product formation or some combination of these steps. When used in the context of' admixing the term “product” refers to the product or any of its components. This admixing step can include a process selected from the step of adding D- ribose to the product, spraying D- ribose on the product, coating D- ribose on the product, suspending the product in D- ribose, painting D- ribose on the product, pasting D- ribose on the product, encapsulating the product with D- ribose, mixing D- ribose with the product and any combination thereof. The D-ribose can be a liquid, dry powder, spray, paste, suspension and any combination thereof.

[0191] 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,

[0192] In certain embodiments, the D-ribose prepared andfor 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.

[0193] 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-ribose 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

[0194] In certain embodiments, the food products to which the D-ribose prepared and / or generated by any of the microorganisms disclosed herein are admixed with comprise, by way of example, the wet soup category1, the dehydrated and cul inary food category, the beverage category,the frozen food category, the snack food category, and seasonings or seasoning blends, described herein.

[0195] In certain embodiments, the D-ribose prepared and / or generated by any of the microorganisms disclosed herein are admixed with one or more confectioneries, chocolate confectionery, tablets, countlines, bagged selfiniesZsoftlines, 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, mipackaged / artisanal bread, pastries, cakes, packaged-industrial cakes, unpackaged / artisanal cakes, cookies, chocolate coated biscuits, sandwich biscuits, filled biscuits, savory biscuits and crackers, bread substitutes, breakfast cereals, rte cereals, family breakfast cereals, flakes, muesli, other rte cereals, children's breakfast cereals, hot cereals, ice cream, impulse ice cream, single portion dairy ice cream, single portion water ice cream, multi-pack dairy ice cream, multi-pack water ice cream, take-home ice cream, take-home daily ice cream, ice cream desserts, bulk ice cream, take-home water ice cream, frozen yoghtirt, artisanal ice cream, dairy products, milk, fresh / pasteurized milk, full fat fresh / pasteurized milk, semi skimmed fresh / pasteurized milk, long-IifeAdit milk, full fat long fifeZuhtmilk, semi skimmed long life / uht milk, fat-free long life / uht milk, goat milk, condensedfevaporated milk, plain condensed / evaporated milk, flavored, functional and other condensed milk, flavored milk drinks, daily only flavored milk drinks, flavored milk drinks with fruit juice, soy milk, sour milk drinks, fermented dairy drinks, coffee whiteuers, 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 yoghtirt, probiotic yoghurt, drinking yoghurt, regular drinking yoghurt, probiotic drinking yoghurt, chilled and shelf-stable desserts, dairy-based desserts, soy-based desserts, chilled snacks, fromage frais and quark, plain fromage frais and quark, flavored fromage frais and quark, savory fromage frais and quark, sweet and savory snacks, fruit snacks, chips / crisps, extruded snacks, tortill&'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, chilied / fresh pasta, noodles, plain noodles, instant noodles, cups / bowl instant noodles, pouchinstant noodles, chilled noodles, snack noodles, canned food, canned meat and meat products, canned frsh / seafood, canned vegetables, canned tomatoes, canned beans, canned fruit, canned ready meals, canned soup, canned pasta, other canned foods, frozen food, frozen processed red meat, frozen processed poultry, frozen processed fish / seafood, frozen processed vegetables, frozen meat substitutes, frozen potatoes, oven baked potato chips, other oven baked potato products, non-oven frozen potatoes, frozen bakery products, frozen desserts, frozen ready meals, frozen pizza, frozen soup, frozen noodles, other frozen food, dried food, dessert mixes, dried ready meals, dehydrated soup, instant soup, dried pasta, plain noodles, instant noodles, cups / bowl instant noodles, pouch instant noodles, chilled food, chilled processed meats, chilled 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 tats, sauces, dressings and condiments, tomato pastes and purees, boudlon / stock cubes, stock cubes, gravy granules, liquid stocks and funds, 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.

[0196] In certain embodiments, the D-ribose 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 general pattern outlined below. In general, a chewing gum composition typically contains a chewable gum base portion that is essentially free of water and is water-insoluble, a water-soluble bulk portion, and flavors that are typically water-insoluble. The water-soluble portion dissipates with a portion of the flavor over a period of time during chewing, The gum base portion is retained in the mouth throughout the chew. The insoluble gum base generally comprises elastomers, elastomer solvents, plasticizers,, waxes, emulsifiers, and inorganic fillers. Plastic polymers, such as polyvinyl acetate, which behave somewhat as plasticizers, are also often included. Other plastic polymers that can be used include polyvinyl laureate, polyvinyl alcohol, and polyvinyl pyrrolidone. Elastomers can include polyisobutylene, butyl rubber, (isobutylene-isoprenecopolymer), 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, aretypically fats and oils, including tallow, hydrogenated and partially hydrogenated vegetable oils, and cocoa buter. 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.

[0197] 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.

[0198] 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.

[0199] The water-soluble portion of the chewing gum can further comprise softeners, sweeteners, flavors, physiological cooling agents, and combinations thereof. The sweeteners often fulfill the role of bulking agents in the gum. The bulking agents typically comprise about 5% to about 95% of the gum composition.

[0200] Softeners are added to the chewing gum in order to optimize the chewability and mouth feel of the gum. Softeners, also known in the art as plasticizers or plasticizing agents, generally constitute between about 0.5% to about 15% of the chewing gum. Softeners contemplated by the presently disclosed subject matter include glycerin, lecithin, and combinations thereof Further, aqueous sweetener solutions such as those containing sorbitol, hydrogenated starch hydrolysate, corn syrup, and combinations thereof can be used, as softeners and binding agents in gum.

[0201] As mentioned above, the D-ribose 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-coutaining 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-ribose prepared and / or generated by any of the microorganisms disclosed herein can also be used in combination with sugarless sweeteners. Generally, sugarless sweeteners include componen ts withsweetening characteristics but which are devoid of the commonly known sugars and comprise, but are not limited to, sugar alcohols such as sorbitol, hydrogenated isomaitulose. mannitol, xylitol, lactitol, erythritol, hydrogenated starch hydrolysate, maltitol and the like alone or in any combination,

[0202] Depending on the particular sweetness release profile and shelf stability needed, coated or nncoated 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, Thaumafin, 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 compositi on, or added as part of the gum base,

[0203] Aqueous syrups, such as corn syrup and hydrogenated coni syrup can be used, particularly if their moisture content is reduced. This can preferably be done by co-evaporating the aqueous syrup with a plasticizer, such as glycerin or propylene glycol, to a moisture content of less than 10%. Preferred compositions include hydrogenated starch hydrolysate solids and glycerin. Such syrups and their methods of preparation are discussed in detail in U.S, Patent No, 4,671,967,

[0204] 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 cuting into sticks, extruding into chunks, or casting into pellets. Generally, the ingredients are mixed by first melting the gum base and adding it to the running mixer. The base can also be melted in the mixer itself Color or emulsifiers can also be added at this time, along with syrup and a portion of the bulking agent. Further portions of the bulking agent can then be added to the mixer. Flavor systems are typically added with the final portion of the bulking agent. If the flavor system is coated or otherwise modified when incorporated into a delivery system to modi fy 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 longermixing times can sometimes be required. Those skilled in the art will recognize that many variations of the above-described procedures can be followed.

[0205] 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 abou t 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.

[0206] The material or syrup that will eventually form the coating is applied or distributed over the gum center tablets. The D-ribose 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 pl urality of coatings or multiple layers of coating. The D-ribose can be added to any' or none of the coatings and / or layers.

[0207] In the panning procedure., syrup is added to the gum center tablets at a temperature range of from about 100°F to about 240ºF. Preferably, die syrup temperature is from about l40oF to about 200ºF. 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,

[0208] 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.

[0209] Each component of the coating on the gum center can be applied in a single layer or a plurality of layers. In general, a plurality of layers is obtained by applying single coats, allowing the layers to dry, and then repeating the process. The amount of solids added by each coating step depends chiefly on the concentration of the coating syrup. Any number of coats can be applied to the gum center tablet. Preferably, no more than about 75 coats are applied to the gum center. More preferably, less than about 60 coats are applied and most preferably, about 30 to about 60 coats are applied. In any event, the presently disclosed subject matter contemplates applying an amount of syrup sufficient to yield a coa ted chewing gum product containing about 10% io about 65% coating. Preferably, the final product will contain from about 20% to about 50% coating.

[0210] Those ski lled 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.

[0211] Once a coating of syrup i s 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 7(FF to about 1 10°F. More preferably, the drying air is in the temperature range of from about SO T to about 100°F. The invention also contemplates that the drying air possesses a relative humidity of less than about 15 percent. Preferably, the relative humidity of the drying air is less than about 8 %.

[0212] The drying air can be passed over and admixed with the syrup coated gam 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.

[0213] The amount of D-ribose 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-ribose 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- ribose prepared and / or generated by any of the microorganisms disclosed herein is generally present in amounts from about 0.02% to about 5%, and preferably from about 0. 1 % to about 2%, and more preferably, from about 0.8% to about .1.8%, by weight of the chewing gum composition.5.2. Sugar confectione

[0214] Another important aspect of the presently disclosed subject matter includes a confectionery composition incorporating the D-ribose prepared and / or generated by any of the microorganisms disclosed herein and a method for preparing the confectionery compositions. The preparation of confectionery formulations is well-known in the art. Confectionery items have been classified as either “hard” confectionery or “soft" confectionery. The D-ribose 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.

[0215] 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 toabout 92% sugar, up to about 55% com syrup, and from about 0.1 % to about 5% water, by weight of the final composition. The syrup component is generally prepared from sucrose and corn syrups but can include other materials. In certain embodiments, the syrup component includes the D- ribose 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.

[0216] 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.

[0217] Fire cookers invol ve 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 14-5° 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.

[0218] A high-speed atmospheric cooker uses a heat-exchanger surface, which invol ves spreading a film of candy on a heat exchange surface, the candy is heated to 165° C to 170* C witbin a few seconds. The candy is then rapidly cooled to 100° C to 120° C and worked as a plastic-like mass enabling incorporation of the additives, such as flavoring agents, colorants, and the like. In vacuum cookers, the carbohydrate bulking agent is boded to 125° C to 132° C, vacuum is applied, and additi onal water is boiled off wi thout extra heating. When cooki ng 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.

[0219] 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.

[0220] Once the candy mass has been properly tempered, it can be cut into workable portions or formed into desired shapes. A variety of forming techniques can be utilized depending upon the shape and size of the final product desired. A general discussion of the composition and preparation of hard confections can be found in H.A. Lieberman, Pharmaceutical Dosage Forms: Tablets, Volume 1 (1989), Marcel Dekker, Inc., New York, N.Y, at pages 419 to 582, which disclosure is incorporated herein by reference.

[0221] 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.

[0222] Similar to hard confectionery, soft confectionery can be utilized in the embodiments of the disclosed subject matter. The preparation of soft confections, such as nougat, involves conventional methods, such as the combination of two primary components, namely (I) a high boiling syrup such as corn syrup, or the like, and (2) a relatively light textured frappe, generally prepared from egg albumin, gum arable, gelatin, vegetable proteins, such as soy-derived compounds, sugaiiess 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.

[0223] 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.

[0224] In general, the frappe component is prepared first and thereafter the syrup component is slowly added under agitation at a temperature of at least about 65® C, and preferably at least about. 100® C. The mixture of components is continued to be mixed to form a uniform mixture, after which the mixture, is cooled to a 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 suitable confectionery shapes,

[0225] In accordance with the present disclosure, amounts of the D-ribose 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-ribose 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-ribose 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 theneed for undue experimentation . In general, the amount of D-ribose 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.

[0226] The presently disclosed subject matter extends to methods for making the improved confections. The D-ribose 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 wel l known in the confectionery manufacturing arts, and therefore the selection of the specific apparatus will be apparent to the artisan.

[0227] In such a method, a composition is made by admixing the D-ribose into the confectionery composition along with the other ingredients of the final desired composition. Other ingredients will usually be incorporated into the composition as dictated by the nature of the desired composition as well known by those having ordinary skill in the art. The ultimate confectionery compositions are readily prepared using methods generally known in the food technology and pharmaceutical arts. Thereafter the confectionery mixture can be formed into desirable confectionery shapes,

[0228] The D-ribose prepared and'or generated by any of the microorganisms disclosed herein can be formulated with conventional ingredients that offer a variety of textures to suit particular applications. Such ingredients can be in the form of hard and soft confections, tablets, toffee, nougat, chewy candy, chewing gum and so forth, center filled candies, both sugar and sugarless. The acceptable ingredients can be selected from a wide range of materials. Without being limited thereto, such materials include diluents, binders and adhesives, lubricants, disin tegrants, bulking agents, humectanis, buffers, and adsorbents. The preparation of such confections and chewing gum products is well known.53. Chocolates and Fillings

[0229] The presently disclosed subject matter is also used with and / or in chocolate products, chocolate-flavored confections, and chocolate flavored compositions. Chocolates also include those containing crumb solids or solids folly 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 Confectioners'; Science and Technology, 2nd edition, AVI Publishing Co., Inc., Westport, Conn. ( 1982), which disclosure is incorporated herein by reference.0230] 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 specfi ica l l y identi fled otherwi se.

[0231] 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,

[0232] 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,

[0233] In certain embodiments, the chocolate can contain D-ribose 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 sweetenersinclude aspartame, cyclamates, saccharin, acesulfeme-K, neohesperidm dihydrochalcone, sucralose, alitame, stevia sweeteners, glycyrrhizin, thaumatin and the like and mixtures thereof. The preferred high potency sweeteners are aspartame, cyclamates, saccharin, and acesulfame-K. Examples of sugar alcohols can be any of those typically used in the art and include sorbitol, mannitol, xylitol, maltitol, isomalt, lactitol and the like.

[0234] The chocolates can also contain bulking agents. The term "‘bulking agents” as defined herein can be any of those typically used in the art and include polydextrose, cellulose and its derivatives, maltodextrin, gum arabic, and the like.

[0235] The chocolate products can contain emulsifiers. Examples of safe and suitable emulsifiers can be any of those typical ly used in the art and include lecithin derived from vegetable sources such as soybean, safflower, corn, etc., fraction ated 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 deri vatives of mono- and diglycerides of edible fats or oils, sorbitau monostearate, hydroxylated lecithin, lactylated fatty acid esters of glycerol and propy lene 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 poiygiycerol polyricinoleate (PGPR), ammonium salts of phospha tidic acid, (e,g., YN) sucrose esters, oat extract, etc., any emulsifier found to be suitable in chocolate or similar lat / solid system or any blend.

[0236] The term ‘‘chocolate-flavored confection” refers to food products., excluding “chocolate'”, having a chocolate flavor / aroma and comprising a cocoa fraction. These products are stable at ambient temperatures for extended periods of time (e.g., greater than I week) and are characterized as microbiologically shelf-stable at 18-30° C under normal atmospheric conditions. Examples include chocolate-flavored hard candies, chewables, chewing gums, etc.

[0237] 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,).54. Savory Goods and Other FoodProdu

[0238] In certain embodiments, the D-ribo-se 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.

[0239] In certain embodiments, the D-ribose 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 l ike a beverage). Soup may be used as an ingredient for preparing other meal components and may range from broths (consomme) to sauces (cream or cheese-based soups).

[0240] In certain embodiments, the D-ribose prepared and / or generated by any of the microorganisms disclosed herein is incorporated into a dehydrated and culinary food category of food products, which comprises (i) cooking aid products such as: powders, granules, pastes, concentrated liquid products, including concentrated bouillon, bouillon and bouillon like products in pressed cubes, tablets or powder or granulated form, which are sold separately as a finished product or as an ingredient within a product, sauces and recipe mixes (regardless of technology): (ii) meal solutions products such as: dehydrated and freeze dried soups, including dehydrated soup mixes, dehydrated instant soups, dehydrated ready-to-cook soups, dehydrated or ambient preparations of ready-made dishes, meals and single serve entrees including pasta, potato and rice dishes; and (iii) meal embellishment products such as: condiments, marinades, salad dressings, salad toppings, dips, breading, batter mixes, shelf stable spreads, barbecue sauces, liquid recipe mixes, concentrates, sauces or sauce mixes, including recipe mixes for salad, sold as a finished product or as an ingredient within a product, whether dehydrated, liquid or frozen,

[0241] In certain embodiments, the D-ribose 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 meatball. A simulated meat food product may be combined with a meat food product.

[0242] In certain embodiments^ the D-ribose prepared and / or generated by any of the microorganisms disclosed herein is incorporated into a snack food category food product. In certain embodiments, snack food products include any food that can be a light informal meal including, but not limited to sweet and savory snacks and snack bars. Examples of snack food include, but are not limited to fruit snacks, chips / crisps, extruded snacks, tortilla 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,

[0243] In certain embodiments, the D-ribose prepared and / or generated by any of the microorganisms disclosed herein is incorporated into frozen food products, which comprises chilled or frozen food products, for example, but not limited to, ice cream, impulse ice cream, single portion dairy ice cream, single portion water ice cream, multi-pack dairy ice cream, multipack water ice cream, take-home ice cream, take-home dairy ice cream, ice cream desserts, bulk ice cream, take-home water ice cream, frozen yogurt, artisanal ice cream, frozen ready meals, frozen pizza, chilled pizza, frozen soup, frozen pasta, frozen processed red meat, frozen processed poultry, frozen processed fish / seafood, frozen processed vegetables, frozen meat substitutes, frozen potatoes, frozen bakery products and frozen desserts.5.4. Pharmaceutical

[0244] The D-ribose prepared and / or generated by any of the microorganisms disclosed herein can also be in the form of a pharmaceutical. One non-limiting example of a pharmaceutical form is a suspension. Pharmaceutical suspensions can be prepared by conventional compounding methods. Suspensions can contain adjunct materials employed in formulating the suspensions of the art. The suspensions of the presently disclosed subject matter can comprise preservatives, buffers, suspending agents, antifoaming agents, sweetening agents, flavoring agents, coloring or decoloring agents, solubilizers, and combinations thereof.

[0245] 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 vanil la, 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.

[0246] The pharmaceutical suspensions of the presently disclosed subject matter can be prepared as follows: (i) admix the thickener with water heated from about 40° C to about 95* C, preferably from about 40® C to about 70® C, to form a dispersion if the thickener is not water-soluble or a solution if the thickener is water soluble; ( ii) admix the D-ribose prepared and / or generated byany of the microorganisms disc losed 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, solubilizers, anti-foaming agents, buffers and additional water with the mixture of step (i v) to form the suspension.

[0247] The D-ribose prepared and / br 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 fla voring 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 com 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 1.20® 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 anti) homogeneous at temperatures between about 65® C and about 120® C. The D-ribose prepared and / or generated by any of the microorganisms disclosed herein can then be added to the homogeneous mixture as the temperature is lowered to about 65® C-95® C whereupon additional ingredients can then be added such as flavoring agents and coloring agents. The formulation is further cooled and formed into pieces of desired dimensions,

[0248] 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 I : I to about 20: L 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. Aningestible 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 .

[0249] The ingestible topical flavoring agents can also contain conventional additives normally employed in those products. Conventional additives include a fluorine -providing compound, a sweetening agent, a flavoring agent, a coloring agent, a humectant, a buffer, and an emulsifier, providing the additives do not interfere with the flavoring properties of the composition. The coloring agents and humectants, and the amounts of these additives to be employed, set out above, can be used in the ingestible topical composition. The flavoring agents (flavors, flavorants) that can be used include those flavors known to the skilled artisan, such as natural and artificial flavors. Suitable flavoring agents include mints, such as peppermint, citrus flavors such as orange and lemon, artificial vanilla, cinnamon, various fruit flavors, both individual and mixed, and the like. The amount of flavoring agent employedin the ingestible topical composition is normally a matter of preference subject to such factors as the type of final ingestible composition, the individual flavor employed, and the strength of flavor desired. Thus, the amount of flavoring can be varied in order to obtain the result desired in the final product and such variations are within the capabilities of those skilled in the art without the need for undue experimentation. The flavoring agents, when used, are generally utilized in amounts that can, for example, range in amounts from about 0.05% to about 6%, by weight of the ingestible topical composition.5.5, Pet Food Product

[0250] The D-ribose prepared and / or generated by any of the .microorganisms disclosed herein can be used in a wide variety of pet food products,

[0251] As used herein, the terms “pet. food” or “pet food product” refer to a product or composition that is intended for consumption by a companion animal, such as cats, dogs, guinea pigs, rabbits, birds and horses . For example, but not by way of limitation, the companion animal can be a “domestic” dog, e.g. C anis lupus familiaris. A “pet food” or “pet food product” includes any food, feed, snack, food supplement, liquid, beverage, treat, toy (chewable and / or consumable toys), meal substitute or meal replacement.

[0252] in certain embodiments, the D-ribose prepared and / or generated by any of the microorganisms disclosed herein is directly added io a pet food product. In certain embodiments, the D-ribose 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.

[0253] 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.

[0254] 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 moisture- containing 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.

[0255] In certain embodiments, the pet food product is a pet food snack product. Non-limiting examples of pet food snack products include snack bars, pet chews, crunchy treats, cereal bars, snacks, biscuits and sweet products.5.4. Delivery Systems

[0256] In certain embodiments, the D-ribose 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 testant. Delivery systems can be liquid or solid, aqueous or non-aqueous. Delivery systems are generally adapted to suit the needs of the flavor composition and / or the edible composition into which the D- ribose will be incorporated.

[0257] The D~ ribose can be employed in liquid form, dried form, and / or solid form. When used in dried form, suitable drying means such as spray drying can be used. Alternatively, D- ribose can be encapsulated or absorbed onto water soluble materials, including but not limited to materials such as cellulose, starch, sugar, maltodextrin, gum arable and so forth. The actual techniques for preparing such dried forms are well-known in the art, and can be applied to the presently disclosed subject matter.

[0258] The D- ribose can be used in many distinct physical forms well known in the art to provide an initial burst of taste, flavor and / or texture; and / or a prolonged sensation of taste, flavor and / or texture. Without being limited thereto, such physical forms include free forms, such as spray dried, powdered, and beaded forms, and encapsulated forms, and mixtures thereof

[0259] In certain embodiments, D- ribose is encapsulated. Encapsulating materials and / or techniques can be selected to impro ve the stabili ty of the D- ribose and / or food product. In certain embodiments, the encapsulating materials and / or techniques are selected to modify the release profile of D- ribose.

[0260] 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, polyiactidacid,polyhydroxyalkanoates, ethylcellulose, polyvinyl acetatephthalate, polyethylene glycol esters. metlracrylicacid*co-methylmetliacrylate, efhylene-vinylacetate (EVA) copolymer, and the like, and combinations thereof. Suitable encapsulating techniques can include, bat 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.

[0261] Encapsulated delivery systems for flavoring agents or sweetening agents (e.g., D- ribose) contain a hydrophobic matrix of fat or wax surrounding a sweetening agent or flavoring agent core. The fats can be selected from any number of conventional materials such as fatty acids, glycerides or poly glycerol esters, sorbitol esters, and mixtures thereof. Examples of fatty acids include but are not limited to hydrogenatedandpartially hydrogenated vegetable oils such as palm oil, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, soybean oil, cottonseed oil, sunflower oil, safflower oil, and mixtures thereof. Examples of glycerides include but are not limited to monoglycerides, diglycerides, and triglycerides.

[0262] Waxes useful can be chosen from the group consisting of natural and synthetic waxes, and mixtures thereof Non-limiting examples include paraffin wax, petrolatum, carbowax, microcrystalline wax, beeswax, carnauba wax, candellila wax, lanolin, bayberry wax, sugarcane wax, spermaceti wax, rice bran wax, and mixtures thereof,

[0263] The fats and waxes can be use itKlividually or in combination in amounts varying from about 10 to about 70%, and alternatively in amounts from about 30 to about 60%, by weight of the encapsulated system. When used in combination., the fat. and wax. are preferably present in a ratio from about 70: 10 to 85:15, respectively.

[0264] Typical encapsulated, compositions, flavoring agent or sweetening agent delivery systems are disclosed in U.S. Patent Nos. 4,597,970 and 4,722,845, the disclosures of which are incorporated herein by reference in their entireties,

[0265] Liquid delivery systems can include, but are not limited to, systems with a dispersion of D- ribose, such as in carbohydrate syrups and / or emulsions. Liquid delivery systems can also include extracts where D- ribose is solubilized in a solvent. Solid delivery systems can be created by spray drying, spray coating, spray chilling, fluidized bed drying, absorption, adsorption, coacervation, complexation, or any other standard technique, In some embodiments, the delivery system can be selected to be compatible with or to function in the edible composition. In some embodiments,, the delivery system will include an oleaginous material such as a fat or oil In some embodiments, the delivery system will include a confectionery fat such as cocoa butter, a cocoa butter replacer, a cocoa butter substitute, or a cocoa butter equivalent.

[0266] When used in dried form, suitable drying means such as spray drying may be used. Alternatively, D- ribose may be adsorbed or absorbed onto substrates such as water soluble materials, such as cellulose, starch, sugar, maltodextriu, gum arable and so forth or may be encapsulated. The actual techniques for preparing such dried forms are well known in the art.EXAMPLES

[0267] The presently disclosed subject matter can be better understood by reference to the following. The below examples are exemplary only and should in no way be taken as limiting, Example 1 — Biosynthesisof Ribose from Glucose

[0268] Genetically modified. E. coli is naturally capable of producing trace amounts of D-ribose. In the present example. D-ribose production is improved by overexpressing key genes, removing competing pathway genes, and optimizing the production conditions.

[0269] Additionally, a series of single gene knockouts (KOs) were constructed. (see Table 1 ), The gene pgm encodes for the enzyme phosphoglucomutase (Pgm) EC 5.4.2.), which is an enzyme that transfers a phosphate group on an a-D-glucose monomer from the 1 to the 6 position in the forward direction or the 6 to the 1 position in the reverse direction. The gene pgi encodes glucose - 6-phosphate isomerase (Pgi) (EC 5.3.1.9), which catalyzes the reversible isomerization of glucose-6-phosphate to finctose-6-phosphate. The gene deoB encodes for the enzyme phosphopeiitoinutase (DeoB) (EC 5.4, 2.7) which catalyzes the phosphotransfer between the Cl and C5 carbon atoms of pentose. The genes iktA and fktB encode for the enzymes transketolase 1 (TktA) and (TktB) (EC 2.2.1.1), which catalyze the transfer of a two-carbon ketol group from a ketose donor to an aldose acceptor, via a covalent intermediate with the cofactor thiamine pyrophosphate.

[0270] Table I. Strain listDetermining Ribos production enzymes

[0271] The production pathway starts with the native assimilation of glucose into £. coll via the phosphotransferase system (PTS) or GalP / Glk, which converts glucose to glucose*6«phosphate (G6P) (Figure 2). G6p is then converted to 6-phosphogluconolactonase (6PGL), also known as 6-phospho-D-glucono-l ,5-lactone, by ghicose-6-phosphate dehydrogenase (Zwf) (EC 1 ,1 , 1.363). 6PGL is then converted to D-gluconate 6-phosphate by 6-phosphogluconolactonase (Pgl) (EC 3.1.1.31). D-gluconate 6-phosphate is then converted to D-ribulose 5-phosphate (Ru5P) by 6- phosphogluconate dehydrogenase (God) (EC: 1.1.1.44), Ru5P is then isomerized to D-ribose 5- phosphate (R5P) by ribose 5-phosphate isomerase A (RpiA) or ribose-5-phosphate isomerase B (RpiB) (5.3.1.6), which is then dephosphorylated to free ribose by sugar phosphatase YbiV (YbiV) or sugar phosphatase YidA (YidA) (EC 3.1.3.23) (Figure 1). A phosphatase could dephosphorylate D-ribose 5-phosphate to free ribose, after which it would be excreted from the cell ( Figure 1). A D-ribose production system using phosphorylation and dephosphorylation steps as system driving .forces should be mote efficient than the pathway currently used in industrial production of D-ribose.

[0272] By literature search and genome mining, two isomerases and two phosphatase candidates were identified. Ribose 5-phosphate isomerase A (RpiA) and ribose-5-phosphate isomerase B (RpiB) have shown activity towards ribose. Two phosphatases (Yida and YbiV) have shown promiscuity towards a variety of pentoses.

[0273] Plasmids were constructed to overexpress genes RpiA or RpiR, and either yzdd oryMKDynamic regulation of production

[0274] Balancing carbon flux between glycolysis and production is critical for maximizing both culture health and D-ribose production. During the logarithmic phase of growth, cells need more energy to rigorously grow and divide. Genes, related to glycolysis should be expressed, while genes related to D-ribose production should be suppressed. When the cells enter the stationary phase and are not actively dividing, carbon flux can be diverted from glycolysis to D-ribose production.Increasin glucose uptake using sugar symporter GalP

[0275] Within £. coll, the favored method for glucose uptake is the phosphotransferase system (PTS). The PTS relies on activation by phosphoenolpyruvate. (PEP), which is a downstream product of glycolysis. By inhibiting glycolysis, either through gene knockouts or CRISPRi, we theorize that cellular stock of PEP may become depleted, leading to reduction in glucose consumption. To increase glucose consumption, extra copies of genes galP aniglk, which encode for galactoserH"' symporter (GalP) and glucokinase (Glk), were introduced. GalP transportsglucose into the cell where it is phosphorylated to gl.ucose-6-phosphate by Glk and assimilated into central carbon metabolism.

[0276] Constructing production pathways within a microorganism requires a careful carbon partitioning between essential metabolic processes and production, especially when working around central carbon metabolism. Within the presently disclosed system, dynamically balancing carbon flux between glycolysis and the D-ribose pathway can help maximize both cellular viability and D-ribose production.

[0277] The life cycle of an £. coli culture includes 5 distinct phases: lag, logarithmic, stationary, death, and long-term stationary phase. The lag phase occurs when cells are inoculated into media and adjust their metabolic processes according to their new environment. The cells will then rapidly grow and divide, entering the logarithmic phase. It is at this time that enzymes related to central carbon metabolism are most important, and the transcription of corresponding genes will be upregnlated. Once the cells sense environmental stressors such as scarci ty of media nutrients, their growth and division slows; and the culture enters the stationary phase. Here, culture density plateaus and genes related stress response are expressed. The transcription of these genes is regulated in part by the <j38-subunit of RNA polymerase, which recognizes the promoter region of a gene.

[0278] E. colis native gene regulatory system was utilized to balance carbon flux by affixing the D-ribose production genes, rpiA, rpiB, ybiF and ykiA, downstream of a stationary phase-active promoter. This prevented the production pathway from competing with central carbon metabolism for carbon flux during the logarithmic phase of growth, a time when cells need carbon to rigorously grow and divide.Manipulati glucose utilization and metabolism

[0279] Another source of glucose siphoning away from the D-ribose pathway is through glycogen biosynthesis. Glycogen is stored for usage during times of starvation, which for the presently disclosed subject matter is unnecessary. The deletion of pgm, which encodes for phosphoglucomntase Pgm, prevents £'. coll from producing glycogen. As such, pgm was deleted in the production strains.

[0280] Inhibiting glycogen biosynthesis by knocking out pgm removes a. source of carbon to central metabolism which would normally be used to help cells grow under periods of starvation.Concluti

[0281] The elimination of competing pathways and additional expression of native £. coll genes (rpM, rpiB, ybiV yidA, gaIP and glk) along with the use of static and dynamic gene regulation strategies led to a strain capable of producing D-ribose using a thermodynamically favorablebiosynthetic pathway from a readily available feedstock. Overall, the engineered strain represents a helpfol step in producing D-ribose in a cost-effective manner, offering the food industry a viable source for creating the low-glycemic index products desired by consumersExample 2 - Productio of D-ribose in Escherichia coli

[0282] D-ribose is a 5-carbon monosaccharide present in biologically relevant molecules, such as RNA and ATP. D-ribose has been used for the chemical synthesis of riboflavin, a key precursor for pharmaceuticals, cosmetics, and other applications [1], D-ribose also largely contributes a meatlike aroma when involved in the Maillard reaction [2], making it a desirable food additive for meat alternatives,

[0283] Microbial production of D-ribose has previously been developed [3], Broadly, biological production of D-ribose has been achieved by accumulating and dephosphoiylating D-ribose-5- phospliate, an intermediate in the pentose phosphate pathway. Accumulation of D-ribose-5- phosphate has broadly been achieved by mutating the transketolase enzymes that convert D- ribose-5-phosphate into other bfomolecules. D-ribose was produced solely from glucose in the engineered microorganisms Pseudomonas retilivora* Candida pellieidose^ as well as many Bacillus sub fills species [3],

[0284] Escherichia coll is also a suitable host for D-ribose bioproduction, due to the various engineering tools to modify it. D-ribose has also been produced in transketolase-deficient Escherichia co / i, but requires both D-glucose and D-xylose as a feedstock instead [4], Furthermore, in this engineered strain of E. call, the D-glucose feedstock did not produce D- ribose; only D-xylpse was converted into D-ribose.

[0285] The present example demonstrates that coll can produce D-ribose from D-glucose when removing the genes encoding its D-ribose catabolism machinery, namely the rbsACBDKRZ operon [5] (Figure 2). In the D-ribose pathway, D-glucose is converted into D-ribose-5-phosphate by following the pentose phosphate pathway, then dephosphorylated by a phosphatase (Figure 2). D-ribose production was improved by additionally expressing the native D-ribose pathway genes and minimizing carbon flux toward competing pathways, including glycolysis, glycogen biosynthesis, and the downstream pentose phosphate pathway. D-glucose uptake was improved using the galactose proton symporter GalP [6], then D-glucose is then phosphorylated by glucokinase Glk [7] Results and Discussion

[0286] Production of D-ribose in E. co / i. The D-ribose moiety is important to Zk cali metabolism, abundantly found in the form of ribonucleic acids and ribose-5-phosphate. Initially, the natural capacity of Ik coll to produce D-ribose was tested (AL1050, Table 2), but It did not produce D- ribose, Thus, it was hypothesized that D-ribose can be produced in E coli by preventing any freeintracellular D-ribose from being assimilated into E. co / i metabolism. Ribokinase (RbsK) is an enzyme that can phosphorylate D-ribose into D-ribose-5-phosphate [8],coJi with a mutated rbsK gene have been, shown to leak D-ribose out of the cell [9], Thus, the rbsABCDR'RZ operon, which is responsible for ribose catabolism [5], was deleted from AL 1050 to generate Strain 1 (AL4588, Table 2), This operon includes the ribose transporter (RbsABC), D-ribose pyranase (RbsD). ribokinase (RbsK), and regulatory protein and sRNA RbsR and RbsZ, respectively. Strain 1 was then grown in M9P supplemented with 10 g L‘!of D-glucose for 24 hours. After 24 h. Strain 1 produced. 0.03 g L-1of D-ribose (Figure 3).Table 2; List of key strains.production, competing pathways were removed and D-ribose production was measured. Glucose can also be diverted away from the D-ribose pathway through glycogen biosynthesis and glycolysis. Glycogen is stored for periods of starvation

[0010] , which may be unnecessary during the D-ribose production. The phosphoglucomutase (Pgm) commits glucose to glycogen biosynthesis by converting G6P to glucose- 1 -phosphate

[0011] . G6P can also be committed to glycolysis via conversion into fmctose-6-phosphate by phosphoglucoisonierase (Pgi)

[0012] ,

[0288] Downstream in the pentose phosphate pathway, ribose- 5 -phosphate can be converted to ribose- 1 -phosphate by phosphopentoniutase ( DeoB)

[0013] , which diverts R5P from being dephosphorylated into D-ribose.

[0289] The pgm ,pgi, and deoB genes were deleted in Strain 1 to generate Strain 2 (AL4614, Table 2). The deletion of these genes slightly improved D-ribose production (0.05 gL-1, Figure 3).

[0290] R5P can also be utilized in the pentose phosphate pathway by transketolase 1 and 2 (TktA and TktB) [14,15]. Deletion of these enzymes may reduce flux of R5P towards the pentose phosphate pathway. Because a Aid double mutant requires other nutrients for growth

[0016] ,only the tfatA gene was deleted in Strain 2 to produce Strain 3 (AL4590, Table 2). Strain 3 further improved D-ribose production to 0.20 g L-1(Figure 3).

[0291] The erirfgene encodes for phosphogluconate dehydratase, by which phosphogluconate, an intermediate for the D-ribose production pathway, can be degraded

[0017] , The edd gene was deleted in Strain 3 to produce Strain 4 (AL4615, Table 2). Strain 4 further improved D-ribose production to 0.86 g L-1(Figure 3).

[0292] E. col cian produce D-psicose

[0018] . The oZvZi' gene encodes for psicose-6-phosphate epimerase, which epimerizes fructose- 6-phosphate into psicose-6-phosphate, could divert flux from the D-ribose production pathway. Thegene was deleted in Strain 4 to generate Strain 5 (Figure 4).

[0293] Carbon that makes it through the pentose phosphate pathway past D-ribose production is converted into tnictose-6-phosphate. The geneencodes phosphofructokinase, by which F6P is committed to glycolysis [19.20]. To minimize carbon flux toward this pathway, / yt.4 was deleted in Strain 5 to produce Strain 6 (AL4528, Table 2). However, both Strain 5 and 6 produced less D-ribose than Strain 4 (0.84 g L-1and 0.77 g L-1respectively. Figure 2).

[0294] Increasing..Prribose.Production.by. Pathway..Enzyme Expression, Continuous import and phosphorylation of D-glucose is vital to D-ribose production. A limitation of decreasing metabolites toward glycolysis is decreasing the pools of phosphoenolpyruvate (PEP). PEP is utilized by the PTS to import and phosphorylate extracellular D-glucose to .intracellular G6P, and the reduction of PEP pools may decrease glucose uptake [21,22]. G6P pools were enhanced through an alternative D-glucose import and phosphorylation mechanism. GalP is a galactose proton symporter capable of importing glucose [6], and Gik is a glucokinase that phosphorylates glucose to G6.P using ATP [7] (Figure 2). A plasmid was constructed containingand gik under the Puacoi promoter (pAL2263, Table 3).Table 3. list of key plasmids.[0295 j G6P is used in many metabolic processes, including the pentose phosphate pathway, which includes the desired intermediate ribose-5-phosphate

[0023] , Giucose-6-phosphate dehydrogenase (Zwf) is a gatekeeper enzyme that converts G6P into 6-phosphogIuconolactone and commits it to the pentose phosphate pathway

[0023] . Later in the pentose phosphate pathway, 6-phosphogIuconate is converted into ribulose-5-phosphate by 6~phosphogluconate dehydrogenase (Gnd), which is then converted into ribose-5 -phosphate via ribose-5-phosphate isomerase A (RpizX). A plasmid was constructed containing zwf and gW under the / Tueoj promoter (pAL2479, Table 3) and another plasmid containing rplA under the strong stationary phasepromoter (pAL2536, Table 3)

[0018] . When these three plasmids were introduced into Strain 3, D-ribose production increased from 0.20 g L-1to 0.40 g L'!('Figure 3).

[0296] Elucidating Phosphatases to Improve D- Ribose Production . E. call possesses a variety of sugar phosphatases with potential activity towards R5P, which could increase the conversion of R5P to D-ribose. A. coll phosphatases with a wide range of activity toward sugar substrates were tested, hexitol phosphatase A (HxpA), hexitol phosphatase B (HxpB), a-D-glucose- 1 -phosphate phosphatase YiliX, phosphosugar phosphatase YigL, and fructose- 1 -phosphate phosphatase YqaB

[0024] , The sugar phosphatase YbiV and sugar phosphatase YidA were also tested, as they have been used to improve D-ribose bioproduction in £. coll

[0025] , The 5’-nucleotidase / UDP-sugar hydrolase UshA, acid phosphatase / phosphotransferase AphA, and alkaline phosphatase phoA were also tested due to their 5’-phosphosugar dephosphorylate activity [26-28], To test each phosphatase for the conversion of R5P to D-ribose, the gene for each phosphatase was expressed from an expression plasmid under thepromoter, along with rplA (pAL2470, pAL2452, pAL2453, pAL2546, pAL2547, pAL2548, pAL2549, pAL2550, pAL2551, pAL2535, Table 3). The plasmids were introduced into Strain 3, along with the expression plasmids containing galP, glP cw / i and gnd (pAL:2479 and pAL.2263, Table 3). The strains were grown in M9P supplemented with 10 g L-1glucose for 24 hours. After 24 h, all of the strains harboring phosphatase genes Improved ribose production compared to the strain harboring plasmids without a phosphatase gene (Figure 4). The strains harboring the y / 44 and i.y / wT genes increased ribose production the most out of the phosphatases tested (0.86 g £* ’ and 0.85 g L-1of D-ribose, respecti vely).Methods

[0297] Reagents. AH enzymes involved in the molecular cloning experiments were purchased from New England Biolabs (NEB), All synthetic oligonucleotides were synthesized by IntegratedDNA Technologies. Sanger Sequencing was provided by Genewiz. D-ribose was purchased from Sigma- Aldrich. D-Glucose was purchased from Fisher Scientific.

[0298] Strains and plasmids, The relevant strains and plasmids used in this study are listed in Table 2 and 3, respectively.

[0299] Genome modifications such as gene deletion and gene insertion were constructed using CRISPR-Cas9-mediated homologous recombination

[0029] . Linear DNA repair fragments for gene deletions and insertions were constructed by amplifying genomic or plasmid DNA via PCR assembly

[0030] . Plasmids encoding sgRNA for CRISPR-CasO-mediated homologous recombination were constructed using Q5 site-directed mutagenesis (New England Biolabs) using pTargetF plasmid ( Addgene #62226) as a template. All genomic modifications were verified via sequencing.

[0300] Culturing conditions. Overnight cultures were grown at 37 °C in 3 niL of Luria-Bertani (LB) media with appropriate antibiotics. Antibiotic concentrations were as follows: spectinomycin (50 pg mL’!), ampicillin (200 gg mL-1)skanamycin (50 pg mL’’), gentamycm (15 pg niL’1)- M9 minimal media consists of 33.7 mM Na2HPO422 mM KH2PO4, 8.6 mM NaCL 9.4 mM NH4CI, 2 mM MgSCL, 0.1 mM CaCh, A5 trace metals mix (2.86 mg L’1HaBOs, 1.81 mg L‘?MnCIrdHsO, 0,079 mg L ’ CaSO-rSHaO, 49.4 mg L-1CofNOjfyGHsO), varying concentrations of glucose, and appropriate antibiotics. M9P media for the D-ribose production consists of M9 minimal media supplemented with 5 g L-1of yeast extract and appropriate antibiotics. Inducer concentrations are as follows; isopropyl-D-1-thfogalactopyranoside (IPTG) (1 mM), anhydrotetracycline (aTc) (100 ng mL- 1). OD600as measured with a Synergy H 1 hybrid plate reader (BioTek Instruments, Inc.).

[0030] ] D-Ribose production, For regular cell densi ty production experiments, overnight cultures were inoculated at 1% into 3 mL of M9P media. Cells were grown at 37 ®C until the OD^x» described, then induced with IPTG if necessary, and gro wn at 30 °C for 24 h,

[0302] HPLC analysis. Analysis of D-glucose and D-ribose concentrations was performed using HPI..C (Shimadzu) equipped with a refractive index detector (RID) 10 A and sugar alcohol column (Phenomenex). Mobile phase was comprised of 100% MilliQ water. Samples were run with an injection volume of 1 mL at a flow rate of 0.5 mL min ’ for 7 min, with the column oven at 83 °C and RID cell temperature

[0303] To prepare samples for HPLC analy sis, 300 mL of culture was centrifuged at 17,000 g for 5 min. Supernatants were applied to a 0.2 mm PVDF hydrophilic membrane 96 well filter plate and centrifuged at 17,000 g for 2 min into a polystyrene 96 well.[0304 Pertbrmance Anion- Exchange chrornatography (HPAE) Analysis, Analysis of D-ribose concentrations was also performed using HPAE with Pulsed Amperometric Detection(PAD) on an ICS-5000 system with a CarboPac PA 10 4x250mm (Thermo Fisher). The equilibrium phase was comprised of 10 mM NaOH in degassed MilliQ water at a flow rate of 2 ml min-1for 1.0 min. Mobile phase was comprised of 10 mM NaOH in degassed MilliQ water at. a flow rate of 2 mL mm-1for 5 min. The concentration of NaOH was increased over a gradient to 206 mM NaOH over 15 minutes. A 206mM NaOH wash was maintained for 7 minutes. Samples were run with an injection volume of 10 mL. The column oven was maintained at. 30ºC.

[0305] To prepare samples for HPAE-PAD analysis, 300 mL of culture was centrifuged at 17,000 g for 3 min. Supernatants were diluted 10- fold and applied to a 0.2 mm PVDF hydrophilic membrane 96 well filter plate and centrifuged at 17,000 g for 5 min into a polystyrene 96 well.References1. Schwechheimer, S.K., Park, E.Y., Revuelta, J.L., Becker, J., Wittmann, C., 2016. Biotechnology of riboflavin. Applied Microbiology and Biotechnology 100, 2107-2119. hftps: / / doi .oi'g / 10..1007 / s00253-015-7256-z2. Mottram, D.S., Nobrega, I.C.C., 2002, Formation of Sulfur Aroma Compounds in Reaction Mixtures Containing Cysteine and Three Different Forms of Ribose. I Agrie. Food Chem. 50, 4080-4086.3. Wulf, P.D., Vandamme, E.J., 1997. Production of d-ribose by fermentation. Applied Microbiology and Biotechnology 48, 141-148. https: / / doi.org / 10.I007 / s0025300510294. Park, H.-C., Kim, Y.-J., Lee, C.-W., Rho, Y.-T., Kang, J., Lee, D.-IL, Seong, Y.4., Park,Y.-C., Lee, D., Kira, S.-G., 2017. Production of d-ribose by raetabolically engineered Escherichia coli. Process Biochemistry 52, 73-77. lit tps: / / d oi . or g / 10. 1016 / j .procbio.2016.10.0015. Lopilato, J.E., Garwin, J.L., Emr, S.D., Silhavy, T.J., Beckwith, J.R., 1984. D-ribose metabolism in Escherichia coli K-12: genetics, regulation, and transport. J Bacteriol 158, 665-673.6. Henderson, P.J.F., Giddens, R.A., Jones Mortimer, M.C., 1977. Transport of galactose, glucose and their molecular analogues by Escherichia coli K12. The Biochemical journal 162, 309-320. https: / / doi.org / 10.1042 / BJ l 6203097. Meyer, D., Schneider-Fresenius, C., Horlacher, R,, Peist, R., Boos, W., 1997. Molecular characterization of glucokinase from Escherichia coli K-12. J Bacteriol 179, 1298 - 1306. https: / / doi.org / 10. U 28 / jb.179.4.1298- 1306.19978. Boric, P.sSander, C., Valencia. A., 1.993. Convergent evolution of similar enzymatic function on different protein folds: the hexokinase, ribokinase, and galactokinase families of sugar kinases. Protein Sci 2, 31-40,Ryu, K.-S.. Kim, C., Kim, I., Yoo, S., Choi, B.~S„ Park, C., 2004. N.MR ApplicationProbes a Novel and Ubiquitous Family of Enzymes That Alter Monosaccharide Configuration*. Journal of Biological Chemistry 279, 25544—25548. h ftps: ; / / dot .org / 10.1074 / j be, M402016200 .Preiss, J., 2014. Glycogen: Biosynthesis and Regulation. EcoSal Plus 6, 10. 1128 / ecosa1plus.ESP-0015-2014. https: / / doi.org / 10.1128 / ecosalplus.esp-0015-2014 Joshi, J.G., Handler, P., 1964. PHOSPHOGLUCOMUTASE, I. PURIFICATION AND PROPERTIES OF PHOSPHOGLUCOMUTASE FROM ESCHERICHIA COLL J Biol Chem 239, 2741 ---2751, Schreyer, R., Bock, A., 1980. Phosphogiucose isomerase from Escherischia coli K1.0: Purification, properties and formation under aerobic and anaerobic condition. 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Superoxide sensitivity of the Escherichia coll 6- phosphogluconate dehydratase. Journal of Biological Chemistry 266, 1478 -1483, https:7 / doi.org / l0.1016 / S0021-9258(18)523I9-X Taylor, J.E., Palm, D.S.K., Zhang, A., Gonzales, J.N., Arredondo, A., Coulther, T.A., Lechner, A.B.J., Rodriguez, E.P., Fiehn, O., Didzbalis, I, Siegel, J.B., Atsumi, S., 2023. Awakening the natural capability of psicose production in Escherichia coli. npj Sei Food 7, 54. https: / / doi.org / 10.1038 / 541538-023-00231 -0 He! lings, H.W., Evans, P.R., 1985. Nucleotide sequence and high-level expression of the major Escherichia coli phosphofructokinase. European Journal of Biochemistry 149, 363 - 373, https:7 / doi.org / 10.1 111 / j, 1432-1.033.1.985.tb08934.xHollinshead, W.D., Rodriguez, S., Martin, H.G., Wang, G., Baidoo, E.E.K., Sale, K.L., Keasling, J.D.. Mukhopadhyay, A., Tang, Y.J., 2016. Examining Escherichia coli glycolytic pathways, catabolite repression, and metabolite channeling using Apfk mutants. Biotechnology for Biofuels 9. 1-13. htps: / / dai.org / 10J 186 / s 13068-0 I 6-0630- y Brockman. EM., Prather, K.L.J., 2015. Dynamic knockdown of E. coli central metabolism for redirecting fluxes of primary metabolites. Metabolic Engineering 28, 104-113. htps; / / doi.org / l 0,1016 / j.ymben.2O14,12.005 Gosset. G., 2005. Improvement of Escherichia coli production strains by modification of the phosphoenolpyruvate:sugar phosphotransferase system. Microb Cell Fact 4, 14. https: / / doi.arg / ! 0. 1186 / 1475-2859-4-14 Fraenkel, D.G., 1968. Selection of Escherichia coli mutants lacking glucose-6-phosphate dehydrogenase or gluconate~6-phosphate dehydrogenase. Journal of bacteriology 95, 1267-127 I. https: / / doi.Org / l0J l28 / jb.95.4.l267-1271J968 Kuznetsova, F... Proudfoot* M., Gonzalez, C,F„ Brown, G„ Omelchenko, M. V., Borozan, L, 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. Journal of Biological Chemistry 281, 36149-36161. htps: / / doi.org / 10.l074 / jbc.M605449200 Gawand, P., Mahadevan, R., 2014. Engineering Escherichia coli for D-Ribose Production from Glucose-Xylose Mixtures. Industrial Biotechnology 10, 106-114. htps: / / doi .org / 10.1089 / ind.20l 3.0028 Heppel, L.A., Harkness, D.R., Hilmoe, R.J., 1962. A Study of the Substrate Specificity and Other Properties of the Alkaline Phosphatase of Escherichia coli. 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[0306] Although the presently disclosed subject matter and its advantages have been described in detail, if should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the presently disclosed subject matter, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized according to the presently disclosed subject matter. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods , or steps.

[0307] 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:L A recombinant microorganism comprising an exogenous isomerase and an exogenous phosphatase, wherein the recombinant microorganism produces an increased amount of ribose as compared to a naturally occurring microorganism.

2. The recombinant microorganism of claim 1, wherein the isomerase is a ribose 5-phosphate isomerase A (RpiA) or ribose 5-phosphate isomerase B (RpiB).

3. The recombinant, microorganism of claim 1 or 2, wherein the isomerase is an A. co / ?:RpiA or RpiB.

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

5. The recombinant microorganisms of any one of claims 1-4, wherein the isomerase comprises the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3.

6. The recombinant microorganisms of any one of claims 1 -5, wherein the isomerase consists of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3.

7. The recombinant microorganism of any one of claims 1 -6, wherein the phosphatase is a sugar phosphatase YbiV or YidA.

8. The recombinant microorganism of any one of claims 1 -7, wherein the phosphatase is an £ co / i YbiV or YidA.

9. The recombinant microorganisms of any one of claims 1-8, wherein the phosphatase comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 7,10. The recombinant microorganisms of any one of claims 1*9, wherein the phosphatase comprises the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 7.

11. The recombinant microorganisms of any one of claims 1-10, wherein the phosphatase consists of the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 7.

12. The recombinant microorganism of any one of claims 1-11, wherein the recombinant microorganism further comprises at least one exogenous enzyme of the pentose phosphate pathway.

13. The recombinant microorganism of claim 12, wherein the at least one exogenous enzyme of the pentose phosphate pathway comprises glucose-6-phosphate dehydrogenase (Zwf).

14. The recombinant microorganism of c laim 13, wherein the Zwf comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO:

15. The recombinant microorganism of claim 14, wherein the Zwf comprises the amino acid sequence set forth in SEQ ID NO: 51.

16. The recombinant microorganism of claim 12, wherein the at least one exogenous enzyme of the pentose phosphate pathway comprises phosphogluconate dehydrogenase (Gnd).

17. The recombinant microorganism of claim 16, wherein the Gnd comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 53,18. The recombinant microorganism of claim 17, wherein the Gnd comprises the amino acid sequence set forth in SEQ ID NO: 53.

19. The recombinant microorganism, of any one of claims 1-18, wherein the recombinant microorganism further comprises exogenous gaIactose:ET symporter (GalP) and glucokinase (Glk).

20. The recombinant microorganism of claims 19, wherein the GalP Is an £ co / i GalP and the Glk is E. &)ti Glk,21. The recombinant microorganisms of claim 20, wherein the GalP comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth In SEQ ID NO: 36 and the Glk comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 38.

22. The recombinant microorganisms of claim 21 , wherein the GalP comprises the amino acid sequence set forth in SEQ ID NO: 36 and the Glk comprises the amino acid sequence set forth in SEQ) ID NO: 38.

23. The recombinant microorganism of any one of claims 1-22, further comprising a mutation of a gene encoding for an enzyme of the glycogen biosynthesis compared to a naturally occurring microorganism,24. The recombinant microorganism of claim 23, wherein enzyme of the glycogen biosynthesis is phosphoglucornutase (Pgm).

25. The recombinant microorganism of any one of claims 1 -24, further comprising a mutation of a gene encoding for an enzyme of the carbohydrate biosynthesis compared to a naturally occurring microorganism.

26. The recombinant microorganism of claim 25, wherein the enzyme of the carbohydrate biosynthesis is ghicose-6-phosphate isomerase (Pgi),27. The recombinant microorganism of any one of claims I -26, forther comprising a mutation of a gene encoding for an enzyme of the purine / pyrimidine ribonucleosides degradation pathway compared to a naturally occurring microorganism.

28. The recoffibitani microorganism of claim 27, wherein the enzyme of the pentose phosphate pathway is phosphopentomutase (DeoB).

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

30. The recombinant microorganism of claim 29, wherein the enzyme of pentose phosphate pathway is transketolase 1 (TkiA) or transketoloase 2 (TktB),31. The recombinant microorganism of any one of claims 1 -30 further comprising a mutation of a gene encoding for an enzyme of the glycolysis compared to a naturally occurring microorganism.

32. The recombinant microorganism of claim 31, wherein the enzyme of the glycolysis is phosphofructokinase- 1 (PfkA).

33. The recombinant microorganism of any one of claims 1-32 further comprising a mutation of a gene encoding for an epimerase compared to a naturally occurring microorganism .

34. The recombinant microorganism of claim 33, wherein the epimerase allose-6-phosphate- 3-epimierase (AIsE)35. The recombinant microorganism of any one of claims 1-34 further comprising a mutation of a gene encoding for an enzyme of the gluconate degradation pathway compared to a naturally occurring rnicroorgani srn.

36. The recombinant microorganism of claim 35. wherein the enzyme of the gluconate degradation pathway is D-giuconate~6-phosphate ( Edd)37, The recombinant microorganism of any one of claims I -36 further comprising a mutation of at least one gene encoding for an enzyme of the ribose catabolism pathway.

38. 'The recombinant microorganism of claim 37, wherein the at least one gene encoding for an enzyme of the ribose catabolism pathway is selected from D-ribose transporter ATP binding subunit (RbsA), D-ribose transporter periplasmic binding protein (RbsB), D- ribose transporter membrane subunit (RbsC), D-ribose pyranase (RbsD), D-ribose kinase (RbsK), DNA-binding transcriptional dual regulator (RbsR), small regulatory RNA (RbsZ), or combinations thereof39. The recombinant microorganism of any one of claims 1-38, wherein the exogenous isomerase and the exogenous phosphatase are expressed by a stationary phase promoter.

40. The recombinant microorganism of any one of claims 1 -38, wherein the exogenous isomerase and the exogenous phosphatase are by an inducible promoter.

41. A microorganism comprising a recombinant polynucleotide encoding an isomerase and a phosphatase, wherein expression of the isomerase and the phosphatase results in anincreased production of ribose as compared to a microorganism lacking the recombinant polynucleotide.

42. The microorganism of claim 41 , wherein the Isomerase is a ribose 5-phosphate isomerase A (RpiA) or ribose 5-phosphate isomerase B (RpiB).

43. The microorganism of claim 41 or 42, wherein the isomerase is an £. call RpiA or RpiB.

44. The microorganisms of any one of claims 41 -43, wherein the isomerase comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3.

45. The microorganisms of any one of claims 41-44, wherein the isomerase comprises the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO:3.

46. The microorganisms of any one of claims 41-45, wherein the isomerase consists of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO:3.

47. The microorganism of any one of claims 41-46, wherein the phosphatase is a sugar phosphatase YbiV or YidA.

48. The microorganism of any one of claims 41 -47, wherein the phosphatase is an E co / z YbiV or YidA.

49. The microorganisms of any one of claims 41-48, wherein the phosphatase comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 7.

50. The microorganisms of any one of claims 41-49, wherein the phosphatase comprises the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 7.51 . The microorganisms of any one of claims 41 -50, wherein the phosphatase consists of the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 7.

52. 'The recombinant microorganism of any one of claims 41-51 , wherein the recombinant, microorganism further comprises at least, one exogenous enzyme of the pentose phosphate pathway.

53. The recombinant microorganism of claim 52, wherein the at least one exogenous enzyme of the pentose phosphate pathway comprises glucose-6-phosphate dehydrogenase (Zwf).

54. The recombinant microorganism of claim 53, wherein the Zwf comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 51.

55. The recombinant microorganism of claim 54, wherein the Zwf comprises the amino acid sequence set forth in SEQ ID NO: 51.

56. The recombinant microorganisms of claim 52, wherein the at least one exogenous enzyme of the pentose phosphate path way comprises phosphogluconate dehydrogenase (Gnd).

57. The recombinant -microorganism of claim 56, wherein the Gn.d comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 53.

58. The recombinant microorganism of claim 58, wherein the Gnd comprises the amino acid sequence set forth in SEQ ID NO: 53.

59. The microorganism of any one of claims 41-58. further comprising a mutation of a gene encoding for an enzyme of the glycogen biosynthesis.

60. The microorganism of claim 59, wherein enzyme of the glycogen biosynthesis is phosphoglucomutase (Pgm).6.

1. The microorganism of any one of claims 41-60, further comprising a mutation of a gene encoding for an enzyme of the carbohydrate biosynthesis.

62. The microorganism of claim 61 wherein the enzyme of the carbohydrate biosynthesis is glucose- 6-phosphate isomerase (Pgi).

63. The recombinant rnicroorganism of any one of claims 41-62, further comprising a mutation of a gene encoding for an enzyme of the purine / pyrimidme ribonucleosides degradation pathway compared to a naturally occurring microorganism.

64. The recombinant microorganism of claim 63, wherein the enzyme of the pentose phosphate pathway is phosphopentomutase (DeoB).

65. The microorganism of any one of claims 41-64, further comprising a mutation of a gene encoding for an enzyme of pentose phosphate pathway .

66. The microorganism of claim 65. wherein enzyme of pentose phosphate pathway transketolase 1 (Tkt A) or transketolase 2 (TktB).

67. The recombinant microorgani sm of any one of claims 41 -66 further comprising a mutation of a gene encoding for an enzyme of glycolysis compared to a naturally occurring microorganism.

68. The recombinant microorganism of claim 67, wherein the enzyme of glycolysis is phosphofructokinase- 1 (PfkA ) .

69. The recombinant microorganism of any one of claims 41 -68 further comprising a mutation of a gene encoding for an epi merase compared to a naturally occurring microorgani sm.

70. The recombinant microorganism of claim 69, wherein the epimerase alIose-6-phosphate- 3 -ep imierase (AlsE)71 . The .recombinant, microorganism of any one of claims 41-70 further comprising a mutation of a gene encoding for an enzyme of the gluconate degradation pathway compared to a naturally occurring microorganism.

72. The recombinant microorganism of claim 41, wherein the enzyme of the gluconate degradation pathway is D-gluconate-6~phosphate (Edd)73. The recombinant microorganism of any one of claims 41-72 further comprising a mutation of at least one gene encoding for an enzyme of the ribose catabolism pathway.

74. The recombinant microorganism of claim 73. wherein the at least one gene encoding for an enzyme of the ribose catabolism pathway is selected from D-ribose transporter ATP binding subunit (RbsA), D-ribose transporter perip lasmic binding protein (RbsB), D- ribose transporter membrane subunit (RbsC), D-ribose pyranase (RbsD), D-ribose kinase (RbsK), DNA-binding transcriptional dual regulator (RbsR), small regulatory RNA (RbsZ), or combinations thereof.

75. The microorganism of any one of claims 41-74, wherein the exogenous isomerase and the exogenous phosphatase are expressed by a stationary phase promoter.

76. The microorganism of any one of claims 41 -74, wherein the exogenous isomerase and the exogenous phosphatase are expressed by an inducible promoter.

77. A microorganism comprising; a) a recombinant polynucleotide encoding an isomerase and a phosphatase; b) a mutation of a gene encoding for an enzyme of the glycogen biosynthesis,; c) a mutation of a gene encoding for an enzyme of the carbohydrate biosynthesis; and d) a mutation of a gene encoding for an enzyme of pentose phosphate pathway; e) optionally a recombinant polynucleotide encoding GalP, Glk, or both; f) optionally a recombinant polynucleotide encoding Zwf. Gnd, or both; g) optionally a mutation of a gene encoding for an epimerase; h) optionally a mutation of a gene encoding for an enzyme of the gl uconaie degradation pathway ; i) optionally a mutation of a gene encoding for an enzyme of glycolysis; and j) optionally a mutation of at least one gene encoding for the ribose c atabol i sm pathway .

78. A microorganism comprising:a) a recombinant polynucleotide encoding a ribose 5-phosphate isomerase A (RpiA) or ribose-5-phosphate isomerase B (RpiB) and a sugar phosphatase YbiV or YidA; b ) a mutation of phosphoglucomutase (pgro); c) a mutation of glucose-6-phosphate isomerased) a mutation of phosphopentomutase (tfeo / i); e) a mutation of transketolase 1 (Wri), transketolase 2 (IktB), or both; f) optionally a recombinant, polynucleotide encoding GalP, Glk, or both; g) optionally a recombinant polynucleotide encoding Zwf, Gnd, or both; h) optionally a mutation of aHulose-6-phosphate 3-epimerase fo / sh'); i) optionally a mutation of phosphogluconate dehydratase (tW); j) optionally a mutation of pliosphofructokinase-1and k) optionally a mutation of at least one gene encoding for an enzyme of the ribose catabolism pathway selected from D-ribose transporter ATP binding subunit (RbsA), D-ribose transporter periplasmic binding protein (RbsB), D-ribose transporter membrane subunit (RbsC), D-ribose pyranase (RbsD), D-ribose kinase (RbsK), DNA-binding transcriptional dual regulator (RbsR). small regulatory RNA (RbsZ), or combinations thereof.

79. A microorganism comprising; a) a recombinant polynucleotide encoding a ribose 5-phosphate isomerase A (Rpi A ) or ribose 5-phosphate isomerase B (RpiB); b) a recombinant polynucleotide encoding a sugar phosphatase YbiV or YidA; c) a mutation of phosphoglucomutase (pg / n); d) a mutation of glucose-6-phosphate isomerase (pg?); e) a mutation of phosphopentomutase (rise / ?); f) a mutation of transketolase I ( / kiA), transketolase 2 ($$) or both; g) optionally a recombinant polynucleotide encoding GalP, Glk, or both; h) optionally a recombinant polynucleotide encoding Zwf, Gnd, or both; i) optionally a mutation of allulose-6-phosphate 3-epimerase (rz / .v / f); j) optionally a mutation of phosphogluconate dehydratase (eric / ); k ) optionally a mutation of phosphofructokinase- 1 (p / kA'y, and l) optionally a mutation of at least one gene encoding for an enzyme of the ribose catabolism pathway selected from D-ribose transporter ATP binding subunit (RbsA), D-ribose transporter periplasmic binding protein (RbsB), D-ribose transporter membrane subunit (RbsC), D-ribose pyranase (RbsD), D-ribose. kinase (RbsK), DNA-binding transcriptional dual regulator (RbsR), small regulatory RNA (RbsZ), or combinations thereof.

80. The microorganism of any one of claims 23-40 and 59-79, wherein the mutation is a deletion.81 The microorganisms of any one of claims 23-40 and 59-80, wherein the mutation reduces or eliminates expression or activity of the enzyme.

82. The microorganism of any one of claims 1-81 , wherein the microorganism is £. co / / , BcKt / hrs snhri / b or Laemcoccus tectb.

83. A method for producing ribose comprising culturing the microorganism of any one of claims 1-82, under conditions suitable for converting a substrate to ribose.

84. The method of claim 83, wherein the substrate comprises glucose.

85. A method of making a food product comprising ribose comprising a) culturing the microorganism of any one of claims 1 -81 under conditions suitable for converting a substrate to ribose; and b) admixing the ribose with one or more foods to form a food comprising ribose.

86. The method of claim 85, wherein the food product is a beverage, yogurt, ice cream, a baked good, or a nutritional bar.

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