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WO2025054412A1 - Micro-organismes pour la production de d-mannose - Google Patents

Micro-organismes pour la production de d-mannose Download PDF

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Publication number
WO2025054412A1
WO2025054412A1 PCT/US2024/045528 US2024045528W WO2025054412A1 WO 2025054412 A1 WO2025054412 A1 WO 2025054412A1 US 2024045528 W US2024045528 W US 2024045528W WO 2025054412 A1 WO2025054412 A1 WO 2025054412A1
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Prior art keywords
microorganism
certain embodiments
mannose
seq
amino acid
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Inventor
Shota Atsumi
Justin B. Siegel
Jayce Elizabeth TAYLOR
Dileep Sai Kumar PALUR
Bryant LUU
Augustine ARREDONDO
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y503/00—Intramolecular oxidoreductases (5.3)
    • C12Y503/01—Intramolecular oxidoreductases (5.3) interconverting aldoses and ketoses (5.3.1)
    • C12Y503/01008—Mannose-6-phosphate isomerase (5.3.1.8)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03068—2-Deoxyglucose-6-phosphatase (3.1.3.68)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the recombinant microorganism further comprises a mutation of a gene encoding for an enzyme of the glycolysis.
  • the enzyme of the glycolysis is phosphofiructokina.se- 1 (PfkA), phosphofructokinase-2 (Pfkb ), pyruvate kinase (PykF) allulose 6- phosphate 3-epimerase (AlsE), or a combination thereof.
  • the enzyme of the glycolysis is phosphofructokinase-. I (PfkA).
  • the enzyme of the glycolysis is allulose 6-phosphate 3-epimerase (AlsE).
  • the recombinant microorganism comprises a mutation of a gene encoding phosphofructokinase- 1 (PfkA) and a mutation of a gene encoding allulose 6-phosphate 3-epimerase (AlsE).
  • PfkA phosphofructokinase- 1
  • AlsE allulose 6-phosphate 3-epimerase
  • the recombinant microorganism further comprises a mutation of one or more genes encoding proteins involved in the mannose metabolism. In some embodiments, the recombinant microorganism further comprises a mutation of the manX YZ operon.
  • the recombinant .microorganism further comprises (a) exogenous galactose:H ; symporter (GalP), and (b) glucokinase (Glk).
  • the GalP is an E. coli GalP
  • the Glk is E. coli Glk.
  • the GalP comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 28, and (b) the Glk comprises an amino acid sequence at least about 80% identical to the amino acid sequence set forth in SEQ ID NO: 30.
  • the GalP comprises or consists of the amino acid sequence set forth in SEQ ID NO: 28, and (b) the Glk comprises or consists of the amino acid sequence set forth in SEQ ID NO: 30.
  • the exogenous isomerase and the exogenous phosphatase are expressed by a stationary phase promoter or an inducible promoter. In some embodiments, the exogenous isomerase and the exogenous phosphatase are expressed by a stationary phase promoter. In some embodiments, the stationary phase promoter is PgadB,
  • 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 mannose as compared to a microorganism lacking the recombinant polynucleotide.
  • the isomerase is a mannose 6- phosphate isomerase (ManA). In some embodiments, the isomerase is an E. coli .ManA. 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 epimerase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1.
  • the microorganism further comprises a mutation of a gene encoding for an enzyme of the pentose phosphate pathway.
  • the enzyme of the pentose phosphate pathway is ghicose-6-phospha.te 1 -dehydrogenase (Zwt).
  • the microorganism further comprises a mutation of a gene encoding for an enzyme of the glycolysis.
  • the enzyme of the glycolysis is phosphofructokinase- 1 (PfkA), phosphofiticiokinase-2 (P&B), pyruvate kinase (PykF), allulose 6-phosphate 3-epimerase (AlsE), or a combination thereof
  • the enzyme of the glycolysis is phosphofructokinase-1 (PfkA).
  • the enzyme of the glycolysis is allulose 6-phosphate 3-epimerase (AlsE).
  • the microorganism comprises a mutation of a gene encoding phosphofruclokinase-l (PfkA) and a mutation of a gene encoding allulose 6-phosphate 3-epimerase (AlsE ).
  • the microorganism further comprises a mutation of one or more genes encoding proteins involved in the mannose metabolism, In some embodiments, the microorganism further comprises a mutation of the manXYZ operon.
  • the recombinant polynucleotide comprises a stationary phase promoter or an inducible promoter.
  • the stationary phase promoter is PgadB.
  • the present disclosure related to a microorganism comprising (a) an exogenous isomerase; (b) an exogenous phosphatase; (c) a mutat ion of a gene encoding for an enzyme of the pentose phosphate pathway; (d) a mutation of a gene encoding for an enzyme of the glycolysis; and (e) a mutation of a gene encoding for an enzyme of mannose metabolism pathway.
  • the microorganism is E. coli, Bacillus sublilis or Lactococus lactis.
  • the present disclosure also relates to a method for producing mannose comprising culturing the microorganism disclosed herein under conditions suitable for converting a substrate to mannose.
  • the substrate comprises glucose.
  • Figure I illustrates an exemplary pathway of the biosynthesis of D-rnannose.
  • the dephosphoiylation step from 'D-mannose-6- phosphate to D-mannose drives the reaction towards D-.matmose production due to a large negative AG’m under cellular reactant concentrations of 1 mM.
  • Deleted steps are underlined. Overexpressed steps are italicized.
  • PTS the phosphotransferase system
  • hexitol phosphatase B Zwf, NADP+-dependent gIucose-6-phosphate dehydrogenase
  • Pgm phosphoglucornutase
  • PfkA 6- phosphofTuctokinase 1
  • PlkB 6 ⁇ phosphofructokinase 2
  • AlsE D-allulose-6 ⁇ phosphate 3-epimerase
  • ManA mannose-6- phosphate isomerase.
  • FIGs 2 A and 2B show an exemplary pathway for the production of D-mannose capability of E. coli Glucose is imported and phosphorylated to glucose-6-phosphate (G6P) by the phosphotransferase system (PTS) or GalP/Glk, G6P is then isomerized to fhictose-6-phosphate (F6P) by Glucose-6-phosphate isomerase, F6P is isomerized to d-mannose 6-phosphate (M6P) by mannose 6-phosphate isomerase (ManA), which is then dephosphorylated to free mannose by Hexitol phosphatase B (HxpB).
  • G6P glucose-6-phosphate
  • PTS phosphotransferase system
  • GalP/Glk GalP/Glk
  • F6P fhictose-6-phosphate
  • F6P isomerized to d-mannose 6-phosphate
  • ManA mannose 6-phosphate
  • Competing pathways include, glycolysis, which is catalyzed by phosphofructokinases A and B (PfkA and B), or allulose 6-phosphate 3 -epimerase (AlsE), the pentose phosphate pathway, catalyzed by glucose-6-phosphate dehydrogenase (Zwf), the glycogen biosynthesis pathways, catalyzed by phosphogiucomutase (pgm) s and the mannose biosynthesis pathway, catalyzed by mannose- 1 •phosphate guanylyltransferase (ManC) and phosphomannomutase (ManB).
  • glycolysis which is catalyzed by phosphofructokinases A and B (PfkA and B), or allulose 6-phosphate 3 -epimerase (AlsE)
  • the pentose phosphate pathway catalyzed by glucose-6-phosphate dehydrogenase (Zwf)
  • Figures 3 A-3C illustrate D-mannose production capability in E. coli Cells were grown in M9P media with 10 g L -1 glucose at 37 °C to OD600 -0.4, then grown at 30 °C for 24 h. At OD600 -0.4, 1 mM IPTG was added ( Figures 3B and 3C).
  • Figure 3 A shows D-mannose production in AL 1050 (MG1655) AL3755 (ALI050 with and AL4240 (A.L3755 with ⁇ zwf) (Table 1).
  • Figure 3B shows D-mannose production in AL4290 (AL4240 with AmanA) with and without plasmid expression of man A. (Table 1 and Table 2).
  • Figure 3C shows various sugar phosphatases with ManA tested for D-mannose production in AL4290, Error bars indicate s.d. (n TM 3 biological replicates).
  • Figure 4 illustrates modulation of gene expression in £ coU for D-manuose production.
  • Cells were grown in M9P media with 10 g L -1 glucose at 37 °C to OD600 -0.4, then grown at 30 °C for 24 h, At OD600 -0.4, 1 mM 1PTG was added when required, Operon with manA and hxpB were expressed under either the Puscoi or Pgadu (p.AL2303, pAL2630) (Table 2) in AI..4290, AL4314 and AI..4329 (Table 1). Error bars indicate s.d. (n ⁇ 3 biological replicates).
  • Figure 5 illustrates monitoring of D-mannose intake in AL.4329 and AL4592.
  • Cells were grown, in M9P media with 10 g L -1 mannose at 30 °C for 24 h.
  • a) Deletion of D-mannose transporter genes, manXYZ in AL4329 resulted in AL4592 (Table 2). Error bars indicate s.d, (n 3 biological replicates).
  • the present disclosure is based, in pan, on the discovery that microorganisms including specific genetic modifications (e.g., gene deletion and/or gene overexpression) can be made for producing sugars.
  • the sugar is D-mannose.
  • 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, [0043]
  • polypeptide “peptide,” “amino acid sequence” and “protein,” used interchangeably herein, refer to a molecule formed from the linking of at least two amino acids.
  • PfkA is a Lactococcus lactis PfkA.
  • a representative amino acid sequence of L-aclocaccus faciis PfkA is found as LLA12 RS07020: ATP-dependent 6- phosphofructokinase EC2.7.1 J 1 , or as set .forth in SEQ ID NO: 13 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 13, which is provided below:
  • a representative .nucleotide sequence of a Zac/oeoccw kwtix gene is 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:
  • PfkB is an E. coli PfkB (EcoCyc ID: EG 10700; UniProt ID: P06999).
  • a representative nucleotide sequence of gene pJkB is set forth in SEQ ID NO: 17, SEQ ID NO: 17 is provided below:
  • coir PfkB comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%. at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 18.
  • A. coll PfkB comprises the amino acid sequence set forth in SEQ ID NO: 18. SEQ ID NO: 18 is provided below:
  • a representative nucleotide sequence of Bacillus sufiiFE geue ZEE 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:
  • the presently disclosed microorganisms include a mutation of one or more genes encoding proteins involved in the mannose metabolism. In certain embodiments, the presently disclosed microorganisms include a reduced expression of one or more genes encoding proteins involved in the mannose metabolism. In certain embodiments, the presently disclosed microorganisms include a mutation of the operon. In certain embodiments, the presently disclosed microorganisms include a reduced expression of genes of the manXYZ operon. Further details on the manXYZ operon can be found in Gosset, Af/craZ? OZZ Fact 4, 14 (2005).
  • the maciXYZ operon comprises a mauX gene (Ecocyc ID: EG10567; UniProt ID: P69797).
  • the manX gene is a E.coli maitX gene.
  • a representative nucleotide sequence of rnanX gene is set forth in S-EQ ID NO: 50 or at least 90%, 95%, 95%, or 99%: identical to SEQ ID NO: 50.
  • coll ManX comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 51.
  • E. coli ManX comprises the amino acid sequence set forth in SEQ ID NO: 51.
  • SEQ ID NO: 50 and 51 are provided below.
  • the manXYZ operon comprises a man Y gene (EcoCyc ID: EG 10568; UniProtID; P69801).
  • the wwKgene isaE.coli wmKgene.
  • E coli ManY 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: 53.
  • Zc coll ManY comprises the amino acid sequence set forth in SEQ ID NO: 53 , SEQ ID NO: 52 and 53 are provided below.
  • the manXYZ operon comprises a mcwZ gene (EcoCyc ID: EG 10569; UniProt ID: P69805).
  • the manZ gene is a E.coli manZ gene.
  • a representative nucleotide sequence ofnmnZ gene is set forth in SEQ ID NO: 54 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 54.
  • K coU ManZ comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%.
  • E. coll ManZ comprises the amino acid sequence set forth in SEQ ID NO: 55, SEQ ID NO: 54 and 55 are provided below.
  • the presently disclosed microqxganisms include mutation of a gene encoding an enzyma of the mannose biosynthesis pathway. In certain embodiments ⁇ the presently disclosed microorganisms include a reduced expression of a gene encoding an snzyme of the mannose biosynthesis pathway.
  • the enzyme of the manncse biosynthesis pathway is mannose- 1-phosphate guanylyltransferase (MauQ> iEntrea Gene ID: 94 DESO 1 .
  • ManC is involved in the synthesis involved in the biosynthesis of the capsular polysaccharide colanic acid.
  • ManC is an lb coll HanC.
  • a representative nucleotide sequence of gons ma.au is set forth in SEQ ID bid 9 or at least DOS; 951, 95iy or 991 identical to SEO ID PG: 9 f which is provided below,
  • Zi co/.i ManC comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 20
  • E. coli ManC comprises the amino acid sequence set forth in SEQ ID NO: 20.
  • SEQ ID NO: 20 is provided below:
  • the enzyme of the mannose biosynthesis pathway is phosphomannomutase (ManB) (Entrez Gene ID: 946574). ManB is involved in the biosynthesis of the capsular polysaccharide colanic acid. In certain embodiments, ManB is an E. coli ManB.
  • a representative nucleotide sequence of gene manB is set forth in SEQ ID NO: 38 or at least 90%, 95%, 95%, or 99% identical to SEQ ID NO: 38, which is provided below.
  • E. coli ManB 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 hi SEQ ID NO: 39
  • E. coli ManB comprises the amino acid sequence set forth in SEQ ID NO: 39.
  • SEQ ID NO: .39 is provided below:
  • the deletion of a gene comprises a non-franieshift deletion, a frameshift deletion, or a combination thereof.
  • the deletion of a. gene can be achieved by an insertion (e.g,, a non-frameshift insertion, a frameshift insertion, or a combination thereof).
  • the deletion of a gene comprises a nonsense mutation,
  • the present disclosure provides recombinant microorganisms. Any culturable microorganism is suitable for use in the compositions and methods described herein.
  • the microorganism is a bacterium.
  • the microorganism is selected from the group consisting of .Aceiabac/er aeeii, Achrwnobacter, Aeidiphilium. Acineiabac/er. Aciirnnmuiura, A ctinoplanes.
  • Melhanobacterium bryantii Micrabacterhiin imperials, Micrococcus lysadeik ticus, Micmlunaius, Mueotjtn ⁇ anicus, Afycobacieriim, Myrolhecium, Niirobacier, Nilrosomonas. Kocardia, Papaya carica, Pediocaccus. Pediococcus halophilus, Paracoccus parnolrophus, Propiarubacteram, Pseudomonas, fyei/donua/as/luui-'cscens, Pseudomouas deuiirifmaus.
  • the microorganism Is Escherichia mZz
  • the microorganism is Bacillus .su/jrib's. In certain embodiments, the microorganism is Laclocaccus laclis.
  • the E. coli is selected from the group consisting of Enterotoxigenic E. coli (ETEC), Enteropathogenic E. coli (EPEC), Enteroinvasive E. coli (El EC ), Enterohemorrhagic E. coE (EHEC), Uropathogenic E. coli (UPEC), Verotoxin-prodncing E. coli E. coli O157:H7, E. coli 0104:414, E. coliOI2L E. coli 0104:021, E.
  • ETEC Enterotoxigenic E. coli
  • EPEC Enteropathogenic E. coli
  • El EC Enteroinvasive E. coli
  • EHEC Enterohemorrhagic E. coE
  • UPEC Uropathogenic E. coli
  • the E. coli is E. coli K12.
  • the E. call is E. coE B.
  • the E. coE is E. coE C.
  • the E. coli is derived from a strain selected from the group consisting of NCTC 1.2757, 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,
  • the E. coli is derived from a strain selected from the group consisting of BC 4734 (O26:HI 1), BC 4735 (Ol57rH-)> BC 4736 , BC 4737 (n.d,), BC 4738 (Ol57rH7), BC 4945 (O26:H-), BC 4946 (0157:147), BC 4947 (0111:H-), BC 4948 (O157:H), BC 4949 (05), BC 5579 (OI57:H7), BC 5580 (O157:H7), BC 5582 (03 :H), BC 5643 (O2:H5), BC 5644 (0128), BC 5645 (O55;H ⁇ ), BC 5646 (O69;H ⁇ ), BC 5647 (0101 :419), BC 5648 (O103:112), BC 5850 (022:118), BC 5851 (055:H-), BC 5852 (048:1121), BC 5853 (O26:H11), BC 5854 (OI57:H7),
  • the E. coli. is derived from a strain selected from the group consisting of BC 8246 (O152:K-:H-), BC 8247 (O124:K(72):H3) 5 BC 8248 (0124), BC 8249 (0112), BC 8250 (OI36:K(78):B ⁇ ), BC 8251 (0124:11-), BC 8252 (O144:K-:H-), BC 8253 (0143:01-), BC 8254 (0443), BC 8255 (0112), BC 8256 (O28a.e), BC 8257 (O124:H-), BC 8258 (0143), BC 8259 (O167X-:H5), BC 8260 (0128a. c.:H35), BC 8261 (0164), BC 8262 (O164:K-:H-), BC 8263 (0164), and BC 8264 (0124).
  • the K svbtilis is derived from Strain 168.
  • the present disclosure provides a recombinant microorganism comprising an increased production of D-mannose as compared to a naturally occurring microorganism.
  • the recombinant microorganism comprises an exogenous isomerase, an exogenous phosphatase, and a deletion of three genes.
  • the exogenous isomerase is a D-mannose 6-phosphate isomerase (ManA).
  • the exogenous phosphatase is hexitol phosphatase B (HxpB).
  • the three deleted genes are phosphofructokinase- 1 (p/fcl), glucGse-6-phosphate 1 -dehydrogenase (zw/) s and allulose 6- phosphate 3-epimerase (afe£).
  • the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is E. coli.
  • the present disclosure provides a recombinant microorganism comprising an increased production of D-mannose as compared to a naturally occurring microorganism.
  • the recombinant microorganism comprises an exogenous isomerase, an exogenous phosphatase, and a deletion of three genes.
  • the exogenous isomerase is a D-mannose 6-phosphate isomerase (MauA).
  • the exogenous phosphatase is hexitol phosphatase B (HxpB).
  • the three deleted genes are phosphofructokinase- 1 fe/M), glucose-d-phosphate 1 -dehydrogenase (cvv/), and allulose 6- phosphate 3-epimerase ( «&£).
  • the recombinant microorganism is a bacterium.
  • the bacterium is L lactis,
  • the present disclosure provides a recombinant microorganism comprising an increased production of D-mannose as compared to a naturally occurring microorganism.
  • the recombinant microorganism comprises an exogenous isomerase and an exogenous phosphatase.
  • the exogenous isomerase is a mannose 6-phosphate isomerase (ManA).
  • the exogenous phosphatase is hexitol phosphatase B (HxpB).
  • the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is £. coll.
  • the present disclosure provides a recombinant microorganism comprising an increased production of D-mannose as compared to a naturally occurring microorganism.
  • the recombinant microorganism comprises an exogenous isomerase, an exogenous phosphatase, and a deletion of at least one gene.
  • the exogenous isomerase is a D-mannose 6-phosphate isomerase (ManA)
  • the exogenous phosphatase is hexitol phosphatase B (HxpB).
  • the at least one gene is selected from phosphofructokinase-l (pfk-A), glucose-6-phosphate 1 -dehydrogenase (zw)), allulose 6-phosphate 3-epimerase phosphoglucomutase (/zgn-i), mannose-specific PTS enzyme II AB component (ntanX), mannose-specific PTS enzyme IIC component (miml), mannose-specific PTS enzyme HD component or a combination thereof.
  • the recombinant microorganism is a bacterium. In certain embodiments, the bacterium is E. coll.
  • the present disclosure provides a microorganism comprising a recombinant polynucleotide, wherein the microorganism comprises an increased production of D- mannose as compared to a naturally occurring microorganism.
  • the recombinant polynucleotide comprises a nucleotide sequence encoding an exogenous isomerase and an exogenous phosphatase.
  • the exogenous isomerase is a mannose 6- phosphate isomerase (ManA).
  • the exogenous phosphatase is hexitol phosphatase B (HxpB).
  • the recombinant microorganism is a bacterium. In certain, embodiments, the bacterium is E. coll.
  • the present disclosure provides a microorganism comprising a recombinant polynucleotide, wherein the microorganism comprises an increased production of mannose as compared to a naturally occurring microorganism.
  • the recombinant polynucleotide comprises a nucleotide sequence encoding an exogenous isomerase and an exogenous phosphatase.
  • the exogenous isomerase is a mannose 6- phosphate isomerase (ManA)
  • the exogenous phosphatase is hexitol phosphatase B (HxpB).
  • the microorganism further comprises a deletion of at least one gene,
  • the at least one gene is selected front phosphofnictokinase-1 (pfltA'), glucose- 6-phosphate l-dehydrogenase (CM;/), allulose 6-phosphate 3-epimerase (a/x/f), phosphoglucomutase (pgw), mannose-specific PTS enzyme I1AB component (manX). mannose- specific PTS enzyme IIC component (w.w F), mannose-specific PTS enzyme IID component (n?unZ), or a combination thereof.
  • the recombinant microorganism is a bacterium.
  • the bacterium is coli
  • the bacterium is B. subtilis.
  • the bacterium is L. lactis.
  • the present disclosure provides a recombinant microorganism comprising an increased production of D-mannose as compared to a naturally occurring microorganism.
  • the recombinant microorganism comprises an exogenous isomerase, an exogenous phosphatase, an exogenous nuclease, a sgRNA, and a deletion of at least one gene, in certain embodiments, the exogenous isomerase is a mannose 6-phosphate isomerase (ManA), In certain embodiments, the exogenous phosphatase is hexitol phosphatase B (HxpB).
  • the at least one gene is selected from phosphofiuctokinase-1 (/y7c4), glucose-6- phosphate 1 -dehydrogenase allulose 6-phosphate 3-epimerase (u/s/i), phosphoglucomutase (pgn?), .mannose-specific PTS enzyme IIAB component (manX), mannose-specific PTS enzyme IIC component (wuT), mannose-specific PTS enzyme IID component (w»Z), or a combination thereof, [0126]
  • the recombinant microorganism is a bacterium, in certain embodiments, the bacterium is E, coli. In certain embodiments, the bacterium is B, subtilise In certain embodiments, the bacterium is L. lactis.
  • the present disclosure provides a microorganism comprising a recombinant polynucleotide, wherein the microorganism comprises an increased production of D- mannose as compared to a naturally occurring microorganism.
  • 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
  • the exogenous isomerase is a mannose 6-phosphate isomerase (ManA)
  • the exogenous phosphatase is hexitol phosphatase B (HxpB)
  • the microorganism further comprises a deletion of at least one gene.
  • the at least one gene is selected from phosphofructokinase- 1 (pjkA), glucose-6- phosphate 1 -dehydrogenase (*pf), allulose 6-phosphate 3-epimerase (ah'E), phosphoglucomutase mannose-specific PTS enzyme IIAB component (manX), mannose-specific PTS enzyme IIC component (rmeK), mannose-specific PTS enzyme IID component (mariZt or a combination thereof.
  • the microorganism further comprises a sgRNA.
  • the recombinant microorgan. ism is a bacterium.
  • the bacterium is K coll
  • the bacterium is & xubrife
  • the bacterium is L, lactis.
  • the present disclosure also provides methods for preparing and/or generating any of die microorganisms disclosed herein, Many recombinant techniques commonly known in the art may be used to introduce one or more recombinant polynucleotides of the present disclosure into a microorganism, including without limitation protoplast fusion, transfection, transformation, conjugation, and transduction. These techniques include conventional molecular biology techniques recombinant techniques), microbiology , cell biology, and biochemistry, which are wi thin the skill of the art.
  • the recombinant polynucleotides disclosed herein can be stably integrated into a microorganism chromosome, la certain embodiments, the recombinant polynucleotides disclosed herein are stably integrated into a microorganism chromosome using homol ogous recombi nation, transposition-based chromosomal integration, recombinase -mediated cassette exchange (RMCE-; e.g,, using a Cre-lox system), or an integrating plasmid fo.g.. a yeast integrating plasmid).
  • RMCE- recombinase -mediated cassette exchange
  • 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).
  • an extra-chromosomal plasmid e.g., an expression plasmid or vector
  • extra-chromosomal plasmids suitable for a range of microorganisms are known in the art, including without, limitation replicating plasmids fo.g., yeast replicating plasmids that include an autonomously replicating sequence, ARS), centromere plasmids feg., yeast centromere plasmids that include an autonomously replicating sequence, CEN), episomal plasmids fe.g., 2 ⁇ p.m plasmids), and/or artificial chromosomes yeast artificial chromosomes, YACs, or bacterial artificial chromosomes. BACs), 3.1J. Vector#
  • the present disclosure provides vectors including the nucleotide sequences disclosed herein.
  • 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.
  • plasmid refers to a circular double-stranded DMA construct used as a cloning and. or expression vector.
  • plasmids can be extrachromosomal self-replicating genetic elements (e.g., episomal plasmids) when introduced into a microorganism.
  • plasmids can integrate into a microorganism chromosome.
  • 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.
  • the vectors allow the integration of one or more polynucleotides into the genome of a microorganism.
  • a vector disclosed herein includes a promoter.
  • the vector is a bacterial or prokaryotic expression vector.
  • the vector is a yeast or fungal cell expression vector.
  • a vector discloses herein comprises nucleotide sequences in a single operaa.
  • the recombinant polynucleotides disclosed herein include a control sequence, an enhancer, or a promoter.
  • a nucleotide sequence encoding the Afonri gene and/or hpxB gene can be operably linked to a control sequence, enhancer, or promoter.
  • promoter refers to any nucleotide sequence that regulates the initiation of transcription for a particular coding sequence under its control. Biologically, promoters are not transcribed but coordinate the assembly of components that initiate the transcription of other nucleotide sequences. In addition, promoters can limit tins assembly and subsequent transcription to specific prerequisite conditions. For example, but without any limitation, a promoter can allow transcription in response to one or more environmental, temporal or developmental stimuli. Bacterial and fungal cells possess a multitude of proteins that sense external or internal conditions and initiate signaling cascades ending in the binding of proteins to specific promoters and subsequent initiation of transcription of nucle ic 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.
  • the promoter is a constitutive promoter.
  • a constituti ve promoter is a promoter that drives the expression of a nucleotide sequence continuously and without interruption in response to internal or external stimuli.
  • Constitutive promoters are commonly used in recombinant engineering to ensure the continuous expression of a desired nucleotide sequence, Constitutive promoters result in a robust amount of nucleic acid expression, and, as such, are used in many recombinant engineering applications to achieve a high level of recombinant protein and enzymatic activity.
  • Non-limiting examples of constitutive promoters encompassed by the present disclosure include £.
  • the promoter is active in the stationary phase of the microorganism.
  • Exemplary stationary phase promoters can be found in, e.g., Shhnada, ef&L JOURNAL OF BACTERlOLOGYLNiw. 2004, p. 7112-7122; Pletnev at eL , .4C7/I ATI TURAE
  • the promoter is an inducible promoter.
  • An inducible promoter is a promoter that drives the expression of a nucleotide sequence in response to a stimulus.
  • An inducible promoter drives sustained expression upon, exposure to a specific stimulus (e.g., IPTG).
  • an inducible promoter drives a graded level of expression correlated with the amount of stimulus.
  • Nou-limiting examples of stimuli for inducible promoters include heat shock, exogenous compounds or a lack thereof (e.g., a sugar, metal, drug, or phosphate),, salts or osmotic shock,, oxygen, and biological stimuli (e.g., a growth factor or pheromone).
  • Non-limiting examples of inducible promoters include the E coli promoters /W ft JWD, and Pucuv.
  • the recombinant polynucleotide can include multiple promoters.
  • the multiple promoters can be the same.
  • the recombinant polynucleotide can include a nucleotide sequence encoding the mariA gene operably linked to a first promoter and a nucleotide sequence encoding the hpxB gene operably linked to a second promoter, wherein the first and second promoter is the same.
  • the multiple promoters can be different.
  • the recombinant polynucleotide can include a nucleotide sequence encoding the gene operably linked to a first promoter and a nucleotide sequence encoding the hpxB gene operably linked to a second promoter, wherein the first and second promoter are different.
  • the promoter is a AMU promoter.
  • the Ptu-oi promoter comprises the nucleotide sequence set forth in SEQ ID NO: 40.
  • the Ptiscoi promoter consists of the nucleotide sequence set forth in SEQ ID NO; 40,
  • the promoter is a hybrid regulatory region including the promoter ft of phage lambda with the CI binding sites replaced with lacOi, The hybrid design allows for a strong promotion that can be repressed by Lacl, the Lac inhibitor (i.e., repressor) or induced by IPTG.
  • the promoter is a /AtOt promoter.
  • the ftreror promoter comprises the nucleotide sequence set forth in SEQ ID NO: 41.
  • the ft.jEiot promoter consists of the nucleotide sequence set forth in SEQ ID NO: 41.
  • the promoter is a ft? promoter.
  • the ft " promoter comprises the nucleotide sequence set forth in SEQ ID NO: 42.
  • the ftp promoter consists of the nuc leotide sequence set forth in S EQ ID NO: 42.
  • the promoter is a promoter.
  • the /At promoter comprises the nucleotide sequence set forth in SEQ ID NO: 43.
  • the /As promoter consists of the nucleotide sequence set forth in SEQ ID NO: 43.
  • the promoter is a /A ⁇ «® promoter.
  • the /A*® promoter comprises the nucleotide sequence set forth in SEQ ID NO: 44.
  • the Pgi «is promoter consists of the nucleotide sequence set forth in SEQ ID NO: 44.
  • ZA-iasoi promoter nucleotide sequence [0142]
  • the promoter is a stationary phase promoter.
  • 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 upregulated.
  • 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.
  • the presently disclosed recombinant polynucleotides include genetic markers. These genetic markers allow the selection of microorganisms that have one or more desired polynucleotides (e.g., recombinant polynucleotides).
  • the genetic marker is an antibiotic resistance marker selected from the group consisting of Apramycin resistance, Ampicillin resistance, Kanamycin resistance, Specdnomycin resistance, Tetracyclin resistance, Neomycin resistance, Chloramphenicol resistance, Gentamycin resistance. Erythromycin resistance, Carbenicillin resistance, Actinomycin D resistance. Neomycin resistance, Polymyxin resistance, Zeocin resistance, and Streptomycin resistance.
  • the genetic marker includes a coding sequence of an antibiotic resistance protein 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.
  • 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, hi certain embodiments, the genetic marker is an auxotrophic marker.
  • the auxotrophic marker is a gene involved in vitamin, amino acid, fatty acid synthesis, or carbohydrate metabolism.
  • the auxotrophic market is a gene for synthesizing amino acid.
  • 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.
  • the auxotrophic marker is a gene for synthesizing adenosine, biotin, thiamine, leucine, glucose, lactose, or maltose.
  • 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.
  • 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 acti vator-like effector nuclease (TALEN), a Clustered regularly-interspaced short palindromic repeats (CRISPR) system, or a combination thereof.
  • TALEN Transcription acti vator-like effector nuclease
  • CRISPR Clustered regularly-interspaced short palindromic repeats
  • the deletion is generated by a CRISPR system.
  • CRISPR Clustered regularly- interspaced short palindromic repeats
  • the system includes Cas9 (a protein able to modify DNA utilizing crRNA as its guide), CRISPR RNA (crRNA, which contains the RNA used by Cas9 to guide it to the correct section of host DNA along with a region that binds to tracrRN A (generally in a hairpin loop form) forming an active complex with Cas9), trans-activating crRNA (tracrRN A, 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).
  • the CRISPR system comprises base editors.
  • the CRISPR system comprises transposases/recombinases.
  • the CRISPR system comprises prime editors.
  • the CRISPR system comprises an epigenetic modulator.
  • the CRISPR system comprises a CRISPRoff system.
  • the Cas9 is regulated by a promoter (e.g., described in Section 3.1.2).
  • the promoter is an inducible promoter.
  • the promoter is a stationary phase promoter.
  • the CRISPR system includes a small guide RNAs (sgRNA).
  • the sgRNA of the CRISPR system targets a gene encoding an enzyme of a competing pathway.
  • the sgRNA can target the pfkA gene, the zwf gene, the gene, the pgm gene, or the manXYZ operon.
  • the sgRNA can target any portion of a gene.
  • the sgRNA can target a promoter, an operator, or a sequence encoding a protein.
  • the CRISPR system includes a Cas9 and a sgRNA.
  • the Cas9 is regulated by an inducible promoter.
  • the inducible promoter is /At.
  • the deletion is generated by a zinc-finger nuclease.
  • a zinc-finger nuclease 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 DN A-binding domains of individual ZFNs typically contain a plurality of individual zinc finger repeats and can each recognize a plurality of base pairs.
  • the most common method to generate a new zinc-finger domain is to combine smaller zinc-finger ‘'modules’” of known specificity.
  • the most common cleavage domain in ZFNs is the non-specific cleavage domain from the Type Ils restriction endonuclease Fokl .
  • the deletion is generated by a TALEN system.
  • Transcription activatorlike effector nucleases are restriction enzymes that can be engineered to cut specific sequences of DMA. 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 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-bindlng domain can be engineered to bind desired DNA sequence, and thereby guide the nuclease to cut at specific locations in genome.
  • the deletion is generated by a meganuclease.
  • a meganuclease is an endodeoxyribonuclease that recognizes a double-stranded DNA si te of approx. 12 to approx, 40 base pairs that occur only once hi a genome.
  • Meganucleases are some of the most specific natural ty 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.
  • the present disclosure also provides methods to reduce the expression of any of the genes or enzymes disclosed herein, hi 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., zu;/,' a/xE, pgm. and cherry suiBaK.).
  • oligonucleotides include small interference RNA (siRNA), short hairpin RNA (shRNA), and microRNA (miRNA).
  • these oligonucleotides can be at least about 75%, at least about 80%, at least about 85%, at least about 90%.
  • 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 azu/tuRNA sequence. In certain embodiments, these oligonucleotides can be identical to at least a portion of a CH/ mRNA sequence.
  • these oligonucleotides can be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to at least a portion of an aLsE mRN A sequence. In certain embodiments, these oligonucleotides can be identical to at least a portion of a pfkA mRNA sequence.
  • these oligonucleotides can be at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identical to at least a portion of a pgm mRNA sequence. In certain embodiments, these oligonucleotides can be identical to at least a portion of a pgm mRNA sequence.
  • 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 manC mRNA sequence. In certain embodiments, these oligonucleotides can be identical to at least a portion of a manC mRNA sequence.
  • 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 io at least a portion of a manB mRNA sequence. In certain embodiments, these oligonucleotides can be identical to at least a portion of a mem8 mRN A sequence.
  • antisense nucleic acid, shRNA, miRNA, or siRNA molecules can include DNA or atypical or uon-naturally occurring residues, for example, but not limited to, phosphorothioate residues.
  • the reduced expression of genes and enzymes disclosed herein comprises using a CRISPR system.
  • the CRISPR system includes a Cas9.
  • 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: 56,
  • Cas9 comprises the amino acid sequence set forth in SEQ ID NO: 56.
  • Cas9 consists of the amino acid sequence set forth in SEQ ID NO: 56. SEQ ID NO: 56 is provided below:
  • dCas9 comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to the amino acid sequence set forth in SEQ ID NO: 49, In certain embodiments, dCas9 comprises the amino acid sequence set forth in SEQ ID NO: 49. In certain embodiments, dCas9 consists of the amino acid sequence set forth in SEQ ID NO: 49. SEQ ID NO: 49 is provided below;
  • the sgRNA can target the gene, the cw/'gene, the ab’JS gene, thepgw gene, the manC gene, or the manB gene.
  • the sgRNA can target any portion of a gene.
  • the sgRNA can target a promoter, an operator, or a sequence encoding a protein.
  • the present disclosure provides the use of transformation of the plasmids and vectors disclosed herein
  • Vectors and plasmids disclosed herein can be transformed into cells through any known system in the art.
  • the presently disclosed microorganisms can be transformed by particle bombardment, chemical transformation. Agrobacterium transformation, nano-spike transformation, electroporation, and virus transformation.
  • 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 ingorge 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 et al.. Nucleic Adds Res. 27:69-74 (1992); Ito et al, J. BacteroL 153: 163-168 (1983); and Becker and Goarente, Methods in Enzymology 194: 182- 187 (1991)),
  • transformed microorganisms are referred to as recombinant microorganisms.
  • 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.
  • exogenous proteins e.g., nuclease
  • RNA e.g., gRNA
  • DNA e.g., a recombinant polynucleotide disclosed herein
  • the present disclosure also provides homologous recombination systems for editing (e.g., insertion, deletion) in a microorganism.
  • the homologous recombination system can be native to the host cell or introduced to the cell host.
  • genes for the homologous recombination system can be introduced on a plasmid, introduced on a linear DMA fragment, introduced as and translated from RNA or set of RNAs, or introduced as a protein or set of proteins.
  • the methods include a recombinant polynucleotide disclosed herein.
  • the polynucleotide includes sequence homologous (e.g., left and right homology anus) to a region in a nucleic acid (e.g., genome, plasmid, etc.) such that the left and right homology arms are separated by a designed genetic edit (e.g., promoter, insertion, substitution, SNP, terminator, degron, a sequence for a tag, sequence for a degradation signal or deletion).
  • the recombinant polynucleotide includes a genetic marker, a counter selectable genetic marker (e.g., SacB or PheS), and an origin of repl ication (e.g., R6K).
  • 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.).
  • the resulting transformants can be plated on a medium to select for transformants expressing the selectable genetic markers.
  • 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
  • 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).
  • the second type of selective medium allows the selection of cells that comprise the desired genetic editing.
  • 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.
  • the proteins from one or more recombination systems can be introduced as nucleic acids (e.g., as a plasmid, linear DN A or RNA, or integron) and be integrated into the genome of the host cell or be stably expressed from an extrachroniosomal element.
  • the proteins from one or more recombination systems can be introduced as RNA and be translated by the host cell.
  • 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 E, Muyrers J.P.P. and Stewart A.F.
  • the present disclosure also provides methods for producing D-mannose.
  • Cell -free methods e.g., in vitro synthesis
  • the presently disclosed methods for producing D-mannose include culturing microorganisms (e.g., one disclosed in Section 2) and purifying D-mannose.
  • culturing a cell refers to introducing an appropriate culture medium, under appropriate conditions, to promote the growth of a cell.
  • culturing is performed using a liquid or solid growth medium.
  • culturing occurs under aerobic or anaerobic conditions based on the requirements of the microorganism and desired metabolic state of the same.
  • culturing includes specific conditions such as temperature, pressure, light pH, and cell density,
  • the methods for producing methods of producing D-mannose 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.
  • 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.
  • the culture media include a complex extract (e.g., yeast extract).
  • the culture medium is enriched in order to support, rapid growth.
  • the culture medium is modified in order to support slower growth.
  • the culture medium includes an agent that can inhibit the growth of or kill contaminating organisms (e.g., an antibiotic).
  • the culture medium includes an agent that, can activate an inducible promoter or enzyme (e.g., IPTG).
  • IPTG inducible promoter or enzyme
  • Non-limiting examples of culture media encompassed by the present disclosure include M9 medium, Lysogeny Broth (LB), Terrific Broth (TB), and ⁇ T broth.
  • the culture medium comprises a substrate that is converted by the recombinant microorganisms to D-matmose.
  • the substrate is a sugar (e.g., glucose or fructose) that can be phosphorylated by the bacteria via a kinase (e.g., hexokinase) and converted into frtrctose-6-phosphate.
  • the substrate is glucose.
  • glucose can derive from cellulose, Cs sugars, hemicellulose, and/or xylose.
  • the substrate is a constituent of the culture medium.
  • the substrate is supplemented with the culture medium.
  • the substrate is continuously present in the culture medium.
  • the substrate is supplemented during the growth phase. In. certain embodiments, the substrate is supplemented during the stationary phase.
  • the methods of the present disclosure further comprise purifying D- mannose produced by a microorganism of the present disclosure, e.g., from cell culture or cell culture medium.
  • a microorganism of the present disclosure e.g., from cell culture or cell culture medium.
  • a variety of methods known in the art may be used to purify a product from a microorganism or microorganism culture.
  • one or more products may be purified continuously. e.g., from a continuous culture.
  • one or more products may be purified separately from fermentation, e.g., from a batch or fed-batch culture.
  • the specific purification method(s) used may depend upon, inter alia, the microorganism, culture conditions, and/or particular product(s),
  • purifying D-mannose comprises separating or filtering the microorganisms from a cell culture medium, separating the D-mannose from the culture medium (e.g.. by chromatography), concentration of water (e.g,, by evaporation), and lyophilization of the D- matmose.
  • the present disclosure also provides delivery systems methods for use in food products including the D-mannose prepared and/or generated by any of the microorganisms disclosed herein.
  • 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 cooki es, cakes, roll s, pastries, pie dough, brownies, breads, bagels, and the like.
  • the D-mannose prepared and/or generated by any of the microorganisms disclosed herein are also suitable as a component in frozen foods.
  • the food product is prepared by admixing the D-mannose 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.
  • admixing for example, “admixing D-mannose 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.
  • product refers to die product or any of its components.
  • This admixing step can include a process selected from the step of adding D-mannose to the product, spraying D-mannose on the product, coating D-mannose on the product, suspending the product in D-mannose, painting D-mannose on the product, pasting D-mannose on the product, encapsulating the product with D-mannose, mixing D-mannose with the product and any combination thereof.
  • the D-mannose can be a liquid, dry powder, spray, paste, suspension and any combination thereof.
  • the present application relates to the modified edible food products produced by the methods disclosed herein.
  • the food products can be produced by processes for producing comestible products well known to those of ordinary skill in the art.
  • the D-mannose prepared and/or generated by any of the microorganisms disclosed herein can be dissolved in or dispersed in one of many known comestible acceptable liquids, solids, or other carriers, such as water at neutral, acidic, or basic pH, fruit or vegetable juices, vinegar, marinades, beer, wine, natural water/fat emulsions such as milk or condensed milk, whey or whey products, edible oils and shortenings, fatty acids, certain low molecular weight oligomers of propylene glycol, glyceryl esters of fatty acids, and dispersions or emulsions of such hydrophobic substances in aqueous media, salts such as sodium chloride, vegetable flours, solvents such as ethanol, solid edible diluents such as vegetable powders or flours, and the like, and then combined with precursors of the comestible or medicinal products, or applied directly to the comestible or medicinal products,
  • the food products to which the D-mannose prepared and/or generated by any of the microorganisms disclosed herein are admixed with comprise, by way of example, the wet soup category; the dehydrated and culinary food category; the beverage category; the frozen food category, the snack food category, and seasonings or seasoning blends, described herein.
  • the D-mannose prepared and/or generated by any of the microorganisms disclosed herein are admixed with one or more confectioneries, chocolate confectionery, tablets, countlines, bagged selfmies/sofilines, boxed assortments, standard boxed assortments, twist wrapped miniatures, seasonal chocolate, chocolate with toys, allsorts, other chocolate confectionery, minis, 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, paekaged/industrial bread, unpackaged/artisanal bread, pastries, cakes, paekaged/industrial cakes, unpackaged/artisanal cakes, cookies, chocolate coated biscuits, sandwich biscuits, filled biscuits, savory biscuits and crackers, bread substitutes, breakfast cereals, rte cereals, family
  • cereals ice cream, impulse ice cream, single portion dairy ice cream, single portion water ice cream, multi-pack daily ice cream, multi-pack water ice cream, take-home ice cream; take-home dairy ice cream, ice cream desserts, bulk ice cream, take-home water ice cream, frozen yoghurt, artisanal ice cream, dairy products, milk, fresh/pasteurized milk, full fat fresh/pasteuri zed milk, semi skimmed fresh, pasteurized milk, long- life/uht milk, full fat long life/uht milk, semi skimmed long life/uht milk, fat-free long life/uht milk, goat milk, condensed/evaporated. milk, plain condensed/evaporated.
  • the D-mannose 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 patern outlined below.
  • 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-isoprene copolymer ), and styrene butadiene rubber, as well as natural latexes such as chicle.
  • Elastomer solvents are often resins such as terpene resins.
  • Plasticizers sometimes called softeners, are typically fats and oils, including tallow, hydrogenated and partially hydrogenated vegetable oils, and cocoa butter.
  • Commonly employed waxes include paraffin, microcrystalline, and natural waxes such as beeswax and carnauba.
  • Microciy stahine waxes especially those with a high degree of crystallini ty, can be considered bodying agents or textural modifiers.
  • 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 incl udes a filler component.
  • the filler component can be calcium carbonate, magnesium carbonate, talc, dicaleium phosphate, or the like.
  • the filler can constitute between about 5% and about 60% by weight of the gum base.
  • the filler comprises about 5% to 50% by weight of the gum base.
  • 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.
  • the water-soluble portion of the chewing gam 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 compos iti on.
  • 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.
  • aqueous sweetener solutions such as those containing sorbitol, hydrogenated starch hydrolysate, com syrup, and combinations thereof can be used as softeners and binding agents in gum.
  • the D-tnannose prepared and/or generated by any of the microorganisms disclosed herein can be used in low-calorie gum formulations.
  • formulations containing sugar are also within the scope of the invention.
  • Sugar sweeteners generally include saccharide- con mining 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-mannose prepared and/or generated by any of the microorganisms disclosed herein can also be used in combination with sugarless sweeteners.
  • sugarless sweeteners include components with sweetening characteristics but which are devoid of the commonly known sugars and comprise, but are not limited to, sugar alcohols such as sorbitol, hydrogenated isomaliulose, mannitol, xylitol, lactitol, erythritol, hydrogenated starch hydrolysate, maltitol and the like alone or in any combination.
  • sugar alcohols such as sorbitol, hydrogenated isomaliulose, mannitol, xylitol, lactitol, erythritol, hydrogenated starch hydrolysate, maltitol and the like alone or in any combination.
  • coated or uncoated high-intensity sweeteners can be used in the chewing gum composition, or can be used in a coating applied to centers made from those gum compositions.
  • High-intensity sweeteners preferably aspartame
  • 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.
  • the chewing gum composition will preferably comprise about 0.5% to about 90% sweetening agents. .Most typically the sweetening agents will comprise at least one bulk sweetener and. at least one high-intensity sweetener. Optional ingredients such as colors, emulsifiers, and pharmaceutical agents can also be added as separate components of the chewing gum composition, or added as part of the gum base.
  • Aqueous syrups such as corn syrup and hydrogenated corn syrup can be used, particularly if their moisture content is reduced. This can preferably be done by co-evaporating the aqueous syrup with a plasticizer, such as glycerin or propylene glycol, to a moisture content of less than. 1.0%.
  • a plasticizer such as glycerin or propylene glycol
  • 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. 4,67.1 ,967.
  • Methods of manufacturing chewing gum include the sequential addition of the various chewing gum ingredients to any commercially available mixer known in the art. After the ingredients have been thoroughly mixed, the gum is discharged from the mixer and shaped into the desired form such as by rolling into sheets and cutting into sticks, extruding into chunks, or casting into pellets. Generally, the ingredients are mixed by first melting the gum base and adding it to the running mixer. The base can also be melted tn 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.
  • the flavor system is coated or otherwise modified when incorporated into a delivery system to modify its release rate, it will preferably be added after the final portion of the bulking agent has been added.
  • the entire mixing procedure typically takes from five to twenty minutes, but longer mixing times can sometimes be required.
  • the chewing gum composition can be coated.
  • the coating is initially present as a liquid syrup which contains from about 30% to about 80% or 85% sugars or sugar alcohols, and from about 15% or 20% to about 70% of a solvent such as water.
  • 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.
  • the material or syrup that will eventually form the coating is applied or distributed over the gum center tablets.
  • the D-mannose can be added before, during, and after applying the syrup to the gum centers. Once the coating has dried to form a hard surface, additional syrup additions can be made to produce a plurality of coatings or multiple layers of coating.
  • the D-mannose can be added to any or none of the coatings and/or layers.
  • syrup is added to the gum center tablets at a temperature range of from about 100°F to about 240' 3 F.
  • the syrup temperature is from about IdffiF to about 200°F.
  • 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.
  • 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,
  • Each component of the coating on the gum center can be applied in a single layer or a plurality of layers.
  • 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 ofcoats can be applied to the gum center tablet. Preferably, no more than about 75 coats are applied to the gum center. More preferably, less than about 60 coats are applied and most preferably, about 30 to about 60 coats are applied. In any event, the presently disclosed subject matter contemplates applying an amount of syrup sufficient to yield a coated chewing gum product containing about 10% to about 65% coating.
  • the final product will contain from about 20% to about 50% coating.
  • 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.
  • a preferred drying medium comprises air.
  • forced drying air contacts the wet syrup coating in a temperature range of from about 7O°F to about 110°F, More preferably, the drying air is in the temperature range of from abou t 80°F to about l00°F.
  • the invention al so con templa t es tha t the drying air possesses a relative humidity of less than about 15 percent.
  • the relative humidity of the drying air is less than about 8 %.
  • the drying air can be passed over and admixed with the syrup coated gum centers in any way commonly known in the art.
  • 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.
  • 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.
  • the amount of D-mannose 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.
  • the amount of D-niannose 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, hi gum compositions, the D-mannose 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.
  • Another important aspect of the presently disclosed subject matter includes a confectionery composition incorporating the D-mannose 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-mannose 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.
  • Hard confectionery can be processed and formulated by conventional means.
  • hard confectionery has a base composed of a mixture of sugar and other carbohydrate bulking agents kept in an amoiphous or glassy condition.
  • the hard confectionery can also be sugarless.
  • the hard confectionery can also be low-calorie. This form is considered a solid syrap of sugars generally having from about 0.5% to about 1 .5% moisture. Such materials normally contain up to about 92% sugar, up to about 55% corn syrup, and from about 0.1 % to about 5% water, by weight of the final composition.
  • the syrup component is generally prepared from sucrose and com syrups but can include other materials. In certain embodiments, the syrup component includes the D-mannose 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.
  • 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.
  • Fire cookers involve the traditional method of making a candy base, in this method, the desired quantity of carbohydrate bulking agent is dissolved in water by heating the agent in a ketle until the bulking agent dissolves. Additional bulking agents can then be added and cooked until a final temperature of 145° C to 156° C is achieved. The batch is then cooled and worked as a plastic-tike mass to incorporate additives such as flavoring agents, colorants, and the like.
  • A. high-speed atmospheric cooker uses a heat-exchanger surface, which involves spreading a film of candy on a heat exchange surface, the candy is heated to 165° C to 170° C within a few’ seconds. The candy is then rapidly cooled to 100* C to 120° C- and worked as a plastic-like mass enabling incorporation of the additives, such as flavoring agents, colorants, and the like.
  • the carbohydrate bulking agent is boiled to 125° C to 132° C. vacuum is applied, and additional water is boiled off without extra heating.
  • 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.
  • the candy mass 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 I (1989), Marcel Dekker, Inc., New York, N.Y. at pages 419 to 582, which disclosure is incorporated herein by reference.
  • soft confectionery can be utilized in the embodiments of the disclosed subject matter.
  • the preparation of soft confections involves conventional methods, such as the combination of two primary components, namely (1) a high boiling syrup such as com syrup, or the like, and (2) a relatively light textured frappe, generally prepared from egg albumin, gum arabic, gelatin, vegetable proteins, such as soy-derived compounds, sugarless milk- derived compounds such as milk proteins, and mixtures thereof.
  • the frappe is generally relatively light, and can, for example, range in density from about 0,5 to about 0.7 grams/cc.
  • 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.
  • the final nougat composition is prepared by the addition of the “bob syrup” to the frappe under agitation, to form the basic nougat mixlure. 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.
  • 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.
  • the amount of D-mannose 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.
  • the presently disclosed subject matter extends to methods for making the improved confections.
  • the D-mannose prepared and/or generated by any of the microorganisms disclosed herein can be incorporated into an otherwise conventional hard or soft confection composition using standard techniques and equipment known to those skilled in the art.
  • the apparatus useful in accordance with the presently disclosed subject matter comprises mixing and heating apparatus well known in the confectionery manufacturing arts, and therefore the selection of the specific apparatus will be apparent to the artisan.
  • a composition is made by admixing the D-mannose 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.
  • the D-mannose prepared and/or generated by any of the microorganisms discl osed 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, disintegrates, bulking agents, humectants, buffers, and adsorbents. The preparation of such confections and chewing gum products is well known.
  • chocolates also include those containing crumb solids or solids fully or partially made by a crumb process.
  • Various chocolates are disclosed, for example, in U.S. Patent Nos. 7,968,140 and 8,263,168, the disclosures of which are incorporated herein by reference in their entireties.
  • a general discussion of the composition and preparation of chocolate confections can be found in B. W. Minifie, Chocolate, Cocoa and Confectioner: Science and Technology, 2nd edition, AVI Publishing Co,, Inc., Westport, Conn, (1982), which disclosure is incorporated herein by reference.
  • chocolate refers to a solid or semi-plastic food and is intended to refer to all chocolate or chocolate-like compositions containing a fat-based component phase or 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 tow fat chocolates, aerated chocolates, compound coatings, non-standardized chocolates and chocolate-like compositions, unless specifically identified otherwise.
  • Nonstandardized chocolates result when, for example, the nutritive carbohydrate sweetener is replaced partially or completely: or when the cocoa butter, cocoa buter alternative, cocoa butter equivalent, cocoa butter extender, cocoa buter 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.
  • chocolate 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.
  • FDA Food and Drug Administration
  • the chocolate can contain D-mannose 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.
  • Nutriti ve carbohydrate sweeteners with varying degrees of sweetness intensity can be any of those typically used in the art and include, but are not limited to, sucrose, e.g., from cane or beet, dextrose, fructose, lactose, maltose, glucose syrup solids, corn syrup solids, invert sugar, hydrolyzed lactose, honey, maple sugar, brown sugar, molasses and the like. Sugar substitutes can partially replace the nutritive carbohydrate sweetener.
  • High potency sweeteners include aspartame, cyclamates, saccharin, acesulfame-K, neohesperidin dihydrochaicone, sucralose, alitame, stevia sweeteners, glycyrrhizin, thaumatin and the like and mixtures thereof.
  • the preferred high potency sweeteners are aspartame, cyclamates, saccharin, and acesulf'ame-K.
  • sugar alcohols can be any of those typically used in the art and include sorbitol, mannitol, xylitol, maltitol, isomalt, lactitol and the like.
  • the chocolates can also contain bulking agents.
  • 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.
  • the chocolate products can contain emulsifiers.
  • safe and sui table emulsifiers can be any of those typically used in the art and include lecithin derived from vegetable sources such as soybean, safflower, corn, etc., fractionated lecithins enriched in either phosphatidyl choline or phosphatidy l ethanolamine, or both, mono- and digy Icerides, diacetyl tartaric acid esters of mono- and diglycerides (also referred to as DATEM), monosodmm phosphate derivatives of mono- and diglycerides of edible fats or oils, sorbitan monostearate, hydroxylated lecithin, lactylated faty acid esters of glycerol and propylene glycol, polyglycerol esters of fatty acids, propylene glycol mono- and di-esters of fats and faty acids, or emulsifiers that can become approved for the US FDA-defined soft candy
  • emulsifiers that can be used include polyglycerol polyricinoleate (PGPR), ammonium salts of phosphatidic acid, (e.g., YN) sucrose esters, oat extract, etc., any emulsifier found to be suitable in chocolate or similar iat/solid system or any blend,
  • PGPR polyglycerol polyricinoleate
  • ammonium salts of phosphatidic acid e.g., YN
  • sucrose esters e.g., oat extract, etc.
  • any emulsifier found to be suitable in chocolate or similar iat/solid system or any blend emulsifier found to be suitable in chocolate or similar iat/solid system or any blend
  • '‘chocolate-flavored confection” refers to food products, excluding “chocolate”, having a chocolate flavor/aroma and comprising a cocoa fraction. These products are stable at. ambient temperatures for extended periods of time (e,g.. greater than 1 week) and are characterized as microbioiogically shelf-stable at 18-30° C under normal atmospheric conditions. Examples include chocolate- flavored hard candies, chewabies, chewing gums, etc.
  • phrases “chocolate-flavored compositions” refers to chocolate-flavored compositions, excluding “chocolate”, containing a cocoa fraction and having a chocolate fiavor/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.).
  • 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.
  • the D-mannose 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.
  • 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.
  • Soup may be used as an ingredient tor preparing other meal components and may range from broths (consomme) to sauces (cream or cheese-based soups).
  • the D-mannose 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, dehy drated ready-to-cook soups, dehydrated or ambient preparations of ready-made di shes, meals and single serve entrees including pasta, potato and rice dishes; and (in) meal embellishment products such as: condiments, marinades, salad dressings, salad toppings, dips, breading, batter mixes, shelf stable spreads, barbecue sauces, liquid
  • the D-mannose prepared and/or generated by any of the microorganisms disclosed herein is incorporated into a meat food product.
  • 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.
  • the D-mannose prepared and/or generated by any of the microorganisms disclosed herein is incorporated into a snack food category food product.
  • 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.
  • snack food include, but are not limited to fruit snacks, chips/crisps, extruded snacks, tortilla/com chips, popcorn, pretzels, nuts, and other sweet and savory snacks.
  • snack bars include, but are not limited to granola/muesli bars, breakfast bars, energy bars, fruit bars, and other snack bars.
  • the D-mannose 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 daity ice cream, single portion water ice cream, multi-pack dairy ice cream, multi-pack water ice cream, take-home Ice cream, take-home dairy ice cream, ice cream desserts, bulk ice cream, take-home water ice cream, frozen 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.
  • ice cream, impulse ice cream single portion daity ice cream
  • single portion water ice cream multi-pack dairy ice cream
  • multi-pack water ice cream take-home Ice cream
  • take-home dairy ice cream take-home dairy ice cream
  • ice cream desserts bulk
  • the D-mannose prepared and/or generated by any of the microorganisms disclosed herein can also be in the form of a pharmaceutical.
  • 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 mater can comprise preservatives, buffers, suspending agents, antifoaming agents, sweetening agents, flavoring agents, coloring or decoloring agents, solubilizers, and combinations thereof.
  • Flavoring agents such as those flavors well known to the skilled artisan, such as natural and artificial flavors and mints, such as peppermint, menthol, citrus flavors such as orange and lemon, artificial vanilla, cinnamon, and various fruit flavors, both individual and mixed and the like can be utilized in amounts from about 0.01% to about 5%, and more preferably 0.01% to about 0.5% by weight of the suspension.
  • 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-mannose prepared and/or generated by any of the microorganisms disclosed herein with water to form a solution; (iii) admix, if desired, a flavoring agent with the thickener-water admixture to form a uniform thickener-flavoring agent; (iv) combine the sweetener solution with the thickener-flavoring agent and mix until uniform: and (v) admix the optional adjunct materials such as coloring agents, flavoring agents, decolorants, solubilizers, antifoaming agents, buffers and additional water with the mixture of step (iv) to form the suspension.
  • the D-mannose prepared and/or generated by any of the microorganisms disclosed herein can also be in chewable form.
  • considerations include the amount of active substance per tablet, the flavoring agent employed, the degree of compressibility of the tablet, and additional properties of the composition.
  • Chewable pharmaceutical candy is prepared by procedures similar to those used to make soft confectionery. A general discussion of the lozenge and chewable tablet forms of confectionery can be found in H, A. Lieberman and L. Lachman, Pharmaceutical Dosage Forms: Tablets Volume 1. Marcel Dekker, InC, New York, N.Y.
  • a boiled sugar-corn syrup blend is formed to which is added a frappe mixture.
  • the boiled sugar-com syrup blend can be prepared from sugar and corn syrup blended in parts by weight ratio of about 90.TO to about 10:90.
  • the sugar-com syrup blend is heated to temperatures above about 120” C to remove water and to form a molten mass.
  • the frappe is generally prepared from gelatin, egg albumin, milk proteins such as casein, and vegetable proteins such as soy protein, and the like, which are added to a gelatin solution and rapidly mixed at ambient temperature to form an aerated sponge-like mass.
  • the frappe is then added to the molten candy mass and mixed until homogeneous at temperatures between about 65” C and about 120° C.
  • the D-mannose 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 addi tional Ingredients can then be added such as flavoring agents and coloring agents.
  • the formulation is further cooled and formed into pieces of desired dimensions.
  • 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-ethauol mixtures.
  • the water-ethanol mixtures are generally employed in a weight ratio front about 1:1 to about 20:1, preferably from about 3:1 to about 20:1, and most preferably from about 3:1 to about 10: 1 , respectively.
  • the pH value of the ingestible vehicle is generally from about 4 to about 7, and preferably from about 5 to about 6.5.
  • An ingestible topical vehicle having a pH value below about 4 is generally irritating to the ingestible cavity and an ingestible vehicle having a pH value greater than about 7 generally results in an unpleasant mouth feel.
  • 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 include those flavors known to the skilled artisan, such as natural and artificial flavors.
  • Suitable flavoring agents include minis, such as peppermint, citrus flavors such as orange and lemon, artificial vanilla, cinnamon, various fruit flavors, both individual and mixed, and the like.
  • the amount of flavoring agent employed in the ingestible topical composition is normally a matter of preference subject to such factors as the type of final ingestible composition, the individual flavor employed, and the strength of flavor desired. 'Thus., the amount of flavoring can be varied in order to obtain the result desired in the final product and such variations are within the capabilities of those skilled in the art without the need for undue experimentation.
  • the flavoring agents, when used, are generally utilized in amounts that can. for example, range in amounts from about 0.05% to about 6%, by weight of the ingestible topical composition.
  • D-mannose prepared and/or generated by any of the microorganisms disclosed herein can be used in a wide variety of pet food products.
  • 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, rabbi ts, birds arid horses.
  • the companion animal can be a “domestic” dog, e.g., Cawfc
  • 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.
  • the D ⁇ mannose prepared and/or generated by any of the microorganisms disclosed herein is directly added to a pet food product, in certain embodiments, the D-maunose 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.
  • 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.
  • the pet food product is a dry food product.
  • a dry or low tnoisture- containing nutritionariy-complete pet food product can comprise less than about 15% moisture.
  • the pet food product is a wet food product.
  • a wet or high moisture-containing nutritionally-cornplete pet food product can comprise greater than about 50% moisture.
  • the pet food product is a nutritionally complete moist food product
  • a moist e.g,, semimoist or semi-dry or soft dry or soft moist or intermediate or medium moisture containing nutritionally- complete pet food product comprises from about 15% to about 50% moisture.
  • the pet food product is a pet food snack product.
  • pet food snack products include snackbars, pet chews, crunchy treats, cereal bars, snacks, biscuits and sweet products.
  • the D-mannose can be incorporated into a delivery system for use in edible compositions.
  • the composition will comprise another flavor or taste modifier such as a salty, umami, bitter, astringent and/or savory tastant.
  • Delivery systems can be liquid or solid, aqueous or non-aqueous. Delivery systems are general ly adapted to suit the needs of the flavor composition and/or the edible composition into which the D-mannose will be incorporated, [0247]
  • the D-mannose 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.
  • D-mannose can be encapsulated, or absorbed onto water soluble materials, including but not limited to materials such as cellulose, starch, sugar, maltodextrin, gum arabic and so forth.
  • materials such as cellulose, starch, sugar, maltodextrin, gum arabic and so forth.
  • the actual techniques for preparing such dried forms are well-known in the art, and can be. applied to the presently disclosed subject matter.
  • the D-mannose 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.
  • such physical forms include free forms, such as spray dried, powdered, and beaded forms, and encapsulated forms, and mixtures thereof.
  • D-mannose is encapsulated.
  • Encapsulating materials and/or techniques can be selected to improve the stability of the D-mannose and/or food product.
  • the encapsulating materials and/or techniques are selected to modify the release profile of D-mannose.
  • Suitable encapsulating materials can include, but are not. limited to, hydrocolloids such as alginates, pectins, agars, guar gums, celluloses, and the like, proteins, polyvinyl acetate, polyethylene, crosslinked polyvinyl pyrrolidone, polymethylmethacrylate, polylactidacid, .polyhydroxyalkanoates, ethylcellulose, polyvinyl acetatephthalate, polyethylene glycol esters, methacrylicacid-co- methylmethacrylate, ethylene-vinylacetate (EV A) copolymer, and the like, and combinations thereof.
  • hydrocolloids such as alginates, pectins, agars, guar gums, celluloses, and the like
  • proteins polyvinyl acetate, polyethylene, crosslinked polyvinyl pyrrolidone, polymethylmethacrylate, polylactidacid, .polyhydroxyalkanoates,
  • Suitable encapsulating techniques can include, but are not limited to, spray coating, spray drying, spray chilling, absorption, adsorption, inclusion complexing (e.g., creating a flavor/cyclodextrin complex), coacervation, fluidized bed coating, or other process can be used to encapsulate an ingredient with an encapsul a ti ng materi al,
  • Encapsulated delivery systems for flavoring agents or sweetening agents 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.
  • fatty acids include but are not limited to hydrogenated and partially hydrogenated vegetable oils such as palm oil, palm kernel oil, peanut oil, rapeseed oil, rice bran oil, soybean oil, cottonseed oil, sunflower oil. safflower oil, and mixtures thereof.
  • Examples of glycerides include but are not limited to monoglycerides, diglycerides, and triglycerides.
  • Waxes useful can be chosen from the group consisting of natural and synthetic waxes, and mixtures thereof.
  • Non-limiting examples include paraffin wax, petrolatum, carbowax, microcrystalline wax, beeswax, carnauba wax, candelilla wax, lanolin, bayberry wax, sugarcane wax, spermaceti wax, rice bran wax, and mixtures thereof
  • the fats and waxes can be use individually or in combination in amounts varying from about 10 to about 70%, and alternatively in amounts from about 30 to about 60%, by weight of the encapsulated system. When used in combination, the fat and wax are preferably present in a ratio from about 70:10 to 85: 15, respectively.
  • Liquid delivery systems can include, but are not limited to, systems with a dispersion of D- mannose, such as in carbohydrate syrups and/or emulsions. Liquid delivery systems can also include extracts where D-mannose is solubilized in a solvent. Solid deli very 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 delivety 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.
  • suitable drying means such as spray drying may be used.
  • D ⁇ mannose may be adsorbed or absorbed onto substrates such as water soluble materials, such as cellulose, starch, sugar, maltodextrin, gum arabic and so forth or may be encapsulated.
  • substrates such as water soluble materials, such as cellulose, starch, sugar, maltodextrin, gum arabic and so forth or may be encapsulated.
  • E. coli is naturally capable of producing trace amounts of D-mannose.
  • D-mannose production is improved by overexpressing key genes, removing competing pathway genes, and optimizing the production conditions.
  • E. coli possesses enzymes capable of producing D-manuose.
  • D- mannose production was tested in the production strains depicted in Table 1 below.
  • K.Os single gene knockouts
  • These K.Os include &pfkA f Acwf EalsE, and A/upu resulting in strains AL3755, AL4240, AL4314, AL4329, respectively (see Table 1).
  • the genes pfkA and pfldl encode for phosphofructokinase A.
  • the gene SM/ encodes for the enzyme glucose-6-phosphate dehydrogenase (Zwf) (EC 1.1 .1.363), which converts glucose-6-phosphate (G6P) to 6"phospho-D“ghicono-l,5-lactone as the first committed step in the pentose phosphate pathway (PPP).
  • Zwf glucose-6-phosphate dehydrogenase
  • the gene alsE encodes for the enzyme allulose 6-phosphate 3-epimerase (Als'E) (EC 5.1.3). which catalyzes the reversible epimerization of D-allulose 6-phosphate to D-fructose 6-phosphate.
  • the genepgn? encodes for the enzyme phosphoglucomutase (pgm) (EC 5.4.2.6), which catalyzes the conversion of beta D-glucose 1 -phosphate (G1P) to D-glucose 6-phosphate (G6P), forming beta-D- glucose 1 ,6-(bis)phosphate (beta-G16P) as an intermediate.
  • the production pathway starts with the native assimilation of glucose into E. coli via the phosphotransferase system (PTS) or GalP/Glk, which converts glucose to glucose-6-phosphate (G6P) ( Figures 2A and 2B). G6P is then isomerized to F6P via glucose ⁇ 6 ⁇ phosphate isomerase (Gpi) (EC 5.3.1.9). F6P could be converted to mannose-6-phosphate by an isomerase. A phosphatase could dephosplioiylate .mannose-6-phosphate to tree mannose, after which it would be excreted from the cell. A D-mannose production system using phosphorylation and dephosphorylation steps as system driving forces should be more efficient than the pathway currently used in industrial production of mannose.
  • the life cycle of an A, 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 upregulated.
  • the transcription of these genes is regulated in part by the u38-subunit of RNA polymerase, which recognizes the promoter region of a gene.
  • E. coli s native gene regulatory system was utilized to balance carbon flux by affixing the D- mannose production genes, manA and fepB, 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.
  • D-mannose a C2 epimer of glucose
  • D-mannose is a common component of polysaccharide sugar chains in nature (Wang et al, 2021 ). It plays a crucial role in various biological processes, including cell recognition and immune response (Dhanalakshmi et aL, 2023).
  • D-mannose predominantly exists in the pyranose form, with approximately 67% as the sweet- tasting a ⁇ anomer and 33% as the bitter-tasting p-anomer (Sharma et al., 2014).
  • the caloric value of D-mannose is reported to be 3.75 kcal g‘ l and is unique among sugars in that its intensity of sweetness does not follow a power function of concentration (Moskowitz, 1970).
  • D-mannose can be used as a. raw material for synthesizing high-value products, including immunostimulants and therapeutics (Hu et al., 2016).
  • D-mannose has demonstrated efficacy in the treatment of urinary tract infections (Wagenlehner et al., 2022) and holds potential as an antibiotic substitute in poultry industry (Oyofo et aL, 1989).
  • D-mannitoL a well- known low-calorie sweetener, can be produced from D-mannose (Mishra and Hwang, 2013).
  • the high cost of D-mannose presents a significant challenge for its widespread use.
  • Free D-mannose is found in small amounts in fruits such as oranges, apples, and cranberries (Shanna et aL, 2014).
  • the commercial D-mannose is primarily extracted from plants, especially fruits and herbs.
  • these extraction methods often require harsh chemical conditions, and the reagents used are harmful to the environment.
  • Traditional chemical synthesis of D- mannose involves using metal catalysts to convert D-glucose to D-mannose via an isomerization reaction. This method is only suitable for small-scale synthesis due to high production and separation costs.
  • the production of dietary supplements and pharmaceutical chemicals should avoid chemical extractants and by-product contamination.
  • D-mannose production is enzymatic synthesis using isomerases, following the Izumormg strategy.
  • isomerases Four key enzymes have been identified for this purpose: D-lyxose isomerase (Huang et al., 2018), D-mannose isomerase (Saburi et al., 2018), cellobiose 2-epimetase (Huerta et al., 2024), and mannose 2 -epimerase (Saburi et al., 2019).
  • D-lyxose isomerase and D- mannose isomerase convert D-frtictose to D-mannose
  • cellobiose 2-ephnerase and mannose 2-epimerase convert D-glucose to D-mannose.
  • these methods often suffer from a lack of thermodynamic driving force, resulting in low conversion yields. Therefore, to develop a feasible large-scale production method for D-mannose, it is essential to establish a production pathway with a high thermodynamic driving force.
  • D-mannose was produced via enzymatic synthesis via phosphorylation and dephosphorylatton strategy from starch. Dephosphorylation of D-tnannose-6-phosphaie provided the thermodynamic driving force towards D-mannose production (Tian et al, 2022, 2020),
  • the present example shows that Esc/t&ichia coli has the capacity to convert D-giucose to D-mannose.
  • the present example elucidated the native production pathway of D-mannose production. Further, genes involved in the production of D-mannose were overexpressed to enhance titers and deletion of competing pathways removed side product formation. Additionally, competing pathways were deleted to eliminate side product formation. To reduce chemical contamination and production costs, the production module was transitioned to an inducer-free system. Finally, the native D-mannose consumption genes were removed to minimize the reassimiiation of produced D-mannose.
  • strains and Plasmids The relevant strains and plasmids used in this example are l isted, in Tables 2 and 3, respectively. Genome modifications such as gene deletion and gene insertion were constructed using CRISPR-Cas9-mediated homologous recombination (Jiang et al., 2015), Linear DNA repair fragments for gene deletions and insertions were constructed by amplifying genomic or plasmid. DNA via PCR assembly (Xiong et al., 2004).
  • Plasmids encoding sgRNA for CRlSPR-CasP- 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.
  • M9P media consists of M9 minimal media supplemented with 5 g I..' 1 of yeast extract and appropriate antibiotics. Inducer concentrations are as follows; isopropyl-P-D- 1-thiogalactopyranoside (IPTG) (1 mM). OD600 was measured with a Synergy HI hybrid plate reader (BioTek Instruments, Inc.).
  • HPAE High-Performance Anion-Exchange chromatography
  • ManA The mannose-6-phosphate isomerase gene, manA, has been shown to convert F6P to D- marmose-6-phosphaie (M6P) (Gao et al., 2005). It was postulated that dephosphorylation of M6P leads to the production of D-mannose in the A/?/M AZH/ strain (AL4240, Table 2). In order to elucidate the native D-mannose production pathway, the manA gene was deleted in AL4240 (ApfkA Azwf, Table 2) to generate AL4290 (Table 2). D-mannose production was abolished in AL4290, suggesting that D- mannose production proceeds through D-mannose-6-phosphate ( Figure 3B). The additional expression of nwM from a plasmid in AL4290 Azwf AmariA, Table 1 ) restored D-mannose production confirming the role of manA in the production pathway ( Figure 3B).
  • the «£$£' gene encodes D-allulose 6-phosphate 3-epimerasere, which interconverts F6P and D ⁇ psicose-6-phosphate, is responsible for the production of D-psicose (Taylor et al, 2023).
  • the deletion of oAE in AL4240 (Ap/14 Asw£ Table 2) resulted in the creation of AL4314 (Ap/A4 Any/' AoZtE'. Table 2). This deletion improved D-mannose production to 3 J g L'* and eliminated D-psicose production ( Figure 4).
  • D-mannose can serve as a carbon source for the growth of A. coZz ⁇ This is a significant challenge in microbial production of D-mannose, since the produced D-mannose can be reassimilated back into the cell for biomass accumulation thereby decreasing yield.
  • the main mannose transporter operon mauXYZ (Erni and Zanolari, 1985) was deleted in AL4329 resulting in strain AL4592 (Table 2), Deletion of mannose transporter genes significantly reduced the mannose consumption rate of the presently disclosed bacteria ( Figure 5).

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Abstract

La présente divulgation concerne des micro-organismes utiles pour la biosynthèse de mannose. L'invention concerne également des procédés de production du micro-organisme décrit et des procédés de production de mannose.
PCT/US2024/045528 2023-09-07 2024-09-06 Micro-organismes pour la production de d-mannose Pending WO2025054412A1 (fr)

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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4597970A (en) 1984-10-05 1986-07-01 Warner-Lambert Company Chewing gum compositions containing novel sweetener delivery systems and method of preparation
US4671967A (en) 1984-05-18 1987-06-09 Wm. Wrigley Jr. Company Carbohydrate syrups and methods of preparation
US4722845A (en) 1986-12-23 1988-02-02 Warner-Lambert Company Stable cinnamon-flavored chewing gum composition
US6536599B1 (en) 1998-04-30 2003-03-25 Ulrich Carlin Nielsen Method and system for portioning and orientating whole fish or other elongate, non-symetrical articles
US6899911B2 (en) 2000-04-05 2005-05-31 International Flavors & Fragrances, Inc. Ethyl 4-(thioacetoxy)butyrate as a flavoring agent and methods for preparing and using same
US7968140B2 (en) 1997-09-08 2011-06-28 Mars, Incorporated Chocolates and chocolate liquor having an enhanced polyphenol content
US8263168B2 (en) 2006-06-27 2012-09-11 Mars, Incorporated Indulgent edible composition
WO2017002978A1 (fr) * 2015-07-02 2017-01-05 協和発酵バイオ株式会社 Procédé de production de sucre rare
WO2022034069A1 (fr) * 2020-08-10 2022-02-17 Inbiose N.V. Production d'un mélange d'oligosaccharides neutres fucosylés par une cellule

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4671967A (en) 1984-05-18 1987-06-09 Wm. Wrigley Jr. Company Carbohydrate syrups and methods of preparation
US4597970A (en) 1984-10-05 1986-07-01 Warner-Lambert Company Chewing gum compositions containing novel sweetener delivery systems and method of preparation
US4722845A (en) 1986-12-23 1988-02-02 Warner-Lambert Company Stable cinnamon-flavored chewing gum composition
US7968140B2 (en) 1997-09-08 2011-06-28 Mars, Incorporated Chocolates and chocolate liquor having an enhanced polyphenol content
US6536599B1 (en) 1998-04-30 2003-03-25 Ulrich Carlin Nielsen Method and system for portioning and orientating whole fish or other elongate, non-symetrical articles
US6899911B2 (en) 2000-04-05 2005-05-31 International Flavors & Fragrances, Inc. Ethyl 4-(thioacetoxy)butyrate as a flavoring agent and methods for preparing and using same
US8263168B2 (en) 2006-06-27 2012-09-11 Mars, Incorporated Indulgent edible composition
WO2017002978A1 (fr) * 2015-07-02 2017-01-05 協和発酵バイオ株式会社 Procédé de production de sucre rare
WO2022034069A1 (fr) * 2020-08-10 2022-02-17 Inbiose N.V. Production d'un mélange d'oligosaccharides neutres fucosylés par une cellule

Non-Patent Citations (53)

* Cited by examiner, † Cited by third party
Title
"Gene Transfer Vectors for Mammalian Cells", 1987
"Oligonucleotide Synthesis", 1984
"PCR: The Polymerase Chain Reaction", 1994
"Uniprot", Database accession no. 034529
"UniProt", Database accession no. P0AEP1
ANGELA XIN YU ZHANG: "Microbial Production of Natural Sugars for A Healthier Future", DISSERTATION, 1 January 2021 (2021-01-01), XP093166016, Retrieved from the Internet <URL:https://escholarship.org/uc/item/7042d2q4> *
ANZALONE ET AL., NATURE BIOTECHNOLOGY, vol. 38, no. 7, 2020, pages 824 - 844
ARROYA-OLANTE ET AL., MICROORGANISMS, no. 4, 9 April 2021 (2021-04-09), pages 844
B. W. MINIFIE: "Chocolate, Cocoa and Confectionery: Science and Technology", 1982, AVI PUBLISHING CO., INC., pages: 576 - 580
BABA, T.ARA, T.HASEGAWA, M.TAKAI, Y.OKUMURA, Y.BABA, M.DATSENKO, K.A.TOMITA, M.WANNER, B.L.MORI, H.: "Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection", MOL. SYST. BIOL., vol. 2, 2006, XP002628975, DOI: 10.1038/MSB4100050
BECKERGUARENTE, METHODS IN ENZYMOLOGY, vol. 194, 1991, pages 182 - 187
DAVIS, L.DIBNER, M .BATTEY, I., BASIC METHODS IN MOLECULAR BIOLOGY, 1986
DHANALAKSHMI, M.SRUTHI, D.JINURAJ, K.R.DAS, K.DAVE, S.ANDAL, N.M.DAS, J.: "Mannose: a potential saccharide candidate in disease management", MED. CHEM. RES., vol. 32, 2023, pages 391 - 408, XP037939248, DOI: 10.1007/s00044-023-03015-z
DICARLO, J. E. ET AL.: "Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems", NUCLEIC ACIDS RES, 2013
ERNI, B.ZANOLARI, B.: "The mannose-permease of the bacterial phosphotransferase system. Gene cloning and purification of the enzyme IIMan/IllMan complex of Escherichia coli", J. BIOL. CHEM., vol. 260, 1985, pages 15495 - 15503
EYDALLIN, G.VIALE, A.M.MORIN-ZORZANO, M.T.MWLOZ. F.J.MONTERO, M.BAROJA-FERNANDEZ, E.POZUETA-ROMERO, J.: "Genome-wide screening of genes affecting glycogen metabolism in Escherichia coli K-12", FEBS LETT, vol. 581, 2007, pages 2947 - 2953, XP022117025
GAO, H.YU, Y.LEARY, J.A.: "Mechanism and Kinetics of Metalloenzyme Phosphomannose Isomerase: Measurement of Dissociation Constants and Effect of Zinc Binding Using ESI-FTICR Mass Spectrometry", ANAL. CHEM., vol. 77, 2005, pages 5596 - 5603
GIETZ ET AL., NUCLEIC ACIDS RES, vol. 27, 1992, pages 69 - 74
GOSSET, MICROB CELL FACT, vol. 4, 2005, pages 14
HU, X.SHI, Y.ZHANG, P.MIAO, M.ZHANG, T.JIANG, B.: "d-Mannose: Properties, Production, and Applications: An Overview", COMPR. REV. FOOD SCI. FOOD SAF., vol. 15, 2016, pages 773 - 785
HUANG, J.YU, L.ZHANG, W.ZHANG, T.GUANG, C.MU, W.: "Production ofd-mannose from d-glucose by co-expression of d-glucose isomerase and d-I xose isomerase in Escherichia coli", J. SCI. FOOD AGRIC., vol. 98, 2018, pages 4895 - 4902
HUERTA, M.CORNEJO, F.ABURTO, C.ILLANES, A.VERA, C.GUERRERO, C.: "Enzymatic production of rare sugars with a new mutant of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus", TECHNOL, vol. 391, 2024, pages 129936
ITO ET AL., J. BACTEROL., vol. 153, 1983, pages 163 - 168
JIANG ET AL., APPL ENVIRON MICROBIOL, vol. 81, no. 7, April 2015 (2015-04-01), pages 2506 - 14
JIANG, Y.CHEN, B.DUAN, C.SUN, B.YANG, J.YANG, S.: "Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System", ENVIRON. MICROBIOL., vol. 8, no. 1, 2015, pages 2506 - 2514
KUZNETSOVA, E.PROUDFOOT, M.GONZALEZ, C.F.BROWN, G.OMELCHENKO, M.V.BOROZAN, I.CARMEL, L.WOLF, Y.I.MORI, H.SAVCHENKO, A.V.: "Genome-wide Analysis of Substrate Specificities of the Escherichia coli Haloacid Dehalogenase-like Phosphatase Family", J. BIOL. CHEM., vol. 281, 2006, pages 36149 - 36161, XP055038714, DOI: 10.1074/jbc.M605449200
LIANG, S.T. ET AL., JMOÍ. BIOL., vol. 292, no. 1, 1999, pages 19 - 37
LIN, S.STAAHL, B. T.ALLA, R. K.DOUDNA, J. A.: "Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery", ELIFE, vol. 3, 2014, pages 04766
MISHRA, D.K.HWANG, J.-S.: "Selective hydrogenation of d-mannose to d-mannitol using NiO-modified TiO2 (NiO-TiO2) supported ruthenium catalyst", APPL. CATAL. GEN., vol. 453, 2013, pages 13 - 19, XP028976067, DOI: 10.1016/j.apcata.2012.11.042
MOSKOWITZ, H.R.: "Ratio scales of sugar sweetness", PSYCHOPHYS, vol. 7, 1970, pages 315 - 320
MUYRERS JP ET AL.: "Techniques: Recombinogenic engineering-new options for cloning and manipulating DNA", TRENDS BIOCHEM SCI, vol. 26, no. 5, May 2001 (2001-05-01), pages 325 - 31, XP004241877, DOI: 10.1016/S0968-0004(00)01757-6
MUYRERS, J.P.P.ZHANG, Y.TESTA, G.STEWART, A.F.: "Rapid modification of bacterial artificial chromosomes by ET-recombination", NUCLEIC ACIDS RES, vol. 27, 1999, pages 1555 - 1557, XP002153801, DOI: 10.1093/nar/27.6.1555
NUILEZ ET AL., CELL, vol. 184, no. 9, 2021, pages 2503 - 2519
OYOFO, B.A.DELOACH, J.R.CORRIER, D.E.NORMAN, J.O.ZIPRIN, R.L.MOLLENHAUER, H.H.: "Prevention of Salmonella typhimurium Coloni .ation of Broilers with D-Mannose", SCI, vol. 68, 1989, pages 1357 - 1360
PLETTIEV, ACTA NATURAE, vol. 7, no. 4, 2015, pages 27
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, vol. 195, 1997, pages 303 - 311, XP004094665, DOI: 10.1016/S0378-1119(97)00186-8
SABURI, W.JAITO, N.KATO, K.TANAKA, Y.YAO, M.MORI, H.: "Biochemical and structural characterization of Marinonionas mediterranea d-maiinose isomerase Marme 2490 phylogenetically distant from known enzymes", BIOCHIMIE, vol. 144, 2018, pages 63 - 73
SABURI, W.SATO, S.HASHIGUCHI, S.MUTO, H.LIZUKA, T.MORI, H.: "Enzymatic characteristics of d-mannose 2-epimerase, a new member of the acylglucosamine 2-epimerase superfamily", BIOTECHNOL, vol. 103, 2019, pages 6559 - 6570, XP036847471, DOI: 10.1007/s00253-019-09944-3
SÉVIN DANIEL C ET AL: "Nontargeted in vitro metabolomics for high-throughput identification of novel enzymes in Escherichia coli", NATURE METHODS, vol. 14, no. 2, 12 December 2016 (2016-12-12), New York, pages 187 - 194, XP093243343, ISSN: 1548-7091, Retrieved from the Internet <URL:http://www.nature.com/articles/nmeth.4103> DOI: 10.1038/nmeth.4103 *
SHARMA, V.ICHIKAWA, M.FREEZE, H.H.: "MANNOSE MER.ABOLISM: MORE THAN MEETS THE EYE", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 453, 2014, pages 220 - 228
SHIMADA ET AL., JOURNAL, November 2004 (2004-11-01), pages 7112 - 7122
TAYLOR, J.E.PALUR, D.S.K.ZHANG, A.GONZALES, J.N.ARREDONDO, A.COULTHER, T.A.LECHNER, A.B.J.RODRIGUEZ, E.P.FIEHN, O.DIDZBALIS, J.: "Awakening the natural capability of psicose production in Escherichia coli", SCI. FOOD, vol. 7, 2023, pages 54
TIAN, C.YANG, J.LI, Y.ZHANG, T.LI, J.REN, C.MEN, Y.CHEN, P.YOU, C.SUN, Y.: "Artificially designed routes for the conversion of starch to value-added mannosyl compounds through coupling in vitro and in vivo metabolic engineering strategies", METAB. ENG., vol. 61, 2020, pages 215 - 224, XP093143228, DOI: 10.1016/j.ymben.2020.06.008
TIAN, C.YANG, J.LIU, C.CHEN, P.ZHANG, T.MEN, Y.MA, H.SUN, Y.MA, Y.: "Engineering substrate specificity of HAD phosphatases and multienzyme systems development for the thermodynamic-driven manufacturing sugars", NAT. COMMUN., vol. 13, 2022, pages 3582
WAGENLEHNER, F.LORENZ, H.EWALD, O.GERKE, P.: "Why d-Mannose May Be as Efficient as Antibiotics in the Treatment of Acute Uncomplicated Lower Urinary Tract Infections Preliminary Considerations and Conclusions from a Noun-Interventional Study", ANTIBIOTICS, vol. 11, 2022, pages 314
WANG YUYAO ET AL: "Biosynthesis of mannose from glucose via constructing phosphorylation-dephosphorylation reactions in Escherichia coli", ENZYME AND MICROBIAL TECHNOLOGY, vol. 177, 1 June 2024 (2024-06-01), US, pages 110427, XP093242751, ISSN: 0141-0229, DOI: 10.1016/j.enzmictec.2024.110427 *
WANG, P.ZHENG, Y.LI, Y.SHEN, J.DAN, M.WANG, D.: "Recent advances in biotransformation, extraction and green production of D-mannose", CURR. RES., vol. 5, 2021, pages 49 - 56
WU YANCHANG ET AL: "Characterization of a Novel Mannose Isomerase from Stenotrophomonas rhizophila and Identification of Its Possible Catalytic Residues", MOLECULAR BIOTECHNOLOGY, vol. 64, no. 6, 20 January 2022 (2022-01-20), New York, pages 650 - 659, XP093243365, ISSN: 1073-6085, Retrieved from the Internet <URL:https://link.springer.com/article/10.1007/s12033-021-00437-1/fulltext.html> DOI: 10.1007/s12033-021-00437-1 *
XIONG, A.-S.YAO, Q.-H.PENG, R.-H.LI, X.FAN, H.-Q.CHENG, Z.-M.LI, Y.: "A simple, rapid, high-fidelity and cost-effective PCR-based two-step DNA synthesis method for long gene sequences", NUCLEIC ACIDS RES, vol. 32, 2004, pages 98
YONEDA, H.TANTILLO, D.J.ATSUMI, S.: "Biological Production of 2-Butanone in Escherichia coli", CHEMSUSCHEM, vol. 7, 2014, pages 92 - 95
ZHANG ET AL., FRONT, MICROBIOL., 31 March 2021 (2021-03-31)
ZHANG Y.BUCHHOLZ F.MUYRERS J.P.P.STEWART A.F.: "A new logic for DNA engineering using recombination in E. coli", NATURE GENETICS, vol. 20, 1998, pages 123 - 128, XP055890274
ZHANG Y.MUYRERS J.P.P.TESTA G.STEWART A.F.: "DNA cloning by homologous recombination in E. coli", NATURE BIOTECHNOLOGY, vol. 18, 2000, pages 1314 - 1317

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