Production of 2-keto-3-deoxygluconate (KDG) from sucrose

Abstract

The disclosure relates to recombinant microorganisms configured for improved sucrose uptake and / or production of produce 2-keto-3-deoxygluconic acid (KDG) from glucose. In some various embodiments, the recombinant microorganisms are engineered to (a) to non-phosphorylatively transport sucrose and / or (b) produce 2-keto-3-deoxygluconic acid (KDG) from glucose and / or (c) hydrolyze sucrose to glucose and fructose intracellularly and / or (d) isomerize fructose and glucose. The disclosure further relates to methods of non-phosphorylatively transporting sucrose and / or producing KDG using the recombinant microorganisms of the disclosure.

Description

PRODUCTION OF 2-KETO-3-DEOXYGLUCONATE (KDG) FROM SUCROSE1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of United States provisional application no. 63 / 516,408, filed July 28, 2023, the contents of which are incorporated herein in their entireties by reference thereto.2. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on June 17, 2024 is named BPC-012WO_SL.xml and is 56,747 bytes in size.3. BACKGROUND

[0003] Sucrose is a plentiful and inexpensive material that can be isolated from various plant sources, such as sugarcane and sugar beet. Sucrose is a disaccharide comprising one glucose subunit and one fructose subunit. Bacteria, archaea, fungi (e.g., yeast), and other microorganisms have a bewildering array of enzymatic activities and interlocking metabolic pathways that can use sucrose, glucose, and / or fructose for cell growth and survival.

[0004] There is a need in the art for efficient methods of producing industrially important products from natural sources such as sucrose. The present disclosure addresses this need and provides microorganisms that can utilize sucrose, or its constituent monosaccharides glucose and fructose, to produce industrially important molecules, such as 2-keto-3-deoxy- D-gluconate (KDG), that can be used in industrial manufacturing processes.4. SUMMARY

[0005] The present disclosure applies synthetic biology to engineer recombinant microorganisms to impart the capability of extracellular sucrose uptake and / or sucrose utilization for the production of KDG within the cell.

[0006] Generally, the overall pathway beginning with extracellular sucrose and resulting in cellular KDG production has four main components. One component of the overall pathwayis non-phosphorylative transport of sucrose into the cell. This activity can be imparted by engineering a microorganism (e.g., a microorganism lacking this capability) to express a sucrose porin nucleotide sequence (e.g., where the microorganism has an outer membrane) and / or a sucrose permease nucleotide sequence. Examples of microorganisms configured to non-phosphorylatively transport sucrose into the cell are described in Section 6.3.1 , and Group 1 numbered embodiments 1 to 160, and their use is described in Section 6.7.3, and Group 1 numbered embodiments 161 to 188. Examples of sucrose porin nucleotide sequences are described in Section 6.4.1 and Group 1 numbered embodiments 1 to 13. Examples of sucrose permease nucleotide sequences are described in Section 6.4.2 and Group 1 numbered embodiments 14 to 22.

[0007] Further examples of microorganisms configured to non-phosphorylatively transport sucrose into the cell are described in Group 2 numbered embodiments 1 to 8, 42 to 49, and 84 to 85. Further examples of the use of such microorganisms are described in Group 2 numbered embodiments 37 to 41 and 72 to 76. Further examples of sucrose porin nucleotide sequences are described in Group 2 numbered embodiments 3 to 5 and 44 to 46. Further examples of sucrose permease nucleotide sequences are described in Group 2 numbered embodiments 6 to 8 and 47 to 49.

[0008] A second component of the overall pathway is the hydrolysis (e.g., intracellularly) of sucrose to its constituent monosaccharides, fructose and glucose. This activity can be imparted by engineering a microorganism (e.g., a microorganism lacking this capability) to express a sucrose invertase nucleotide sequence, which can be localized to the cytoplasm. Examples of microorganisms configured to hydrolyze sucrose to fructose and glucose are described in Section 6.3.2 and Group 1 numbered embodiments 2 to 160, and their use is described in Section 6.7.4 and Group 1 numbered embodiments 161 to 188. Examples of sucrose invertase nucleotide sequences are described in Section 6.4.3 and Group 1 numbered embodiments 23 to 25.

[0009] Further examples of microorganisms configured to hydrolyze sucrose to fructose and glucose are described in Group 2 numbered embodiments 1 to 2, 9 to 11 , 42, 50 to 52, and 84 to 85. Further examples of sucrose invertase nucleotide sequences are described in Section 6.4.3 and Group 2 numbered embodiments 9 to 11 and 50 to 52.

[0010] A third component of the overall pathway is conversion of glucose to KDG, through the intermediates gluconolactone and gluconic acid or a salt thereof ( / .e. , a gluconate). This activity can be imparted by engineering a microorganism (e.g., a microorganism lacking this capability) to express a glucose dehydrogenase nucleotide sequence and / or a gluconate dehydratase nucleotide sequence. Examples of microorganisms configured to convert glucose to KDG are described in Section 6.3.4 and Group 1 numbered embodiments 3 to 160, and their use is described in Section 6.7.5 and Group 1 numbered embodiments 161 to 169 and 189 to 205. Examples of glucose dehydrogenase nucleotide sequences are described in Section 6.4.5 and Group 1 numbered embodiments 27 to 41. Examples of gluconate dehydratase nucleotide sequences are described in Section 6.4.7 and Group 1 numbered embodiments 42 to 50. This component of the pathway may include the spontaneous conversion of gluconolactone to gluconate. This conversion may be catalyzed by gluconolactonase. In some embodiments, a recombinant microorganism of the disclosure is further engineered to express a gluconolactonase nucleotide sequence. Examples of gluconolactonase nucleotide sequences are described in Section 6.4.6 and Group 1 numbered embodiments 52 to 66.

[0011] Further examples of microorganisms configured to convert glucose to KDG are described in Group 2 numbered embodiments 1 , 12 to 22, and 58 to 69. Further examples of the use of such microorganisms are described in Group 2 numbered embodiments 36 to 41 and 71 to 76. Further examples of glucose dehydrogenase nucleotide sequences are described in Group 2 numbered embodiments 13 to 15 and 60 to 62. Further examples of gluconate dehydratase nucleotide sequences are described in Group 2 numbered embodiments 16 to 18 and 63 to 65. Further examples of gluconolactonase nucleotide sequences are described in Group 2 numbered embodiments 20 to 22 and 67 to 69.

[0012] A fourth component of the overall pathway is isomerization of fructose and glucose. This component of the overall pathway can convert fructose (such as that produced by the activity of sucrose invertase) to glucose (which can be converted to KDG, e.g., by activity of glucose dehydrogenase, a gluconate dehydratase, and optionally gluconolactonase). Doing so can funnel more of the taken-up mass of sucrose into KDG production, and is thereby expected to increase yield. This activity can be imparted by engineering a microorganism (e.g., a microorganism lacking this capability) to express a fructose isomerase nucleotidesequence. Examples of microorganisms configured to isomerize fructose and glucose are described in Section 6.3.3 and Group 1 numbered embodiments 73 to 151 , and their use is described in Section 6.7.5 and Group 1 numbered embodiments 189 to 197.

[0013] Further examples of microorganisms configured to isomerize fructose and glucose are described in Group 2 numbered embodiments 29 to 32 and 53 to 57. Further examples of the use of such microorganisms is described in Group 2 numbered embodiments 77 to 83. Examples of fructose isomerase nucleotide sequences are described in Group 1 numbered embodiments 75 to 149 and Group 2 numbered embodiments 30 to 32 and 55 to 57.

[0014] A microorganism of the disclosure can comprise any one, any two, any three, or all four components of the overall pathway. In some embodiments, a microorganism of the disclosure is produced by engineering a parental microorganism to have the capability of performing one, two, three or all four components of the overall pathway. For example, a parental microorganism may naturally contain or be engineered to contain the machinery (e.g., enzymes, transporters and the like) capable of performing one or two components of the pathway, and any polypeptides necessary to perform one or two additional components of the pathway are further engineered into a parental microorganism.

[0015] In some embodiments, a parental microorganism may naturally contain the non- phosphorylative sucrose transport component of the overall pathway and be engineered to contain the sucrose hydrolysis component of the pathway, the fructose isomerization component of the pathway, the component of the pathway converting glucose to KDG, or any two or all three thereof. In some embodiments, a parental microorganism may naturally contain the sucrose hydrolysis component of the pathway and be engineered to contain the non-phosphorylative sucrose transport component of the pathway, the fructose isomerization component of the pathway, the component of the pathway converting glucose to KDG, or any two or all three thereof. In some embodiments, a parental microorganism may naturally contain the component of the pathway converting glucose to KDG and be engineered to contain the non-phosphorylative sucrose transport component of the pathway, the sucrose hydrolysis component of the pathway, the fructose isomerization component of the pathway, or any two or all three thereof. In some embodiments, a parental microorganism may naturally contain the component of the pathway isomerizing fructoseand glucose and be engineered to contain the non-phosphorylative sucrose transport component of the pathway, the sucrose hydrolysis component of the pathway, the component of the pathway converting glucose to KDG, or any two or all three thereof.

[0016] In some embodiments, a parental microorganism may naturally contain the non- phosphorylative sucrose transport component of the pathway and the sucrose hydrolysis component of the pathway, and be engineered to contain the component of the pathway converting glucose to KDG. In some embodiments, a parental microorganism may naturally contain the non-phosphorylative sucrose transport component of the pathway and the component of the pathway converting glucose to KDG, and be engineered to contain the sucrose hydrolysis component of the pathway. In some embodiments, a parental microorganism may naturally contain the sucrose hydrolysis component of the pathway and the component of the pathway converting glucose to KDG, and be engineered to contain the non-phosphorylative sucrose transport component of the pathway.

[0017] KDG produced by the microorganisms of the disclosure can subsequently be utilized in chemical or biochemical processes to produce further products, e.g., terpenoids. These subsequent processes are described in Section 6.7.7 and Group 1 numbered embodiments 170 to 173.

[0018] A microorganism of the disclosure can also further be engineered to have reduced phosphorylation of sucrose, fructose, and / or glucose. Doing so can reduce the mass of sugars shunted into other pathways, and is thereby expected to increasing the yield of KDG. Examples of microorganisms configured to have reduced phosphorylation of sucrose, fructose, and / or glucose are described in Section 6.3.5 and Group 1 numbered embodiments 67 to 72.

[0019] Further examples of microorganisms configured to have reduced phosphorylation of sucrose, fructose, and / or glucose are described in Group 2 numbered embodiments 23 to 28.

[0020] Microorganisms of the disclosure can be engineered from parental microorganisms using known engineering techniques. Examples of parental microorganisms are described in Section 6.5 and Group 1 numbered embodiments 87 to 160. Examples of engineering techniques are described in Section 6.6.1.

[0021] Further examples of parental microorganisms are described in Group 2 numbered embodiments 33 to 35.5. BRIEF DESCRIPTION OF THE FIGURES

[0022] FIG. 1 shows an exemplary pathway by which a recombinant microorganism according to some aspects of the present disclosure can transport extracellular sucrose into the cell without phosphorylating the sucrose. Extracellular sucrose can be transported into the periplasmic space by sucrose porin (activity A) and sucrose can be transported from the periplasmic space into the cell by sucrose permease (activity B).

[0023] FIG. 2 shows an exemplary pathway by which a recombinant microorganism according to some aspects of the present disclosure can hydrolyze sucrose to fructose and glucose, isomerize fructose and glucose, and produce 2-keto-3-deoxygluconate (KDG) starting from glucose as precursor. Sucrose can be hydrolyzed to fructose and glucose by a sucrose invertase (reaction 1). Fructose can be isomerized to glucose by a fructose isomerase (reaction 2). Glucose can be converted to gluconolactone by a glucose dehydrogenase (GDH) (reaction 3). Gluconolactone can spontaneously convert to gluconic acid, or this conversion can be catalyzed by a gluconolactonase (reaction 4). A gluconate dehydratase (GAD) can convert gluconic acid to KDG (reaction 5).

[0024] FIG. 3 shows an exemplary pathway by which a recombinant microorganism according to some aspects of the present disclosure can non-phosphorylatively transport extracellular sucrose into the cell, hydrolyze sucrose to fructose and glucose by a sucrose invertase, isomerize fructose and glucose, and produce 2-keto-3-deoxygluconate (KDG) starting from glucose as precursor. Activities A and B and reactions 1-5 are as shown in FIG. 1 and FIG. 2.

[0025] FIG. 4 shows a schematic of plasmid pTrcHis2b-cscA.cscB.scrY. cscA: E. coli strain W sucrose invertase; cscB: E. coli strain W sucrose permease; scrY: Salmonella thyphimurium sucrose porin.

[0026] FIG. 5 schematically represents a pathway from glucose to gluconate (reactions 3 and 4 shown in FIG. 2). As described in Example 2, because E. coli SuA7.1 lacks glucose dehydrogenase activity, it cannot grow on glucose as its carbon source unless a glucose dehydrogenase (GDH) activity is engineered into it.

[0027] FIG. 6 shows a schematic of plasmid pTrcHis2b_GDH+gluconolactonase. Bs_GDH, Bacillus subtilis glucose dehydrogenase.

[0028] FIG. 7 shows cell growth (as optical density at 600 nm, ODeoo) of E. coli SuA7.1 expressing four different heterologous GDH nucleotide sequences in M9 media with 1% glucose in 3 days.

[0029] FIG. 8 shows cell growth (as OD6oo) of E. coli SuA7.1 expressing heterologous Bacillus subtilis GDH only or Bacillus subtilis GDH + four different gluconolactonase nucleotide sequences, in M9 media with 1% glucose in 2 days.6. DETAILED DESCRIPTION6.1. Definitions

[0030] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0031] Various saccharides and compounds generated therefrom are chiral compounds, i.e., have D- and L- stereoisomers. Given that essentially all naturally-occurring saccharides have D- stereochemistry, saccharides may be referred to herein with or without the “D-” prefix. In other words, unless the context dictates otherwise, the term “glucose” as used herein refers to D-glucose.

[0032] Fructose isomerase: The term “fructose isomerase” refers to an enzyme capable of catalyzing the isomerization of fructose and glucose. Some enzymes capable of catalyzing the isomerization of fructose and glucose, and thus correspond to fructose isomerases in the context of the present disclosure, have other activities, such as isomerization of xylose and xylulose, and thus may be known by other names, such as xylose isomerases. Irrespective of its scientific name, any enzyme that is capable of catalyzing the isomerization of fructose and glucose is encompassed by the term “fructose isomerase.”

[0033] Heterologous: The term “heterologous”, as used herein in relationship to a polypeptide (or amino acid sequence) or nucleic acid (or nucleotide sequence), refers to polypeptide (or amino acid sequence) or nucleic acid (or nucleotide sequence) that has been engineered into a microorganism. For example, in relation to a nucleic acid or nucleotide sequence, the nucleic acid or nucleotide sequence is deemed to be “heterologous” to a recombinant microorganism when the nucleic acid does not include a coding region having a nucleotide sequence not found in the parental microorganism of the recombinant microorganism, when a coding region encodes an amino acid sequence not found in the parental microorganism, and / or the nucleic acid includes a coding region operably linked to a regulatory region to which it is not operably linked in the parental microorganism. Similarly, in relation to a polypeptide or amino acid sequence, the polypeptide or amino acid sequence is deemed to be “heterologous” to a recombinant microorganism when the polypeptide is not found in the parental microorganism of the recombinant microorganism or the polypeptide has an amino acid sequence that is not found in the parental microorganism.

[0034] Nucleic Acid: The term “nucleic acid” is used herein interchangeably with the term “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form.

[0035] Operably linked: In the context of transcriptional regulation, the term “operably linked” refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter, operator, or other regulatory region is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate recombinant cell or other expression system.

[0036] Parental cell, parental microorganism: The terms “parental cell” or “parental microorganism” are used interchangeably to refer to unicellular organisms which can be engineered to express one or more heterologous polypeptides or heterologous nucleic acids. A parental microorganism can be a bacterium, an archaeon, a fungus (e.g., a yeast), or any other unicellular organism. The adjective “parental” indicates that a recombinant cell or recombinant microorganism can be engineered by the introduction into a parental cell or parental microorganism of a heterologous nucleic acid or plurality of heterologous nucleic acids, such as nucleic acid(s) each comprising a coding region or plurality of coding regions each encoding a heterologous polypeptide. A parental microorganism can be a microorganism found in nature or a microorganism that is non-naturally occurring. In other words, a parental microorganism can comprise one or more genetic modifications (e.g., insertion, deletion, or modification of one or more coding regions and / or regulatory regions) relative to a strain thereof found in nature. In relationship to a recombinant microorganism of the disclosure generated through a series of engineering steps, the terms “parental cell” and “parental microorganism” can refer to an ancestral cell or organism incorporating any of the engineering steps, as well as a cell or microorganism without any of the engineering steps. Sometimes, for ease of reference and comparison, the terms “parental cell” and “parental microorganism” refer to a cell or microorganism which, if having genetic modifications, the genetic modification(s) do not relate to any of the pathway components specifically described herein.

[0037] Polypeptide, peptide, and protein: The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. A polypeptide herein may be identified by a name or by a percentage of sequence identity to a reference amino acid sequence. When a polypeptide is identified by a name indicative of an activity performed or enabled by the polypeptide, the name refers to any polypeptide capable of performing or enabling the activity.

[0038] Recombinant cell, recombinant microorganism: The terms “recombinant cell” and “recombinant microorganism” are used interchangeably to refer to a cell that has been genetically engineered. It should be understood that this term refers not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, a recombinant counterpart of a parental cell or parental microorganism includes progeny that are not identical to the initial recombinant cell or microorganism engineered from the parent cell or parental microorganism, but are still included within the scope of the terms “recombinant cell” or “recombinant microorganism” as used herein.

[0039] Sequence identity: “Sequence identity” in relation to nucleotide or amino acid sequence of a nucleic acid or polypeptide molecule, refers to the overall relatedness between two such sequence. Calculation of the percent sequence identity (nucleotide or amino acid sequence identity) of two sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid or amino acid sequence for optimal alignment). The nucleotides or amino acids at corresponding positions are then compared. When a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. Percent sequence identity can be determined manually once an alignment of nucleotide or amino acid sequences is generated. An alignment of query nucleotide or amino acid sequence and a reference nucleotide or amino acid sequence can be generated using the computer program ClustalW (version 1 .83, default parameters), which allows alignments of nucleic acid or protein sequences to be carried out across their entire length (global alignment). ClustalW calculates the best match between a query and one or more reference sequences and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a query sequence, a reference sequence, or both, to maximize sequence alignments. For fast pair wise alignment of nucleotide sequences, the following default parameters are used: word size: 2; window size: 4; scoring method: percentage; number of top diagonals: 4; and gap penalty: 5. For fast pairwise alignment ofamino acid sequences, the following parameters are used: word size: 1 ; window size: 5; scoring method: percentage; number of top diagonals: 5; gap penalty: 3. Unless indicated otherwise, the percent sequence identity between a reference nucleotide or amino acid sequence (e.g. a sequence with a defined SEQ ID NO: as disclosed herein) and a query nucleotide or amino acid sequence is calculated across the entire length of the reference sequence.

[0040] Transformation: The term “transformation” refers to the introduction of nucleic acid molecules into cells, e.g., into prokaryotic cells. In the context of the present disclosure, the term “transformation” encompasses any method known to the skilled person for introducing nucleic acid molecules into cells, e.g., into prokaryotic cells, such as into bacterial cells.Such methods encompass, for example, electroporation, calcium phosphate precipitation, or nanoparticle-based transformation, among other techniques known to the person of ordinary skill in the art having the benefit of the present disclosure.6.2. Pathways

[0041] The present disclosure provides recombinant microorganisms configured to non- phosphorylatively transport sucrose; hydrolyze (e.g., intracellularly) sucrose to glucose and fructose; isomerize fructose and glucose, optionally at a mesophilic temperature; produce 2- keto-3-deoxygluconic acid (KDG) from glucose; or any two, three, or all four thereof.Accordingly, a recombinant microorganism of the disclosure may comprise means for non- phosphorylative transport of sucrose; means for hydrolyzing intracellularly sucrose to glucose and fructose; means for isomerizing fructose and glucose, optionally at a mesophilic temperature; means for producing KDG from glucose; or any two, any three, or all four thereof.

[0042] The non-phosphorylative transport of sucrose can enhance a recombinant microorganism’s ability to survive or grow in media comprising sucrose. The non- phosphorylative transport of sucrose can also provide a substrate for a sucrose invertase or otherwise provide fructose and / or glucose for biochemical processes of a recombinant microorganism. Those biochemical processes can include production of KDG from glucose, as well as the production of compounds derived from KDG.

[0043] In some embodiments, the non-phosphorylative transport of sucrose includes activity of one or both of a sucrose porin (e.g., where a microorganism has an outer membranethrough which sucrose is to be transported), which is schematically depicted in FIG. 1 as activity (A) and described in Section 6.4.1, and a sucrose permease, which is schematically depicted in FIG. 1 as activity (B) and described in Section 6.4.2.

[0044] The hydrolysis of sucrose to fructose and glucose can enhance a recombinant microorganism’s ability to survive or grow in media comprising sucrose. It can also provide fructose and / or glucose for biochemical processes of a recombinant microorganism. Those biochemical processes can include production of KDG from glucose.

[0045] In some embodiments, the hydrolysis of sucrose to glucose and fructose includes the activity of a sucrose invertase enzyme, which is schematically depicted in FIG. 2 as reaction [1] and described in Section 6.4.3.

[0046] The production of KDG from glucose is independent of the source of the glucose. For example, glucose can be provided by uptake by a recombinant microorganism from a medium comprising glucose. For another example, glucose can be provided by non- phosphorylative transport of sucrose into the cell followed by hydrolysis to fructose and glucose.

[0047] In some embodiments, the production of KDG from glucose includes the activity of a glucose dehydrogenase enzyme, which is schematically depicted in FIG. 2 as reaction [3] and described in Section 6.4.5, and a gluconate dehydratase enzyme, which is schematically depicted in FIG. 2 as reaction [5] and described in Section 6.4.7. In some embodiments, the production of KDG from glucose further includes the activity of a gluconolactonase enzyme, which is schematically depicted in FIG. 2 as reaction [4] and described in Section 6.4.6.

[0048] Optionally, a recombinant microorganism is configured to have a fructose isomerase activity, which is schematically depicted in FIG. 2 as reaction [2] and described in Section 6.4.4.

[0049] Also optionally, a recombinant microorganism of the disclosure is configured to have reduced phosphorylation of sucrose, glucose, and / or fructose, as described in Section 6.3.5.

[0050] Recombinant microorganisms configured to non-phosphorylatively transport sucrose; hydrolyze sucrose to glucose and fructose; isomerize fructose and glucose, optionally at a mesophilic temperature; produce KDG from glucose; or any two, any three, or all fourthereof include those described in Section 6.3. A recombinant microorganism can comprise nucleic acids comprising coding regions encoding one or more polypeptides described in Section 6.4 wherein the polypeptides are heterologous to the recombinant microorganism. A recombinant microorganism can be engineered starting from a parental microorganism described in Section 6.5, by techniques including those described in Section 6.6.1.

[0051] Recombinant microorganisms of the disclosure can be used in methods to non- phosphorylatively transport sucrose; hydrolyze sucrose to glucose and fructose; isomerize fructose and glucose, optionally at a mesophilic temperature; produce KDG from glucose; or any two, any three, or all four thereof, as described in Section 6.7. KDG produced by the methods can be used as a feedstock in other methods, such as those described in Section 6.7.7.

[0052] Further details of recombinant microorganisms, polypeptides, pathways, methods, and uses of the disclosure are presented below.6.3. Recombinant Microorganisms

[0053] In some aspects, the present disclosure relates to a recombinant microorganism configured to perform one, two, three, or all four of non-phosphorylative sucrose transport; hydrolyze sucrose to glucose and fructose; isomerize fructose and glucose, optionally at a mesophilic temperature; and / or produce KDG from glucose. A recombinant microorganism can optionally have reduced phosphorylation of sucrose, fructose, and / or glucose.

[0054] In some embodiments, a recombinant microorganism of the disclosure is configured to perform one or more of these activities at mesophilic temperatures (e.g., from 20°C to 40°C).

[0055] A recombinant microorganism of the disclosure can be engineered from a parental microorganism known or hereafter discovered to be suitable for use in bioindustrial processes. Appropriate parental microorganisms include those described in Section 6.5, and suitable engineering techniques include those described in Section 6.6.1.

[0056] Generally, a recombinant microorganism of the disclosure will differ from a parental microorganism by the ability to perform one, two, three, or all four of non-phosphorylative sucrose transport; hydrolyze sucrose to glucose and fructose; isomerize fructose and glucose, optionally at a mesophilic temperature; and / or produce KDG from glucose. Thiscan be achieved by engineering a parental microorganism to express, by for example introducing one or more nucleotide sequences encoding one or more heterologous polypeptides, e.g., as described in Section 6.4, that impart the ability to perform one, two, three, or all four of non-phosphorylative transport of sucrose; hydrolyze sucrose to glucose and fructose; isomerize fructose and glucose, optionally at a mesophilic temperature; and / or produce KDG from glucose. Concurrently, a parental cell can be engineered to increase yield of glucose and / or KDG, for example by deleting or disrupting one or more kinases and / or phosphotransferases that phosphorylate sucrose, fructose, or glucose.

[0057] The particular coding regions desirable for engineering into a particular parental microorganism to yield a particular recombinant microorganism can vary based on a number of factors, including but not necessarily limited to the activity for which it is desired that the recombinant microorganism be configured; any sub-activities the parental microorganism is capable of performing, if any; other features of the parental microorganism that may be relevant; or two or more thereof, among other factors that will be apparent to the person of ordinary skill in the art having the benefit of the present disclosure. Examples of such factors are discussed in Sections 6.3.1-6.3.5, both in general terms and with particular reference to exemplary parental microorganisms: E. coli K12 (American Type Culture Collection (ATCC) Accession Number 29425), E. coli MV (ATCC 9637), Bacillus subtilis (e.g., B. subtilis 168, ATCC 23857), Pseudomonas putida (e.g., P. putida Migula, ATCC 12633), Klebsiella oxytoca (e.g., K. oxytoca (Flugge) Lautrop, ATCC 13182), Pantoea ananatis (e.g., P. ananatis (Serrano) Mergaert et al., ATCC TSD-232), Tatumella citrea (e.g., T. citrea (Kageyama et al.) Brady et al., ATCC 31623), Zymomonas mobilis (e.g., Z. mobilis subsp. mobilis (Lindner) Kluyver and van Niel, ATCC 10988), and Corynebacterium glutamicum (e.g., C. glutamicum (Kinoshita et al.) Abe et al., ATCC 13032). A skilled artisan can readily apply the teachings provided with respect to the exemplary parental microorganisms to other species and strains of microorganisms.6.3.1. Recombinant Microorganisms Configured to Non- Phosphorylatively Transport Sucrose

[0058] The non-phosphorylative transport of sucrose from the extracellular space into cells of a recombinant microorganism can be engineered into a recombinant microorganism by incorporating nucleic acid(s) comprising coding region(s) encoding a sucrose porin (the“sucrose porin nucleotide sequence”), a sucrose permease (the “sucrose permease nucleotide sequence”), or both.

[0059] Features of a parental microorganism that can be considered when engineering a recombinant microorganism to gain or improve the function of a sucrose porin or a sucrose permease include whether the parental microorganism has a single membrane (e.g., the parental microorganism is a Gram-positive bacterium) or has an outer membrane and an inner membrane (e.g., the parental microorganism is a Gram-negative bacterium). In parental microorganisms, sucrose porins are generally found in the outer membranes of parental microorganisms having two membranes, and sucrose permeases are generally found in the inner membranes of parental microorganisms having two membranes or in the membranes of parental microorganisms having a single membrane.

[0060] In some embodiments, this general pattern can be mimicked, wherein a recombinant microorganism having outer and inner membranes can be engineered to localize a sucrose porin to the outer membrane, and a recombinant microorganism having one or two membranes can be engineered to localize a sucrose permease to the inner membrane or single membrane. In other embodiments, a recombinant microorganism can be engineered to localize a sucrose porin to an inner membrane or single membrane of a recombinant microorganism, to localize a sucrose permease to an outer membrane when a recombinant microorganism comprises outer and inner membranes, to localize both a sucrose porin and a sucrose permease to the same membrane or membranes, etc.

[0061] Of the exemplary parental microorganisms, parental E. coli K12 and parental P. putida lack both sucrose porin and sucrose permease activities. Parental Z. mobilis lacks sucrose permease activity and is not known to have sucrose porin activity. In some embodiments, a recombinant E. coli K12, P. putida, or Z. mobilis can be engineered to include a nucleic acid encoding a sucrose porin, a nucleic acid encoding a sucrose permease, or nucleic acid(s) encoding both.

[0062] Parental E. coli W lacks sucrose porin activity. In some embodiments, a recombinant E. coli W can be engineered to include a nucleic acid encoding a sucrose porin.

[0063] Parental 8. subtilis and C. glutamicum are Gram-positive, i.e., both lack an outer membrane, and both have a sucrose permease activity coupled to sucrose phosphorylation. In some embodiments, a recombinant B. subtilis or C. glutamicum can be engineered tolocalize a sucrose porin to its single membrane, to supplement or replace its endogenous phosphorylation-coupled sucrose permease activity with a sucrose permease activity that does not phosphorylate sucrose, or both.

[0064] Parental K. oxytoca has both sucrose porin and sucrose permease activity. In some embodiments, a recombinant K. oxytoca can be engineered to localize a sucrose porin to its inner membrane, to localize a sucrose permease to its outer membrane, to operably link either or both of a sucrose porin coding region and a sucrose permease coding region to a regulatory region with which it is not operably linked in parental K. oxytoca, or two or more thereof.

[0065] Parental P. ananatis and T. citrea are not known to have sucrose porin activity. Both have a sucrose permease activity coupled to sucrose phosphorylation. In some embodiments, a recombinant P. ananatis or T. citrea can be engineered to include a nucleic acid encoding a sucrose porin, to supplement or replace its endogenous phosphorylation- coupled sucrose permease activity with a sucrose permease activity that does not phosphorylate sucrose, or both.6.3.2. Recombinant Microorganisms Configured to Hydrolyze Sucrose to Fructose and Glucose

[0066] The hydrolysis of sucrose to glucose and fructose can be engineered into a recombinant microorganism by incorporating a nucleic acid comprising a coding region encoding a sucrose invertase (the “sucrose invertase nucleotide sequence”).

[0067] Features of a parental microorganism that can be considered when engineering a recombinant microorganism to gain or improve the function of a sucrose invertase include whether the parental microorganism has sucrose invertase activity (at a desired level) and / or whether the sucrose invertase activity is intracellular or extracellular.

[0068] A recombinant microorganism can have extracellular sucrose invertase activity. In further embodiments, a recombinant microorganism can further be engineered to non- phosphorylatively transport extracellular fructose, glucose, or both into the cell, by the engineering into a recombinant microorganism of a porin and / or a permease that transports fructose and / or glucose.

[0069] Preferably, a recombinant microorganism has intracellular sucrose invertase activity.

[0070] Turning to the exemplary parental microorganisms, parental E. coli K12, B. subtilis, P. putida, P. ananatis, T. citrea, and C. glutamicum do not or are not known to have sucrose invertase activity. In some embodiments, a recombinant E. coli K12, B. subtilis, P. putida, P. ananatis, T. citrea, or C. glutamicum can be engineered to include a sucrose invertase activity.

[0071] Parental E. coli W and K. oxytoca are known to have sucrose invertase activity. In some embodiments, a recombinant E. coli W or K. oxytoca can be engineered to operably link a coding region encoding a sucrose invertase to a regulatory region to which the coding region is not operably linked in the parental microorganism.

[0072] Parental Z. mobilis is known to have extracellular sucrose invertase activity. In some embodiments, a recombinant Z. mobilis can be engineered to include an intracellular sucrose invertase activity, to operably link a coding region encoding a sucrose invertase to a regulatory region to which the coding region is not operably linked in the parental microorganism, or both.6.3.3. Recombinant Microorganisms Configured to Isomerize Fructose and Glucose

[0073] The isomerization of fructose and glucose can be engineered into a recombinant microorganism by incorporating a nucleic acid comprising a coding region encoding a nucleotide sequence encoding a fructose isomerase (the “fructose isomerase nucleotide sequence”).

[0074] Features of a parental microorganism that can be considered when engineering a recombinant microorganism to gain or improve the function of a fructose isomerase include whether the parental microorganism has fructose isomerase activity (at a desired level).

[0075] Turning to the exemplary parental microorganisms, all of them lack fructose isomerase activity. In some embodiments, a recombinant E. coli K12, E. coli W, B. subtilis, P. putida, K. oxytoca, P. ananatis, T. citrea, Z. mobilis, or C. glutamicum can be engineered to include a fructose isomerase activity.6.3.4. Recombinant Microorganisms Configured to Produce KDG from Glucose

[0076] The production of KDG from glucose can be engineered into a recombinant microorganism by incorporating nucleic acid(s) comprising coding region(s) encoding a nucleotide sequence encoding a glucose dehydrogenase (the “glucose dehydrogenase nucleotide sequence”), and / or a nucleotide sequence encoding a gluconate dehydratase (the “gluconate dehydratase nucleotide sequence”), and optionally a nucleotide sequence encoding a gluconolactonase (the “gluconolactonase nucleotide sequence”).

[0077] Features of a parental microorganism that can be considered when engineering a recombinant microorganism to gain or improve the function of a glucose dehydrogenase, a gluconate dehydratase, and a gluconolactonase include whether the parental microorganism has one or more of these activities (at a desired level), the location of the activity (e.g., in the periplasm and / or in the cytoplasm) and which electron acceptors are available to receive electrons from glucose dehydrogenase activity. Another feature to be considered is the rate of spontaneous conversion of D-glucono-y-lactone into gluconate in the parental microorganism, which may be sufficiently high that the engineering of gluconolactonase activity into a recombinant microorganism can be optional.

[0078] Of the exemplary parental microorganisms, parental E. coli \2, E. coli \N, P. putida, K. oxytoca, P. ananatis, and T. citrea have periplasmic glucose dehydrogenase activity with pyrroloquinoline quinone (PQQ) as the electron acceptor. Recombinant E. coli K12, E. coli W, P. putida, K. oxytoca, P. ananatis, or T. citrea can be engineered to have cytoplasmic glucose dehydrogenase activity and / or glucose dehydrogenase activity dependent on nicotinamide adenine dinucleotide (NAD+) or nicotinamide adenine dinucleotide phosphate (NADP+) (collectively, NAD(P)+) as the electron acceptor.

[0079] Parental B. subtilis has NAD(P)+-dependent glucose dehydrogenase activity. Recombinant B. subtilis can be engineered to have PQQ-dependent glucose dehydrogenase activity and / or periplasmic glucose dehydrogenase activity.

[0080] Parental Z. mobilis lacks and parental C. glutamicum is not known to have glucose dehydrogenase activity. Recombinant Z. mobilis or C. glutamicum can be engineered to have PQQ-dependent and / or NAD(P)+-dependent glucose dehydrogenase activity that is cytoplasmic, periplasmic, or both.

[0081] All of the exemplary parental microorganisms lack or are not known to have gluconate dehydratase activity. In some embodiments, a recombinant E. coli K12, E. coli W, B. subtilis, P. putida, K. oxytoca, P. ananatis, T. citrea, Z. mobilis, or C. glutamicum can be engineered to include a gluconate dehydratase activity.

[0082] Exemplary parental microorganisms E. coli K12, E. coli \N, K. oxytoca, P. ananatis, T. citrea, Z. mobilis, and C. glutamicum lack or are not known to have gluconolactonase activity. In some embodiments, a recombinant E. coli K12, E. coli W, K. oxytoca, P. ananatis, T. citrea, Z. mobilis, or C. glutamicum is engineered to include a gluconolactonase activity.

[0083] Parental B. subtilis and P. putida have gluconolactonase activity. In some embodiments, a recombinant B. subtilis or P. putida is engineered to operably link a coding region encoding a gluconolactonase to a regulatory region to which the coding region is not operably linked in the parental microorganism, to delete (partially or fully) a coding region encoding the parental gluconolactonase, to reduce or inhibit transcription of the parental gluconolactonase, or combinations thereof.

[0084] In some embodiments, e.g., when a parental microorganism has low levels of a particular activity, it may be supplemented, e.g., by engineering expression of a heterologous polypeptide capable of performing the activity, or by engineering improved activity of a native polypeptide (e.g., through increasing the expression of the native polypeptide).

[0085] Table 1 summarizes what is known of the seven activities discussed above in the exemplary parental microorganisms. It will be apparent that no parental microorganism has all seven activities. Furthermore, any polypeptide activity known in a parental microorganism can be replaced by or supplemented with the corresponding activity provided by a heterologous polypeptide, or a coding region for a homologous polypeptide can be operably linked to a regulatory region to which it is not operably linked in the parental microorganism.6.3.5. Recombinant Microorganisms Configured to Have Reduced Phosphorylation of Sucrose, Glucose, and / or Fructose

[0086] Phosphorylation of sucrose, glucose, and / or fructose are common processes in many parental microorganisms. For example, a parental microorganism may retain intracellular glucose-6-phosphate better than intracellular glucose. For another example, phosphorylation of glucose to glucose-6-phosphate is the first step of the Embden- Meyerhof- Parnas pathway, culminating in the production of pyruvate for entry into the TCAcycle. While phosphorylation of sucrose, glucose, and / or fructose can be beneficial to parental microorganisms, phosphorylation of these sugars by a recombinant microorganism described herein under controlled conditions is undesirable, especially when the production of KDG is intended. For example, phosphorylation of glucose to glucose-6-phosphate in the Embden-Meyerhof-Parnas pathway eventually yields 2-keto-3-deoxy-6-phosphogluconate (KDPG), which is not desirable when the production of KDG is intended.

[0087] Hence, in some embodiments, a recombinant microorganism of the present disclosure can further comprise one or more genetic modifications which reduce phosphorylation of sucrose, glucose, and / or fructose. These modifications can include reduction of fructokinase activity, e.g., by deleting or disrupting a coding region encoding a fructokinase catalyzing the phosphorylation of fructose to fructose-1 -phosphate. For example, if a parental microorganism is an E. coli strain K12, the mak gene can be deleted or disrupted.

[0088] Another modification can be reduction of glucose phosphotransferase (PTS) activity, e.g., by deleting or disrupting a coding region encoding one or more proteins involved in the uptake of extracellular glucose with concomitant transfer of a phosphate group to the glucose to yield a glucose-6-phosphate. For example, if a parental microorganism is an E. coli or a B. subtilis, one or more of the genes encoding Enzyme I, Enzyme IIA, Enzyme IIB, Enzyme IIC, or Histidine Protein, can be deleted or disrupted. Similar modifications include reduction of fructose PTS activity and mannose PTS activity. Still another modification can be reduction of gluconate kinase activity, e.g., by deleting or disrupting a coding region encoding a gluconokinase catalyzing the phosphorylation of gluconate to 6- phosphogluconate. For example, if a parental microorganism is an E. coli strain K12, the gntK gene can be deleted or disrupted.

[0089] As should be apparent, the reduction of activity in a recombinant microorganism is determined relative to a parental microorganism.6.4. Heterologous Polypeptides6.4.1. Sucrose Pori n

[0090] In aspects, the present disclosure provides a recombinant microorganism engineered to express a sucrose porin. A sucrose porin is a beta barrel protein which, whenpresent in the outer membrane of Gram-negative bacteria, permits passive diffusion of extracellular sucrose into the periplasmic space. This activity is schematically represented in FIG. 1 as activity (A). A porin that permits the passive diffusion of other molecules as well as sucrose is a sucrose porin as described herein, provided it permits the diffusion of sucrose. A porin that permits the passive diffusion of extracellular sucrose through the single membrane of a Gram-positive bacterium is also a sucrose porin as described herein.

[0091] In some embodiments, a sucrose porin of the present disclosure has an activity identified by Transport Classification Database (TCDB) number 1.B.3.1.2 or 1.B.3.1.1.

[0092] In some embodiments, a sucrose porin of the present disclosure comprises an amino acid sequence having at least 90% sequence identity, such as at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, to the mature sequence of Salmonella thyphimurium sucrose porin (scrY) having the UniProt identifier P22340 (SEQ ID NO:1), Klebsiella pneumonia sucrose porin (scrY) having the UniProt identifier P27218 (SEQ ID NO:2), or E. coli strain K-12 maltoporin (lamB) having the UniProt identifier P02943 (SEQ ID NO:3), or to the C-terminal portion of SEQ ID NO:1 , SEQ ID NO:2, or SEQ ID NO:3 minus the N-terminal signal sequence of each described below.

[0093] Known sucrose porins comprise from about eight to about 35 p strands. For example, S. thyphimurium sucrose porin comprises 21 p strands. E. coli K-12 maltoporin comprises 25 p strands. S. thyphimurium and K. pneumoniae sucrose porins also include an N-terminal 22-mer signal sequence (residues 1-22 of SEQ ID NO:1 and SEQ ID NO:2), and E. coli K-12 maltoporin has a comparable 25-mer N-terminal signal sequence (residues 1-25 of SEQ ID NO:3). Known sucrose porins form homotrimers. Accordingly, in some embodiments, a sucrose porin of the present disclosure retains p strands and the ability to homotrimerize. Alternatively, in some embodiments efficiency of localization to the outer membrane of a sucrose porin of the present disclosure can be increased by providing an alternative signal sequence to those of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0094] Exemplary sucrose porin amino acid sequences are provided in Table 2:6.4.2. Sucrose Permease

[0095] In aspects, the present disclosure provides a recombinant microorganism engineered to express a sucrose permease. A sucrose permease is a member of major facilitator superfamily (MFS) and is a membrane protein which actively transports sucroseacross the membrane, such as from the periplasmic space through the inner membrane of Gram-negative bacteria or from the extracellular space through the cell membrane of Grampositive bacteria and other microorganisms. This activity is schematically represented in FIG. 1 as activity (B). A permease that actively transports other molecules as well as sucrose is a sucrose permease as described herein, provided it actively transports sucrose.

[0096] The present disclosure provides a recombinant microorganism engineered to express a sucrose permease. In bacteria, sucrose can be transported by at least 2 different mechanisms. One involves a PTS system that transports and simultaneously phosphorylate sucrose to produce sucrose 6-phosphate. This activity is identified in general by EC number 2.7.1.69, and in particular, by EC number 2.7.1.211.

[0097] A different mechanism to transport sucrose is carried out by sucrose permeases which transport sucrose into the cells without modifying the sucrose molecule.

[0098] In some embodiments, sucrose permeases of the present disclosure have an activity identified by Transport Classification Database (TCDB) numbers: 2A.1.5.3, 2A.1.5.6, 3.A.1.1.8, 3.A.1.1.17, 3.A.1.1.25, 3.A.1.1.28, 3.A.1.1.32, 3.A.1.1.41 , 3.A.1.1.52, 2A.2.4.1 , 2. A.123.2.13, 2.A.123.2.14, and / or 2.A.123.2.16.

[0099] In some embodiments, a sucrose permease can be generated by mutagenesis of other permeases that normally do not transport sucrose, for example mutants of the E.coli lactose permease lacY (TCDB number2A.1.5.1) have been shown to transport sucrose (King, S. and Wilson T. 1990, J. Biol. Chem. 265: 9638-9644).

[0100] In some embodiments, a sucrose permease of the present disclosure comprises an amino acid sequence having at least 90% sequence identity, such as at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, to the mature sequence of E.coli strain W sucrose permease (cscB) having the UniProt identifier E0IXR1 (SEQ ID NO:4), E. coli 017:K52:H 18 (strain UMN026 / ExPEC) sucrose permease (cscB) having the UniProt identifier B7N5W1 (SEQ ID NO:5), or Corynebacterium glyciniphilum AJ 3170 sucrose permease (cscB) having the UniProt identifier X5E4R9 (SEQ ID NO:6).

[0101] Known sucrose permeases typically comprise twelve transmembrane helices formed by N and C terminal domains connected by a cytoplasmic loop. Accordingly, in someembodiments, a sucrose permease retains this overall structure. The cytoplasmic loop of the E.coli strain W sucrose permease is located at residues 191-218 of SEQ ID NO:4. Amino acid substitutions or deletions in the cytoplasmic loop that do not substantially change the hydrophilicity or conformational flexibility of the loop are generally tolerable in the cytoplasmic loop of a sucrose permease of the present disclosure. Also, in some embodiments, a sucrose permease retains sufficient structure corresponding to the MFS profile, e.g., a sucrose permease of the present disclosure comprises an amino acid sequence having at least 90% sequence identity, such as at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to positions 223-415 of SEQ ID NO:5.

[0102] The replacement of native Cys residues of E. coli strain W cscB with Ser increased sucrose transport activity. Sahin-Toth et al., 2000, Biochemistry, 39(20):6164-9. Also, the site-specific substitution Cys -> Ser substitution rendered mutant cscB insensitive to inhibition by N-ethylmaleimide (NEM). Accordingly, in some embodiments, a sucrose permease of the disclosure comprises an amino acid sequence having one, two, three, four, five, six, or seven Cys — > Ser substitutions corresponding to C83S, C126S, C198S, C274S, C305S, C328S, or C389S in SEQ ID NO:4, or comparable Cys — > Ser substitutions in SEQ ID NO:5 or SEQ ID NO:6.

[0103] Exemplary sucrose permease amino acid sequences are provided in Table 3:6.4.3. Sucrose Invertase

[0104] In one aspect, the present disclosure provides a recombinant microorganism engineered to express a sucrose invertase. A sucrose invertase is an enzyme that catalyzes the hydrolysis of sucrose to its component monosaccharides, fructose and glucose. This activity is schematically represented in FIG. 2. as activity [1], Sucrose invertase is generally located in the cytoplasm of cells. A sucrose invertase may also be termed a sucrose hydrolase. A sugar invertase or sugar hydrolase that hydrolyzes disaccharides other than sucrose as well as sucrose is a sucrose invertase as described herein, provided it hydrolyses sucrose to fructose and glucose.

[0105] In some embodiments, a sucrose invertase of the present disclosure has an activity identified by EC number 3.2.1 .26.

[0106] In some embodiments, a sucrose invertase of the present disclosure comprises an amino acid sequence having at least 90% sequence identity, such as at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity, to the mature sequence of E.coli strain W sucrose invertase (cscA), UniProt Identifier E0IXQ9 (SEQ ID NO:7), Vibrio cholerae serotype 01 (strain ATCC 39541 1 Classical Ogawa 3951 0395) sucrose invertase (cscA) having the UniProt identifier A5EZZ8 (SEQ ID N0:8), or Staphylococcus aureus (strain COL) sucrose invertase (cscA) having the UniProt identifier A0A0H2WWU4 (SEQ ID N0:9).

[0107] In some embodiments, a sucrose invertase retains residues known to be in the substrate binding sites of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, such as residues 36-39, 55, 63, 98-99, 160-161 , 215, or 298 of SEQ ID NO:7; or residues 105-108, 124,167- 168, 228-229, or 283 of SEQ ID NO:8. In other embodiments, a sucrose invertase retains residues known to be in the active sites of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, such as residue 39 of SEQ ID NO:7 or residue 108 of SEQ ID NO:8. In some embodiments, a sucrose invertase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, or 100% sequence identity to SEQ ID NO:7 and is identical to SEQ ID NO:7 at positions 36-39, 55, 63, 98-99, 160-161 , 215, and 298. In some embodiments, a sucrose invertase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, or 100% sequence identity to SEQ ID NO:8 and is identical to SEQ ID NO:8 at positions 105-108, 124,167-168, 228-229, and 283.

[0108] Exemplary sucrose invertase amino acid sequences are provided in Table 4:6.4.4. Fructose Isomerase

[0109] In one aspect, the present disclosure provides a recombinant microorganism engineered to express a fructose isomerase. A fructose isomerase of the present disclosure is an enzyme that catalyzes the isomerization of fructose and glucose. An isomerase that can act on other monosaccharides in addition to fructose and glucose can be a fructose isomerase as described herein, provided it catalyzes the isomerization of fructose and glucose.

[0110] In certain embodiments, when expressed by recombinant microorganisms, fructose isomerases of the present disclosure can isomerize fructose to glucose in vivo at mesophilic temperatures. Mesophilic temperatures are 20°C-45°C and include subranges thereof (e.g.,20°C-25°C, 25°C-30°C, 30°C-35°C, 35°C-40°C, 40°C-45°C, or any combination of the foregoing) and particular temperatures therein, such as 37°C.

[0111] In some embodiments, a fructose isomerase of the present disclosure has an activity identified by EC numbers 5.3.1.5, 5.3.1.8, 5.3.1.9, 5.3.1.12, 5.3.1.14, and / or 5.3.1.17. In some embodiments, a fructose isomerase of the present disclosure has an activity identified by EC number 5.3.1.5.

[0112] Examples of such enzymes include xylose isomerase (which is known to catalyze isomerization between D-xylose and D-xylulose), also known as “XylA.” Exemplary XylAs include those from Actinoplanes missouriensis (UniProt identifier P12851), Escherichia coli (UniProt identifier P00944), Clostridium thermosulfurogenes (UniProt identifier P19148), Anoxybacillus kamchatkensis (UniProt identifier M4HQI7), Bacillus licheniformis (UniProt identifier P77832), B. coagulans (UniProt identifier G2TH70), Streptomyces rubiginosus (UniProt identifier P24300), S. olivochromogenes (UniProt identifier P15587), Thermotoga neapolitana (UniProt identifier P45687), Arthrobacter sp. (UniProt identifier P12070), Actinoplanes sp. (UniProt identifier p10654), and an uncultured bacterium (NCBI Protein Database ID AEL74969).

[0113] Examples of such enzymes also include L-rhamnose isomerase (which is known to catalyze isomerization between L-rhamnose and L-rhamnulose), also known as “L-Rhi”. An exemplary L-Rhi is that from Pseudomonas stutzeri (UniProt identifier Q75WH8).

[0114] Other examples of such enzymes include glucose-6-phosphate isomerase (GPI), also known as phosphoglucose isomerase / phosphoglucoisomerase (PGI) or phosphohexose isomerase (PHI), which is known to catalyze the isomerization of glucose-6- phosphate and fructose-6-phosphate. Exemplary PGIs or putative PGIs include those from Rhizobium meliloti (strain 1021) (UniProt identifier Q92UI1 and UniProt identifier Q92MQ8), E. coli MG1655 (UniProt identifier P0A6T1), Salmonella enterica serovar typhimurium (strain LT2 I SGSC1412 I ATCC 700720) (UniProt identifier Q8ZMP7), Archaeoglobus fulgidus (UniProt identifier 028778), Methanosarcina mazei (UniProt identifier Q8PVJ5), and Pyrococcus furiosus (UniProt identifier P83194).

[0115] Further examples of such enzymes include mannose-6-phosphate isomerase (ManA), also known as phosphomannose isomerase (PM I), which is known to catalyze the interconversion of fructose 6-phosphate and mannose-6-phosphate, an example of which isManA from E. coli MG1655 (UniProt identifier P00946); D-glucoronate / D-galacturonate isomerase (UxaC), which is known to catalyze the interconversion of D-glucoronate and D- fructuronate, an example of which is UxaC from E. coli MG1655 (UniProt identifier P0A8G3); and 5-dehydro-4-deoxy-glucuronate isomerase (Kdul), which is understood to catalyze the interconversion of 5-dehydro-4-deoxy-D-glucuronate to 3-deoxy-D-glycero-2,5- hexodiulosonate, an example of which is Kdul from E. coli MG1655 (UniProt identifier Q46938).

[0116] In some embodiments, when fructose isomerases are heterologous to and are expressed by recombinant organisms that otherwise lack the ability to metabolize fructose, a recombinant organism expressing a fructose isomerase has greater growth than a nonexpressing control organism, as determined by the optical density at 600 nm (OD6oo) after 3 days of growth in a medium containing fructose at the optimal temperature for growth of the organism.

[0117] In some embodiments, fructose isomerases of the present disclosure comprises an amino acid sequence having at least 90% sequence identity, such as at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, or 100% sequence identity, to the mature sequence of at least one of Actinoplanes missouriensis xylose isomerase (XylA) having UniProt identifier P12851 (SEQ ID NO:21), Escherichia coli xylose isomerase (XylA) having UniProt identifier P00944 (SEQ ID NO:22), Clostridium thermosulfurogenes xylose isomerase (XylA) having UniProt identifier P19148 (SEQ ID NO:23), Anoxybacillus kamchatkensis xylose isomerase (XylA) having UniProt identifier M4HQI7 (SEQ ID NO:24), Bacillus licheniformis xylose isomerase (XylA) having UniProt identifier P77832 (SEQ ID NO:25), Bacillus coagulans xylose isomerase (XylA) having UniProt identifier G2TH70 (SEQ ID NO:26), Streptomyces rubiginosus xylose isomerase (XylA) having UniProt identifier P24300 (SEQ ID NO:27), Streptomyces ol ivoch romogenes xylose isomerase (XylA) having UniProt identifier P15587 (SEQ ID NO:28), Thermotoga neapolitana xylose isomerase (XylA) having UniProt identifier P45687 (SEQ ID NO:29), uncultured bacteria xylose isomerase (XylA) having NCBI identifier AEL74969 (SEQ ID NQ:30), Pseudomonas stutzeri L-rhamnose isomerase (L-Rhi) having UniProt identifier Q75WH8 (SEQ ID NO:31), Arthrobacter sp. xylose isomerase (XylA) having UniProt identifier P12070 (SEQ ID NO:32), Rhizobium meliloti (strain 1021) putativeglucose-6-phosphate isomerase (pgiA2) having UniProt identifier Q92UI1 (SEQ ID NO:33), E. coli MG1655 mannose-6-phosphate isomerase (ManA) having UniProt identifier P00946 (SEQ ID NO:34), Pseudomonas stutzeri L-rhamnose isomerase - E. coli optimized (IDT) (L- Rhi) having UniProt identifier Q75WH 8 (SEQ ID NO:35), E. coli MG1655 glucose-6- phosphate isomerase (PGI) having UniProt identifier P0A6T1 (SEQ ID NO:36), E. coli MG1655 D-glucoronate / D-galacturonate isomerase (UxaC) having UniProt identifier P0A8G3 (SEQ ID NO:37), Rhizobium meliloti (strain 1021) putative glucose-6-phosphate isomerase (pgiA1) having UniProt identifier Q92MQ8 (SEQ ID NO:38), Salmonella enterica serovar typhimurium (strain LT2 / SGSC1412 / ATCC 700720) glucose-6-phosphate isomerase (PGI) having UniProt identifier Q8ZMP7 (SEQ ID NO:39), E. coli MG1655 5-dehydro-4- deoxy-glucuronate isomerase (Kdul) having UniProt identifier Q46938 (SEQ ID NQ:40), Archaeoglobus fulgidus glucose-6-phosphate isomerase (PGI) having UniProt identifier 028778 (SEQ ID NO:41), Methanosarcina mazei glucose-6-phosphate isomerase (PGI) having UniProt identifier Q8PVJ5 (SEQ ID NO:42), Pyrococcus furiosus glucose-6- phosphate isomerase (PGI) having UniProt identifier P83194 (SEQ ID NO:43), or Actinoplanes sp. xylose isomerase (XylA) having UniProt identifier p10654 (SEQ ID NO:44).

[0118] The active site of Actinoplanes missouriensis xylA comprises residues 54 and 57; and the binding site for Mg2+(cofactor) comprises residues 181 , 217, 220, 245, 255, 257, and 292. In some embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to SEQ ID NO:21 and is identical to SEQ ID NO:21 at positions 54, 57, 181 , 217, 220, 245, 255, 257, and 292.

[0119] In some embodiments, a fructose isomerase of the present disclosure comprises the amino acid sequence of SEQ ID NO:21.

[0120] The active site of E. coli xylA comprises residues 101 and 104; and the binding site for Mg2+(cofactor) comprises residues 232, 268, 271 , 296, 307, 309, and 339. In some embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to SEQ ID NO:22 and is identical to SEQ ID NO:22 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339.

[0121] In some embodiments, a fructose isomerase of the present disclosure comprises the amino acid sequence of SEQ ID NO:22.

[0122] The active site of Clostridium thermosulfurogenes xylA comprises residues 101 and 104; the binding site for Co2+(cofactor) comprises residues 232, 268, 271 , 296, 307, 309, and 339. Mutagenesis of residue 101 abolishes activity. Site-specific substitutions W139F and V186T enhance activity. In some embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, or 100% sequence identity to SEQ I D NO:23; is identical to SEQ I D NO:23 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339; and comprises one or more site-specific substitutions W139F and 186T.

[0123] In some embodiments, a fructose isomerase of the present disclosure comprises the amino acid sequence of SEQ ID NO:23 with the amino acid substitutions W139F and V186T.

[0124] The active site of Anoxybacillus kamchatkensis xylA comprises residues 99 and 102; and the binding site for Mg2+ (cofactor) comprises residues 230, 266, 269, 294, 305, 307, and 337. In some embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to SEQ ID NO:24 and is identical to SEQ ID NO:24 at positions 99, 102, 230, 266, 269, 294, 305, 307, and 337.

[0125] In some embodiments, a fructose isomerase of the present disclosure comprises the amino acid sequence of SEQ ID NO:24.

[0126] The active site of Bacillus licheniformis xylA comprises residues 99 and 102; and the binding site for Mg2+ (cofactor) comprises residues 230, 266, 269, 294, 305, 307, and 337. In some embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to SEQ ID NO:25 and is identical to SEQ ID NO:25 at positions 99, 102, 230, 266, 269, 294, 305, 307, and 337.

[0127] In some embodiments, a fructose isomerase of the present disclosure comprises the amino acid sequence of SEQ ID NO:25.

[0128] The active site of Bacillus coagulans xylA comprises residues 100 and 103; and the binding site for Mg2+ (cofactor) comprises residues 231 , 267, 270, 295, 306, 308, and 338. In some embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to SEQ ID NO:26 and is identical to SEQ ID NO:26 at positions 100, 103, 231 , 267, 270, 295, 306, 308, and 338.

[0129] In some embodiments, a fructose isomerase of the present disclosure comprises the amino acid sequence of SEQ ID NO:26.

[0130] The active site of Streptomyces rubiginosus xylA comprises residues 54 and 57; and the binding site for Mg2+ (cofactor) comprises residues 181 , 217, 220, 245, 255, 257, and 287. In some embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to SEQ ID NO:27 and is identical to SEQ ID NO:27 at positions 54, 57, 181 , 217, 220, 245, 255, 257, and 287.

[0131] In some embodiments, a fructose isomerase of the present disclosure comprises the amino acid sequence of SEQ ID NO:27.

[0132] The active site of Streptomyces olivochromogenes xylA comprises residues 54 and 57; and the binding site for Mg2+ (cofactor) comprises residues 181 , 217, 220, 245, 255, 257, and 287. In some embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to SEQ ID NO:28 and is identical to SEQ ID NO:28 at positions 54, 57, 181 , 217, 220, 245, 255, 257, and 287.

[0133] In some embodiments, a fructose isomerase of the present disclosure comprises the amino acid sequence of SEQ ID NO:28.

[0134] The active site of Thermotoga neapolitana xylA comprises residues 101 and 104; the binding site for Co2+ (cofactor) comprises residues 232, 268, 271 , 296, 307, 309, and 339. In some embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to SEQ ID NO:29 and is identical to SEQ ID NO:29 at positions 101 , 104, 232, 268, 271 , 296, 307, 309, and 339.

[0135] In some embodiments, a fructose isomerase of the present disclosure comprises the amino acid sequence of SEQ ID NO:29 with amino acid substitutions V186T, L283P, and F187S.

[0136] The cofactor binding site of L-rhamnose isomerase of Pseudomonas stutzeri comprises residues 219, 254, 257, 281 , 289, 291, 298, and 327. In some embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to SEQ ID NO:31 or SEQ ID NO:35 and is identical to SEQ ID NO:31 or SEQ ID NO:35 at positions 219, 254, 257, 281 , 289, 291 , 298, and 327.

[0137] In some embodiments, a fructose isomerase of the present disclosure comprises the amino acid sequence of SEQ ID NO:31 or SEQ ID NO:35.

[0138] The active site of Arthrobacter sp. (strain NRRL B3728) xylA comprises residues 54 and 57; and the binding site for Mg2+ (cofactor) comprises residues 181 , 217, 220, 245, 255, 257, and 293. In some embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to SEQ ID NO:32 and is identical to SEQ ID NO:32 at positions 54, 57, 181 , 217, 220, 245, 255, 257, and 293.

[0139] In some embodiments, a fructose isomerase of the present disclosure comprises the amino acid sequence of SEQ ID NO:32.

[0140] The binding sites of both Rhizobium meliloti pgiA1 and pgiA2 for Fe cation (cofactor) comprise residues 92, 94, 101 , and 140. In some embodiments, a fructose isomerase of thepresent disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to SEQ ID NO:33 or SEQ ID NO:38 and is identical to SEQ ID NO:33 or SEQ ID NO:38 at positions 92, 94, 101 , and 140.

[0141] In some embodiments, a fructose isomerase of the present disclosure comprises the amino acid sequence of SEQ ID NO:33 or SEQ ID NO:38.

[0142] The binding site of E. coli manA for Zn2+ (cofactor) comprises residues 97, 99, 134, and 255, and the active site is expected on the basis of similarity studies to comprise residue 274. In some embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to SEQ ID NO:34 and is identical to SEQ ID NO:34 at positions 97, 99, 134, 255, and 274.

[0143] In some embodiments, a fructose isomerase of the present disclosure comprises the amino acid sequence of SEQ ID NO:34.

[0144] The active site of E. coli PGI comprises residues 355, 386, and 514. In some embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to SEQ ID NO:36 and is identical to SEQ ID NO:36 at positions 355, 386, and 514.

[0145] In some embodiments, a fructose isomerase of the present disclosure comprises the amino acid sequence of SEQ ID NO:36.

[0146] The binding site of E. coli Kdul for Zn2+ (cofactor) comprises residues 196, 198, 203, and 245. In some embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to SEQ ID NQ:40 and is identical to SEQ ID NQ:40 at positions 196, 198, 203, and 245.

[0147] In some embodiments, a fructose isomerase of the present disclosure comprises the amino acid sequence of SEQ ID NQ:40.

[0148] The binding site of Pyrococcus furiosus PGI for Fe cation (cofactor) comprises residues 88, 90, 97, and 146. The site-specific substitution T85Q may impart higher fructose isomerase activity. In some embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to SEQ ID NO:43; is identical to SEQ ID NO:43 at positions 88, 90, 97, and 146; and comprises the site-specific substitution T85Q.

[0149] In some embodiments, a fructose isomerase of the present disclosure comprises the amino acid sequence of SEQ ID NO:43 with the amino acid substitution T85Q.

[0150] The active site of Actinoplanes sp. (strain ATCC 31351 / 3876) xylA comprises residues 54 and 57; and the binding site for Mg2+ (cofactor) comprises residues 181 , 217, 220, 245, 255, 257, and 292. In some embodiments, a fructose isomerase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to SEQ ID NO:44 and is identical to SEQ ID NO:44 at positions 54, 57, 181 , 217, 220, 245, 255, 257, and 292.

[0151] In some embodiments, a fructose isomerase of the present disclosure comprises the amino acid sequence of SEQ ID NO:44.

[0152] Exemplary fructose isomerase amino acid sequences are provided in Table 5:Table 5Table 5Table 5Table 5Table 5Table 56.4.5. Glucose Dehydrogenase

[0153] In one aspect, the present disclosure provides a recombinant microorganism engineered to express a glucose dehydrogenase. A glucose dehydrogenase of the present disclosure is an enzyme that catalyzes the conversion of glucose to D-glucono-1 ,5-lactone, schematically represented in FIG. 2. as activity [3], Examples of glucose dehydrogenases are: ^-D-glucose: NAD(P)+ 1-oxidoreductase, D-glucose:ubiquinone oxidoreductase, -o- glucose:oxygen 1-oxidoreductase, and pyranose:oxygen 2-oxidoreductase. A dehydrogenase that has oxidoreductase activity and can act on CH-OH moieties ofmolecules other than glucose is a glucose dehydrogenase as described herein, provided it catalyzes the conversion of glucose to gluconolactone.

[0154] In some embodiments, a glucose dehydrogenase of the present disclosure has an activity identified by EC number 1.1.1.47.

[0155] In some embodiments, a glucose dehydrogenase of the present disclosure comprises an amino acid sequence having at least 90% sequence identity, such as at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity, to the mature sequence of at least one of Priestia megaterium glucose dehydrogenase (gdhlV) having the UniProt identifier P39485 (SEQ ID NO:10); Bacillus pumullis glucose dehydrogenase having the UniProt identifier A8FEX4 (SEQ ID NO:11); Bacillus subtilis glucose dehydrogenase (gdh) having the UniProt identifier P12310 (SEQ ID NO: 12); and Lysinibacillus sphaericus glucose dehydrogenase (glcDH) having the UniProt identifier C5IFU0 (SEQ ID NO:13).

[0156] Glucose dehydrogenases generally comprise a binding site for an electron acceptor (typically NAD(P)+), a binding site for the substrate (glucose), and an active site. For both SEQ ID NQ:10 and SEQ ID NO:12, these sites are NAD(P)+binding site, residues 11-35; glucose binding site, residue 145; active site, residue 158. Accordingly, in some embodiments, a glucose dehydrogenase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, or 100% sequence identity to either SEQ ID NQ:10 or SEQ ID NO:12 and is identical thereto at positions 11-35, 145, and 158.

[0157] Exemplary glucose dehydrogenase amino acid sequences are provided in Table 6:6.4.6. Gluconolactonase

[0158] In one aspect, the present disclosure provides a recombinant microorganism engineered to express a gluconolactonase. A gluconolactonase of the present disclosure is an enzyme that catalyzes the hydrolysis of D-glucono-1 ,5-lactone to D-gluconate. This activity is schematically represented in FIG. 2. as activity [4], A hydrolase that can act on ester bonds of other organic molecules in addition to that of D-glucono-1 ,5-lactone is a gluconolactonase as described herein, provided it catalyzes the hydrolysis of gluconolactonase.

[0159] In some embodiments, a gluconolactonase of the present disclosure has an activity identified by EC numbers 3.1.1.17, 3.1.1.31, and / or 3.1.1.-.

[0160] In some embodiments, a gluconolactonase of the present disclosure comprises an amino acid sequence having at least 90% sequence identity, such as at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity, to the mature sequence of at least one of Zymomonas mobilis gluconolactonase (gnl) having the UniProt identifier Q01578 (SEQ ID NO:14); E.coli K12 gluconolactonase (pg / ) having the UniProt identifier P52697 (SEQ ID NO:15); Pseudomonas putida gluconolactonase (ppgL) having the UniProt identifier Q88LB4 (SEQ ID NO:16); and Bacillus subtilis 168 gluconolactonase (yvrE) having the UniProt identifier 034940 (SEQ ID NO:17).

[0161] In parental Z. mobilis, gnl is located in the periplasm and contains an N-terminal signal peptide (residues 1-35). Also, Z. mobilis gnl is found as a homodimer. In some embodiments, a gluconolactonase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, or 100% sequence identity to the C-terminal portion of SEQ ID NO:14 minus residues 1-35 of SEQ ID NO:14 and retains structural features that enable homodimerization.

[0162] E.coli K12 pgl is cytoplasmic and contains an N6-acetyl modification of L287 that may represent an evolutionarily conserved role for lysine acetylation in stress responses in metabolic enzymes. Zhang et al., 2009, Mol Cell Proteomics, 8(2):215-225. In a particular embodiment, a gluconolactonase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, or 100% sequence identity to SEQ ID NO: 15 and retains residue L287.

[0163] P. putida ppgL is believed to have an N-terminal signal peptide (residues 1-22). In some embodiments, a gluconolactonase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, or 100% sequence identity to a the C-terminal portion of SEQ ID NO:16 minus residues 1-22 of SEQ ID NO:16.

[0164] B. subtilis 168 yvrE is cytoplasmic and is believed to contain three binding sites for divalent metal cation cofactors, at residues 15, 146, and 196. In a particular embodiment, a gluconolactonase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, or 100% sequence identity to SEQ ID NO: 17 and is identical to SEQ ID NO:17 at positions 15, 146, and 196.

[0165] Exemplary gluconolactonase amino acid sequences are provided in Table 7:6.4.7. Gluconate Dehydratase

[0166] In one aspect, the present disclosure provides a recombinant microorganism engineered to express a gluconate dehydratase. A gluconate dehydratase of the present disclosure is an enzyme that catalyzes the conversion of D-gluconate to 2-keto-3-deoxygluconate (“KDG”), schematically represented in FIG. 2. as activity [5], An enzyme that can break carbon-oxygen bonds of other molecules in addition to gluconate can be a gluconate dehydratase as described herein, provided it catalyzes the conversion of gluconate to KDG.

[0167] In some embodiments, a gluconate dehydratase of the present disclosure has an activity identified by EC number 4.2.1.9 and / or 4.2.1.-.

[0168] In some embodiments, a gluconate dehydratase of the present disclosure comprises an amino acid sequence having at least 90% sequence identity, such as at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, to the mature sequence of at least one of Achromobacter sp. having the NCBI protein identifier WP_054458272 (SEQ ID NO: 18), Achromobacter sp., gluconate dehydratase (ilvD_T) having the UniProt identifier A0A0M7KL98 (SEQ ID NO: 19), or Achromobacter veterisilvae gluconate dehydratase (HvD_3) having the UniProt identifier A0A446D025 (SEQ ID NQ:20).

[0169] The binding site of Achromobacter ilvD_1 comprises residues 50, 82, 124, 125, and 447; the active site comprises residue 473; and it is believed K125 is N6-carboxylated. In some embodiments, a gluconate dehydratase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, or 100% sequence identity to SEQ ID NO: 19 and is identical to SEQ ID NO:19 at positions 50, 82, 124, 125, 447, and 473.

[0170] The binding site of Achromobacter veterisilvae ilvD_3 comprises residues 81 , 123, 124, and 494; the active site comprises residue 520, and it is believed K124 is N6- carboxylated. In some embodiments, a gluconate dehydratase of the present disclosure comprises an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity, or 100% sequence identity to SEQ ID NQ:20 and is identical to SEQ ID NQ:20 at positions 81 , 123, 124, 494, and 520.

[0171] Exemplary gluconate dehydratase amino acid sequences are provided in Table 8:6.5. Nucleic acids

[0172] In some embodiments, the present disclosure relates to nucleic acids comprising one or more of a sucrose porin nucleotide sequence, a sucrose permease nucleotide sequence, a sucrose invertase nucleotide sequence, a glucose dehydrogenase nucleotide sequence, a gluconate dehydratase nucleotide sequence, a gluconolactonase nucleotide sequence, or a fructose isomerase nucleotide sequence. In some embodiments, a nucleic acid of the present disclosure is a vector or a plasmid.

[0173] A nucleic acid can further comprise regulatory region(s) operably linked to coding region(s), generally with a 1 :1 correspondence of regulatory region to coding region.

[0174] A nucleic acid can further comprise one or more nucleic acid sequences that permit or enhance a construct’s ability to be introduced into a cell of a parental microorganism to yield a recombinant microorganism, to be selected for after introduction into the cell, to replicate independently of the genome of a recombinant microorganism, to be integrated into the genome of a recombinant microorganism, or two or more thereof. Hence, nucleic acids include but are not limited to plasmids.

[0175] In some embodiments, a coding region in a nucleic acid can be modified (e.g., to add a peptide sequence to the N- or C-terminus of the transcribed product or to replace apeptide sequence in the transcribed product with a substitute peptide sequence, to localize the translated product of the coding region to a particular cellular location, to add or remove multimerization sites, to render the translated product more or less sensitive to interactions with molecules other than its intended substrate, or the like). For example, a sucrose porin nucleotide sequence can be modified to reduce localization of a sucrose porin to the outer membrane of a Gram-negative bacterium and increase localization thereof to the inner membrane of a Gram-negative bacterium or the single membrane of a Gram-positive bacterium.

[0176] Any nucleotide sequence encoding a polypeptide of interest can be optimized to increase the percentage of codons that are preferred by a recombinant microorganism, i.e. , the codon from among each group of synonymous codons that is most prevalent in coding regions of a recombinant microorganism’s genome. Several methods for codon optimization are known in the art. In addition to increasing the percentage of preferred codons in the optimized nucleotide sequence, preferably, codon optimized sequences avoid nucleotide repeats and restriction sites.

[0177] In addition to a nucleotide sequence encoding a polypeptide described herein, a coding region in a nucleic acid can further comprise a nucleotide sequence encoding one or more amino acid sequences each in a position that is N-terminal or C-terminal to the polypeptide amino acid sequence. Such other amino acid sequences can include His tags, polypeptide domains for inclusion in fusion proteins, and linkers, among others known to the person of ordinary skill in the art.

[0178] When a nucleic acid comprises multiple coding regions, each coding region may have a unique regulatory region, or two or more coding regions may have identical or substantially identical regulatory regions. Each distinct regulatory region can comprise a constitutive promoter or an inducible promoter. In embodiments wherein one or more regulatory regions comprises an inducible promoter, a nucleic acid can further comprise coding region(s) each encoding a repressor polypeptide, wherein the repressor polypeptide is involved in regulation of an inducible promoter. Additionally or alternatively, the repressor polypeptide can be natively expressed by a recombinant microorganism. In embodiments comprising an inducible promoter, the repressor polypeptide can be controlled by aninducing agent, such as a sugar {e.g., lactose) or an organic acid or a salt thereof (e.g., gluconate).

[0179] In some embodiments, a nucleic acid can comprise a transposable region, such that one or more coding regions (and optionally an operably-linked regulatory region of each) can be integrated into the chromosome of a recombinant microorganism.

[0180] A nucleic acid can comprise a selectable marker or reporter gene, which can be used to identify cells which retain the selectable marker / reporter gene (and a coding region encoding a polypeptide described herein) in a non-integrated nucleic acid and / or integrated into the genome of a recombinant microorganism. Common selectable markers include genes encoding antibiotic resistance, fluorescence markers, enzymes catalyzing formation of a product that can be readily detected, and enzymes or cofactors required for cell survival or growth, among others.

[0181] In some embodiments, a coding region encoding a sucrose porin, a sucrose permease, a sucrose invertase, a glucose dehydrogenase, a gluconate dehydratase, a gluconolactonase, or a fructose isomerase as described herein can also function as a selectable marker or reporter gene. In some embodiments, a recombinant microorganism may inherit from its parent a phenotype such that it can only survive or grow if it expresses one or more of a sucrose porin, a sucrose permease, a sucrose invertase, a glucose dehydrogenase, a gluconate dehydratase, a gluconolactonase, or a fructose isomerase. In some embodiments, a recombinant microorganism can be cultured under conditions such that, if it expresses one or more of a sucrose porin, a sucrose permease, a sucrose invertase, a glucose dehydrogenase, a gluconate dehydratase, a gluconolactonase, or a fructose isomerase, a product can be detected.

[0182] A nucleotide sequence encoding an amino acid sequence of any heterologous polypeptide can be codon optimized for a recombinant microorganism, i.e., the nucleotide sequence can comprise one or more codons which lead to more rapid translation and / or translation with fewer errors, which reduce the likelihood of a transcript forming secondary structures, or provide other advantages in expression of the polypeptide in a recombinant microorganism. In some embodiments, a codon optimized nucleotide sequence can be generated using the Integrated DNA Technologies (IDT) algorithm (www.idtdna.com / pages / tools / codon-optimization-tool).

[0183] Each nucleic acid can be a vector, such as a plasmid (e.g., a pTrcHis2B plasmid). A pTrcHis2B plasmid comprises an origin of replication (ori), a lacl coding region operably linked to a laclq promoter, a site for incorporation of a coding region of interest (i) operably linked to a trc promoter (ii) in frame with a coding region encoding a 6xHis tag (SEQ ID NO:45) and (iii) and the T1 and T2 transcription terminators from E. coli rrnB, and an AmpR coding region operably linked to an AmpR promoter.

[0184] A particular plasmid, pTrcHis2b-cscA.cscB.scrY, has the schematic structure shown in FIG. 4, wherein cscA is a coding region encoding a protein having 100% identity to SEQ ID NO:7 (E. coli strain W sucrose invertase), cscB is a coding region encoding a protein having 100% identity to SEQ ID NO:4 (E. coli strain W sucrose permease), and scrY is a coding region encoding a protein having 100% identity to SEQ ID NO:1 (Salmonella thyphimurium sucrose porin).6.6. Parental Microorganisms

[0185] A parental microorganism that can be engineered into a recombinant microorganism of the disclosure can be any unicellular organism (e.g., a bacterium, an archaeon, or a fungus (e.g., a yeast), among others), in particular such an organism known or discovered to be suitable for use in one or more of non-phosphorylatively transporting sucrose, hydrolyzing sucrose to fructose and glucose, or converting glucose to KDG.

[0186] In some embodiments, the microorganism is E. coli. In particular aspects, the E. coli is E. coli-K^ or a strain derived therefrom, such as E. coli MG1655. In other aspects, the E. coli is E. coli W.

[0187] In some embodiments, the microorganism is Bacillus subtilis.

[0188] In some embodiments, the microorganism is Pseudomonas putida.

[0189] In some embodiments, the microorganism is Klebsiella oxytoca.

[0190] In some embodiments, the microorganism is Pantoea ananatis.

[0191] In some embodiments, the microorganism is Tatumella citrea.

[0192] In some embodiments, the microorganism is Zymomonas mobilis.

[0193] In some embodiments, the microorganism is Corynebacterium glutamicum.

[0194] In some embodiments, the microorganism is E. coli SuA6 (parent strain K12 MG1655, APTS fruBKA, APTS manXYZ, APTS Hlcrr, Amak, AgntK, AidnK).

[0195] In some embodiments, the microorganism is E. coli SuA7.1 (parent strain K12 MG1655, APTS fruBKA, APTS manXYZ, APTS Hlcrr, Amak, AidnK, Aglk).

[0196] In some embodiments, the microorganism is E. coli SuA5_KmR (Apts, AgntK, AidnK, Aglk, AkdgK::KmR).6.6.1. Engineering Methods

[0197] A parental microorganism can be engineered using techniques known in the art. For example, nucleic acid(s) comprising a coding region encoding a polypeptide specifically described herein can be introduced into a parental microorganism via techniques known in the art.

[0198] In some embodiments, a nucleic acid can be introduced into the microorganism by any appropriate transformation technique. The nucleic acid can be extrachromosomal, on a vector (typically a plasmid), such as a low copy number vector, an intermediate copy number vector, or a high copy number vector. The nucleic acid may be maintained episomally and thus comprise a sequence for autonomous replication, such as an autosomal replication sequence. Alternatively, the nucleic acid can be integrated in one or more copies into the genome of the cell. Integration into the cell’s genome can occur at random by non-homologous recombination, or at selected locations by homologous recombination, as is well known in the art.

[0199] Moreover, in some embodiments, a nucleic acid comprises a regulatory region and a coding region which are operably linked. Such a nucleic acid can be referred to as a “recombinant expression vector” or “expression vector.”

[0200] Various genome editing techniques, including but not limited to homologous recombination, CRISPR-Cas, zinc finger nucleases, and transcription activator-like effector nucleases (TALENs), can be used to delete or disrupt genes in a parental microorganism or to operably link a coding region to a regulatory region to which it is not operably linked in a parental microorganism (which may change promoter strength, change whether a promoter is constitutive or inducible, or change which inducer molecule induces transcription of a coding region from an inducible promoter).

[0201] Additionally or alternatively, other techniques can be used in engineering a parental microorganism to yield a recombinant microorganism. Non-specific mutagens can be used to delete or disrupt genes in a parental microorganism, and cells can be screened for a phenotype indicative of deletion or disruption of a gene of interest. In some embodiments, the gene of interest is a gene involved in phosphorylation of sucrose, fructose, or glucose. Cells found to have the desired phenotype can then receive a heterologous nucleic acid encoding a polypeptide specifically described herein.6.7. Methods of use

[0202] The present disclosure also relates to the use of a recombinant microorganism described herein in one or more methods. Specific methods include the non-phosphorylative transport of sucrose, the intracellular hydrolysis of sucrose to fructose and glucose, and the production of KDG from glucose.6.7.1. Culture media

[0203] Generally, the methods comprise culturing a recombinant microorganism in a medium comprising a feedstock molecule of interest. Culturing can be in a batch mode or a continuous mode.

[0204] Examples of media that can be used in batch mode culturing include M9 medium and Hi-Def medium. The M9 medium can comprise the following: sodium phosphate dibasic heptahydrate, 1.28 w / v%; potassium phosphate monobasic, 0.3 w / v%; sodium chloride, 0.05 w / v%; ammonium chloride, 0.1 w / v%; glucose, 0.4 w / v%; MgSO4, 0.024 w / v%; and CaCI2, 0.001 w / v%. The Hi-Def medium can comprise ingredients known to the person of ordinary skill in the art, and it is commercially available (Teknova Inc. Hollister, CA).

[0205] In some embodiments, a culture medium comprises sucrose, glucose, and / or fructose. A culture medium can comprise at least 0.1 w / v% sucrose, glucose, and / or fructose. In some embodiments, a culture medium comprises at least 0.1 w / v% sucrose, at least 0.2 w / v% sucrose, at least 0.3 w / v% sucrose, at least 0.4 w / v% sucrose, at least 0.5 w / v% sucrose, at least 0.6 w / v% sucrose, at least 0.7 w / v% sucrose, at least 0.8 w / v% sucrose, at least 0.9 w / v% sucrose, or at least 1 w / v% sucrose. A culture medium typically comprises less than 5 w / v% sucrose, more typically less than 2 w / v% sucrose (e.g., a culture medium can comprise from 0.1 w / v% to 5 w / v% sucrose; from 0.1 w / v% to 2 w / v%sucrose; from 0.1 w / v% to 1 w / v% sucrose; or from 1 w / v% to 2 w / v% sucrose, among other possible ranges).

[0206] In some embodiments, a culture medium comprises at least 0.1 w / v% glucose, at least 0.2 w / v% glucose, at least 0.3 w / v% glucose, at least 0.4 w / v% glucose, at least 0.5 w / v% glucose, at least 0.6 w / v% glucose, at least 0.7 w / v% glucose, at least 0.8 w / v% glucose, at least 0.9 w / v% glucose, or at least 1 w / v% glucose. A culture medium typically comprises less than 5 w / v% glucose, more typically less than 2 w / v% glucose (e.g., a culture medium can comprise from 0.1 w / v% to 5 w / v% glucose; from 0.1 w / v% to 2 w / v% glucose; from 0.1 w / v% to 1 w / v% glucose; or from 1 w / v% to 2 w / v% glucose, among other possible ranges).

[0207] In some embodiments, a culture medium comprises at least 0.1 w / v% fructose, at least 0.2 w / v% fructose, at least 0.3 w / v% fructose, at least 0.4 w / v% fructose, at least 0.5 w / v% fructose, at least 0.6 w / v% fructose, at least 0.7 w / v% fructose, at least 0.8 w / v% fructose, at least 0.9 w / v% fructose, or at least 1 w / v% fructose. A culture medium typically comprises less than 5 w / v% fructose, more typically less than 2 w / v% fructose (e.g., a culture medium can comprise from 0.1 w / v% to 5 w / v% fructose; from 0.1 w / v% to 2 w / v% fructose; from 0.1 w / v% to 1 w / v% fructose; or from 1 w / v% to 2 w / v% fructose, among other possible ranges).

[0208] In some embodiments, a culture medium can be M9 medium or Hi-Def medium supplemented with sucrose, glucose, and / or fructose. Such a medium may be referred to herein as a “production medium.”

[0209] In some embodiments, a production medium comprises sucrose. In some embodiments, a production medium comprises fructose. In some embodiments, a production medium comprises glucose. In some embodiments, a production medium comprises any two or all three of sucrose, fructose, or glucose.

[0210] In some embodiments, wherein a culture medium comprises two or more carbon sources, the culture medium comprises at least 0.5 w / v% total carbon sources, at least 0.6 w / v% total carbon sources, at least 0.7 w / v% total carbon sources, at least 0.8 w / v% total carbon sources, at least 0.9 w / v% total carbon sources, or at least 1 w / v% total carbon sources. A culture medium typically comprises less than 5 w / v% total carbon sources, more typically less than 2 w / v% total carbon sources (e.g., a culture medium can comprise from0.1 w / v% to 5 w / v% total carbon sources; from 0.1 w / v% to 2 w / v% total carbon sources; from 0.1 w / v% to 1 w / v% total carbon sources; or from 1 w / v% to 2 w / v% total carbon sources, among other possible ranges).

[0211] In some embodiments, a production medium comprises an inducer, i.e., a molecule which binds to a repressor and thereby induces translation of a coding region regulated by an inducible promoter.

[0212] In some embodiments of some methods described herein, it may be desirable to allow growth of a recombinant microorganism without expression of one or more polypeptides required to non-phosphorylatively transport sucrose; hydrolyze sucrose to glucose and fructose; and / or produce 2-keto-3-deoxygluconic acid (KDG) from glucose until a desired biomass of the recombinant microorganism has been reached. This can be effected by use of a growth medium comprising a carbon source other than glucose, fructose, or sucrose. In some embodiments, a growth medium comprises glycerol, such as at least 0.1 w / v% glycerol, at least 0.2 w / v% glycerol, at least 0.3 w / v% glycerol, at least 0.4 w / v% glycerol, at least 0.5 w / v% glycerol, at least 0.6 w / v% glycerol, at least 0.7 w / v% glycerol, at least 0.8 w / v% glycerol, at least 0.9 w / v% glycerol, or at least 1 w / v% glycerol. A growth medium typically comprises less than 5 w / v% glycerol, more typically less than 2 w / v% glycerol (e.g., a growth medium can comprise from 0.1 w / v% to 5 w / v% glycerol; from 0.1 w / v% to 2 w / v% glycerol; from 0.1 w / v% to 1 w / v% glycerol; or from 1 w / v% to 2 w / v% glycerol, among other possible ranges).

[0213] Although glycerol can provide a carbon source for growth of a recombinant microorganism in a growth medium, glycerol can be included in a production medium. Typically, glycerol is included in a production medium at the same or lower concentration than in a growth medium.

[0214] In particular embodiments, a growth medium lacks added glucose, fructose, and / or sucrose, i.e., one or more of these sugars is not intentionally included in a growth medium. In particular embodiments, a growth medium comprises no more than 0.1 w / v% each of glucose, fructose, and / or sucrose.

[0215] The selection of particular concentrations of sucrose, glucose, fructose, and / or glycerol to include in a production medium and / or a growth medium can be made by theperson of ordinary skill in the art having the benefit of the present disclosure as a routine matter.

[0216] For fed-batch and / or continuous mode culturing, the ranges of sucrose, glucose, fructose, glycerol, or combinations thereof given above can be initially provided to the medium. The consumption of the carbon source(s) during culturing can be repeatedly or continuously monitored and additional carbon source(s) can be provided as needed to sustain a desired respiratory coefficient, growth rate, or a rate of production of desired compound(s). The feed rate may be adjusted to avoid accumulation of carbon source(s), which may maximize output of desired compound(s) and minimize waste of carbon source(s). The person of ordinary skill in the art having the benefit of the present disclosure can select the medium composition and the amount of carbon source added thereto during the process to enable the production of desired product(s) to a desired concentration, such as at least 20 g / L, at least 50 g / L, or at least 100 g / L.6.7.2. Culture conditions

[0217] Recombinant cells of the disclosure may be cultured under suitable conditions in a medium, such as a medium described in Section 6.7.1. In some embodiments, recombinant cells of the disclosure undergo fermentation. Fermentation conditions include batch, fed- batch and continuous fermentation. Classical batch fermentation is a closed system, wherein the composition of the medium is not subject to artificial alterations during fermentation. In fed-batch fermentation, the substrate is added in increments as fermentation progresses. In both classical batch fermentation and batch-fed fermentation, the product(s) remain in the bioreactor until the end of the process. Batch and fed-batch fermentation are common and well-known in the art. In continuous fermentation, a defined medium is added continuously to the bioreactor and an equal volume of product containing medium is removed simultaneously. Continuous fermentation aims to maintain steady state growth conditions. Methods for modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology. The fermentation process is typically an aerobic fermentation process.

[0218] The fermentation process is typically run at a temperature that is optimal for growth of a recombinant microorganism. Fermentation for a mesophilic microorganism is typicallycarried out at a temperature within the range of from 20°C to 45°C, from 25°C to 40°C, from 35°C to 40°C, or from 30°C to 37°C. In embodiments wherein a recombinant microorganism is derived from one of the exemplary microorganisms described herein, culturing can comprise maintaining the recombinant microorganism at a mesophilic temperature. In some embodiments, the mesophilic temperature is selected from any of the foregoing ranges.

[0219] Fermentation is typically carried out at a pH in the range of 4 to 8, in the range of 5 to 7, or the range of 5.5 to 6.5. In certain embodiments, fermentation is carried out for a period of time within the range of from 8 to 240 hours, from 12 hours to 168 hours, from 16 hours to 144 hours, from 20 hours to 120 hours, from 24 hours to 72 hours, or from 46 to 48 hours.6.7.3. Methods Of Non-Phosphorylatively Transporting Sucrose

[0220] The present disclosure also relates to methods for non-phosphorylatively transporting sucrose ( / .e., from a medium into a cell). In some embodiments, the transporting methods comprise culturing a recombinant microorganism as described herein in a production medium comprising sucrose as described herein. In some embodiments, a production medium comprises at least 0.1 w / v% sucrose, at least 0.5 w / v% sucrose, or at least 1 w / v% sucrose.

[0221] A recombinant microorganism of the disclosure that can be used in transporting methods can comprise (e.g., be engineered to express) one or more nucleic acids comprising a sucrose porin nucleotide sequence and / or a sucrose permease nucleotide sequence. Optionally, a recombinant microorganism can further comprise one or more nucleic acids comprising other nucleotide sequences encoding other polypeptides as described herein. Further optionally, a recombinant microorganism can further comprise one or more genetic modifications which reduce phosphorylation of sucrose, glucose, and / or fructose.

[0222] Transporting methods of the disclosure can further comprise growing a recombinant microorganism in a growth medium comprising a carbon source other than sucrose, prior to culturing in a production medium. In some embodiments, a growth medium comprises glycerol. In some embodiments, a growth medium comprises at least 0.5 w / v% glycerol or at least 1 w / v% glycerol. In some embodiments, a growth medium lacks added glucose, fructose, and sucrose. In some embodiments, a growth medium comprises no more than 0.1 % each of glucose, fructose, and sucrose.

[0223] Transporting methods of the disclosure typically yield non-phosphorylated sucrose in cells of a recombinant microorganism. The intracellular non-phosphorylated sucrose can be used in one or more natively-occurring or engineered metabolic processes of a recombinant microorganism. In some embodiments, intracellular non-phosphorylated sucrose can be hydrolyzed to glucose and fructose. In some further embodiments, glucose can be used to produce KDG.6.7.4. Methods Of Hydrolyzing Sucrose to Fructose and Glucose

[0224] The present disclosure also relates to methods for hydrolyzing sucrose to glucose and fructose. In some embodiments, the hydrolyzing methods comprise culturing a recombinant microorganism as described herein in a production medium comprising sucrose as described herein. In some embodiments, a production medium comprises at least 0.1 w / v% sucrose, at least 0.5 w / v% sucrose, or at least 1 w / v% sucrose.

[0225] A recombinant microorganism of the disclosure that can be used in hydrolyzing methods can comprise a nucleic acid comprising a sucrose invertase nucleotide sequence. Optionally, a recombinant microorganism can further comprise one or more nucleic acids comprising a sucrose porin nucleotide sequence and a sucrose permease nucleotide sequence. Such a recombinant microorganism may be capable of the non-phosphorylative transport of sucrose into the cell from a production medium comprising sucrose, followed by hydrolysis of sucrose to glucose and fructose. Optionally, a recombinant microorganism can further comprise one or more nucleic acids comprising other nucleotide sequences encoding other polypeptides as described herein. For example, a recombinant microorganism can further comprise a fructose isomerase nucleotide sequence. Further optionally, a recombinant microorganism can further comprise one or more genetic modifications which reduce phosphorylation of sucrose, glucose, and / or fructose.

[0226] Hydrolyzing methods of the disclosure can further comprise growing a recombinant microorganism in a growth medium comprising a carbon source other than sucrose, glucose, or fructose, prior to culturing in a production medium. In some embodiments, a growth medium comprises glycerol. In some embodiments, a growth medium comprises at least 0.5 w / v% glycerol or at least 1 w / v% glycerol. In some embodiments, a growth medium lacks added glucose, fructose, and sucrose. In some embodiments, a growth medium comprises no more than 0.1% each of glucose, fructose, and sucrose.

[0227] Hydrolyzing methods of the disclosure typically yield fructose and glucose which each can be used in one or more natively-occurring or engineered metabolic processes of a recombinant microorganism. In some embodiments, the glucose can be used to produce KDG.6.7.5. Methods of Isomerizing Fructose and Glucose

[0228] The present disclosure also relates to methods for isomerizing fructose and glucose. In some embodiments, the methods comprise culturing a recombinant microorganism as described herein in a production medium comprising sucrose, fructose, and / or glucose as described herein.

[0229] Although fructose and glucose spontaneously interconvert, the rate at which this occurs may be undesirably low for some desired purposes. The interconversion can be catalyzed by a fructose isomerase. Accordingly, a recombinant microorganism of the disclosure that can be used in isomerizing methods can comprise one or more nucleic acids comprising a fructose isomerase nucleotide sequence. In some embodiments, a recombinant microorganism can further comprise one or more nucleic acids comprising a sucrose porin nucleotide sequence, a sucrose permease nucleotide sequence, and / or a sucrose invertase nucleotide sequence. Such a recombinant microorganism may be capable of isomerizing fructose and glucose in embodiments wherein a production medium comprises sucrose.

[0230] All else being equal, isomerization of fructose and glucose will tend toward a 50:50 equilibrium by weight or by mole parts between these two monosaccharides. The equilibrium can be driven in favor of one of the monosaccharides if other metabolic processes irreversibly or essentially irreversibly convert that monosaccharide into other products. For example, if glucose is converted into other products, such as KDG via pathways described herein, then the equilibrium will be driven in favor of glucose (in other words, spontaneous conversion or isomerization catalyzed by a fructose isomerase will convert more fructose to glucose than the reverse). Catalysis by a fructose isomerase can thus increase flux into metabolic pathways making use of glucose, such as the production of KDG, thereby increasing the yield of the pathway product (e.g., KDG) as described herein.6.7.6. Methods Of Producing 2-Keto-3-Deoxygluconate

[0231] The present disclosure also relates to methods for producing 2-keto-3- deoxygluconate (“KDG”). In some embodiments, the methods comprise culturing a recombinant microorganism as described herein in a production medium comprising sucrose, fructose, and / or glucose as described herein.

[0232] A recombinant microorganism of the disclosure that can be used in producing methods can comprise one or more nucleic acids comprising a glucose dehydrogenase nucleotide sequence and a gluconate dehydratase nucleotide sequence. In some embodiments, a recombinant microorganism can further comprise one or more nucleic acids comprising a gluconolactonase nucleotide sequence. Such a recombinant microorganism may be capable of producing KDG in embodiments wherein a production medium comprises glucose.

[0233] Optionally, a recombinant microorganism can comprise one or more nucleic acids comprising a sucrose porin nucleotide sequence, a sucrose permease nucleotide sequence, or both, and / or a sucrose invertase nucleotide sequence. Further optionally, a recombinant microorganism can comprise a nucleic acid comprising a fructose isomerase nucleotide sequence. Such a recombinant microorganism may be capable of producing KDG in embodiments wherein a production medium comprises sucrose.

[0234] In some embodiments, a recombinant microorganism can comprise a nucleic acid comprising a fructose isomerase nucleotide sequence. Such a recombinant microorganism may be capable of producing KDG in embodiments wherein a production medium comprises fructose.

[0235] Optionally, in any producing methods of the disclosure, a recombinant microorganism can further comprise one or more genetic modifications which reduce phosphorylation of sucrose, glucose, and / or fructose.

[0236] Producing methods of the disclosure can further comprise growing a recombinant microorganism in a growth medium comprising a carbon source other than glucose or sucrose, prior to culturing in a production medium. In some embodiments, a growth medium comprises glycerol. In some embodiments, a growth medium comprises at least 0.5 w / v% glycerol or at least 1 w / v% glycerol. In some embodiments, a growth medium lacks addedglucose, fructose, and sucrose. In some embodiments, a growth medium comprises no more than 0.1 % each of glucose, fructose, and sucrose.

[0237] Producing methods of the disclosure typically yield KDG. In some embodiments, producing methods produce KDG at a yield of 60%. The yield of KDG is calculated as the ratio of the weight of KDG produced to the weight of substrate consumed. Weight of substrate consumed is calculated by subtracting the amount of substrate present in the supernatant at the end of the reaction from the amount of substrate present in the supernatant at the beginning of the reaction. In certain embodiments, the substrate for KDG production is glucose. Thus, in certain embodiments, at least 0.6 g of KDG is produced for every 1 g of glucose consumed by a recombinant cell. In certain embodiments, the yield of KDG is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. KDG concentration (or weight) may be determined using conventional methods known in the art, e.g. column chromatography (see e.g. US 7,125,704). KDG concentration (or weight) may be determined using ion chromatography.6.7.7. Methods of Using KDG

[0238] KDG produced by the methods described in Section 6.7.5 can be used for any desired purpose. In some embodiments, KDG can be isolated from a recombinant microorganism, purified, and provided as a feedstock to non-biological chemical processes. In other embodiments, KDG can be supplied to biochemical processes for the production of further products. The biochemical processes can be intracellular to a recombinant microorganism or can be performed in other organisms, e.g., KDG can be isolated from a recombinant microorganism, purified, and used by other organisms.

[0239] In embodiments, the KDG can be used for the production of pyruvate and / or glyceraldehyde; the production of isopentenyl pyrophosphate (IPP) and / or dimethylallyl pyrophosphate (DMAPP); and / or the production of terpenoids. Further genetic modifications that may be desirable for a recombinant microorganism to increase the yield of one or more of these products from KDG include those described in W02021016220.6.8. Numbered Embodiments

[0240] The present disclosure is exemplified by the numbered embodiments below.

[0241] Various aspects of the present disclosure are described in the embodiments set forth in the following numbered paragraphs of Group 1.

[0242] Group 1 :1. A recombinant microorganism configured to non-phosphorylatively transport sucrose.2. A recombinant microorganism, which is optionally the recombinant microorganism of embodiment 1 , configured to hydrolyze sucrose to glucose and fructose.3. A recombinant microorganism, which is optionally the recombinant microorganism of embodiment 1 or embodiment 2, configured to produce 2-keto-3- deoxygluconic acid (KDG) from glucose.4. A recombinant microorganism, which is optionally the recombinant microorganism of any one of embodiments 1 to 3, comprising one or more nucleic acids comprising:(a) a nucleotide sequence encoding a sucrose porin (the “sucrose porin nucleotide sequence”),(b) a nucleotide sequence encoding a sucrose permease (the “sucrose permease nucleotide sequence”), and(c) a nucleotide sequence encoding a sucrose invertase (the “sucrose invertase nucleotide sequence”), wherein at least one of the sucrose porin nucleotide sequence, the sucrose permease nucleotide sequence, and the sucrose invertase nucleotide sequence is heterologous to the microorganism.5. The recombinant microorganism of embodiment 4, wherein the sucrose porin nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:1.6. The recombinant microorganism of embodiment 4 or embodiment 5, wherein the sucrose porin nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:1.7. The recombinant microorganism of any one of embodiments 4 to 6, wherein the sucrose porin nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:1.8. The recombinant microorganism of embodiment 4, wherein the sucrose porin nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:2.9. The recombinant microorganism of embodiment 4 or embodiment 8, wherein the sucrose porin nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:2.10. The recombinant microorganism of any one of embodiments 4 or 8 to 9, wherein the sucrose porin nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:2.11 . The recombinant microorganism of embodiment 4, wherein the sucrose porin nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:3.12. The recombinant microorganism of embodiment 4 or embodiment 11 , wherein the sucrose porin nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:3.13. The recombinant microorganism of any one of embodiments 2 or 11 to 12, wherein the sucrose porin nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:3.14. The recombinant microorganism of any one of embodiments 2 to 13, wherein the sucrose permease nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:4.15. The recombinant microorganism of any one of embodiments 2 to 14, wherein the sucrose permease nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:4.16. The recombinant microorganism of any one of embodiments 2 to 15, wherein the sucrose permease nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:4.17. The recombinant microorganism of any one of embodiments 2 to 13, wherein the sucrose permease nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:5.18. The recombinant microorganism of any one of embodiments 2 to 13 and 17, wherein the sucrose permease nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:5.19. The recombinant microorganism of any one of embodiments 2 to 13 and 18, wherein the sucrose permease nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:5.20. The recombinant microorganism of any one of embodiments 2 to 13, wherein the sucrose permease nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:6.21 . The recombinant microorganism of any one of embodiments 2 to 13 and 20 wherein the sucrose permease nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:6.22. The recombinant microorganism of any one of embodiments 2 to 13 and 21 , wherein the sucrose permease nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:6.23. The recombinant microorganism of any one of embodiments 2 to 22, wherein the sucrose invertase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:7.24. The recombinant microorganism of any one of embodiments 2 to 23, wherein the sucrose invertase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:7.25. The recombinant microorganism of any one of embodiments 2 to 24, wherein the sucrose invertase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:7.26. A recombinant microorganism, which is optionally the recombinant microorganism of any one of embodiments 1 to 25, comprising one or more nucleic acids comprising:(a) a nucleotide sequence encoding a glucose dehydrogenase (the “glucose dehydrogenase nucleotide sequence”), and(b) a nucleotide sequence encoding a gluconate dehydratase (the “gluconate dehydratase nucleotide sequence”), wherein at least one of the glucose dehydrogenase nucleotide sequence and the gluconate dehydratase nucleotide sequence is heterologous to the microorganism.27. The recombinant microorganism of embodiment 26, wherein the glucose dehydrogenase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to any one of SEQ ID NO: 10- 13.28. The recombinant microorganism of embodiment 12 or embodiment 27, wherein the glucose dehydrogenase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to any one of SEQ ID NO:10-13.29. The recombinant microorganism of any one of embodiments 12 to 28, wherein the glucose dehydrogenase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to any one of SEQ ID NQ:10-13.30. The recombinant microorganism of embodiment 12 or embodiment 27, wherein the glucose dehydrogenase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 10.31 . The recombinant microorganism of any one of embodiments 12 to 28 and 30, wherein the glucose dehydrogenase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 10.32. The recombinant microorganism of any one of embodiments 12 to 31 , wherein the glucose dehydrogenase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 10.33. The recombinant microorganism of embodiment 12 or embodiment 27, wherein the glucose dehydrogenase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:11.34. The recombinant microorganism of any one of embodiments 12 to 28 and 33, wherein the glucose dehydrogenase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:11.35. The recombinant microorganism of any one of embodiments 12 to 29 and 33 to 34, wherein the glucose dehydrogenase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:11.36. The recombinant microorganism of embodiment 12 or embodiment 27, wherein the glucose dehydrogenase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 12.37. The recombinant microorganism of any one of embodiments 12 to 28 and 36, wherein the glucose dehydrogenase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 12.38. The recombinant microorganism of any one of embodiments 12 to 29 and 36 to 37, wherein the glucose dehydrogenase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 12.39. The recombinant microorganism of embodiment 12 or embodiment 27, wherein the glucose dehydrogenase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 13.40. The recombinant microorganism of any one of embodiments 12 to 28 and 39, wherein the glucose dehydrogenase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 13.41 . The recombinant microorganism of any one of embodiments 12 to 29 and 39 to 40, wherein the glucose dehydrogenase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 13.42. The recombinant microorganism of any one of embodiments 12 to 41 , wherein the gluconate dehydratase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 18.43. The recombinant microorganism of any one of embodiments 12 to 42, wherein the gluconate dehydratase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 18.44. The recombinant microorganism of any one of embodiments 12 to 43, wherein the gluconate dehydratase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 18.45. The recombinant microorganism of any one of embodiments 12 to 41 , wherein the gluconate dehydratase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 19.46. The recombinant microorganism of any one of embodiments 12 to 41 and 45, wherein the gluconate dehydratase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 19.47. The recombinant microorganism of any one of embodiments 12 to 41 and 45 to 46, wherein the gluconate dehydratase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 19.48. The recombinant microorganism of any one of embodiments 12 to 41 , wherein the gluconate dehydratase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NQ:20.49. The recombinant microorganism of any one of embodiments 12 to 41 and 48, wherein the gluconate dehydratase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NQ:20.50. The recombinant microorganism of any one of embodiments 12 to 41 and 48 to 49, wherein the gluconate dehydratase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NQ:20.51 . A recombinant microorganism, which is optionally the recombinant microorganism of any one of embodiments 1 to 50, further comprising one or more nucleic acids comprising a nucleotide sequence encoding a gluconolactonase (the “gluconolactonase nucleotide sequence”).52. The recombinant microorganism of embodiment 51 , wherein the gluconolactonase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to any one of SEQ ID NO: 14- 17.53. The recombinant microorganism of embodiment 19 or embodiment 52, wherein the gluconolactonase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to any one of SEQ ID NO:14-17.54. The recombinant microorganism of any one of embodiments 19 to 53, wherein the gluconolactonase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to any one of SEQ ID NO:14-17.55. The recombinant microorganism of embodiment 19 or embodiment 52, wherein the gluconolactonase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to any one of SEQ ID NO:14.56. The recombinant microorganism of any one of embodiments 19 to 53 and 55, wherein the gluconolactonase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to any one of SEQ ID NO:14.57. The recombinant microorganism of any one of embodiments 19 to 56, wherein the gluconolactonase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to any one of SEQ ID NO:14.58. The recombinant microorganism of embodiment 19 or embodiment 52, wherein the gluconolactonase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to any one of SEQ ID NO: 15.59. The recombinant microorganism of any one of embodiments 19 to 53 and 58, wherein the gluconolactonase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to any one of SEQ ID NO: 15.60. The recombinant microorganism of any one of embodiments 19 to 54 and 58 to 59, wherein the gluconolactonase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to any one of SEQ ID NO: 15.61 . The recombinant microorganism of embodiment 19 or embodiment 52, wherein the gluconolactonase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to any one of SEQ ID NO:16.62. The recombinant microorganism of any one of embodiments 19 to 53 and 61 , wherein the gluconolactonase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to any one of SEQ ID NO:16.63. The recombinant microorganism of any one of embodiments 19 to 54 and 61 to 62, wherein the gluconolactonase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to any one of SEQ ID NO:16.64. The recombinant microorganism of embodiment 19 or embodiment 52, wherein the gluconolactonase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to any one of SEQ ID NO: 17.65. The recombinant microorganism of any one of embodiments 19 to 53 and 64, wherein the gluconolactonase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to any one of SEQ ID NO: 17.66. The recombinant microorganism of any one of embodiments 19 to 54 and 64 to 65, wherein the gluconolactonase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to any one of SEQ ID NO: 17.67. The recombinant microorganism of any one of embodiments 1 to 66, which further has reduced glucokinase activity relative to a parental microorganism.68. The recombinant microorganism of any one of embodiments 1 to 67, which further has reduced fructokinase activity relative to a parental microorganism.69. The recombinant microorganism of any one of embodiments 1 to 68, which further has reduced glucose phosphotransferase (PTS) activity relative to a parental microorganism.70. The recombinant microorganism of any one of embodiments 1 to 69, which further has reduced fructose PTS activity relative to a parental microorganism.71 . The recombinant microorganism of any one of embodiments 1 to 70, which further has reduced mannose PTS activity relative to a parental microorganism.72. The recombinant microorganism of any one of embodiments 1 to 71 , which further has reduced gluconate kinase activity relative to a parental microorganism.73. A recombinant microorganism, which is optionally the recombinant microorganism of any one of embodiments 1 to 72, configured to isomerize fructose and glucose, optionally at a mesophilic temperature.74. The recombinant microorganism of embodiment 73, wherein the recombinant microorganism comprises one or more nucleic acids comprising a nucleotide sequence encoding a fructose isomerase (the “fructose isomerase nucleotide sequence”), wherein the fructose isomerase nucleotide sequence is heterologous to the recombinant microorganism.75. The recombinant microorganism of embodiment 74, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to any one of SEQ ID NO:21-44.76. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to any one of SEQ ID NO:21-44.77. The recombinant microorganism of any one of embodiments 74 to 76, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to any one of SEQ ID NO:2144.78. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:21.79. The recombinant microorganism of any one of embodiments 74 to 76 and 78, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:21.80. The recombinant microorganism of any one of embodiments 74 to 79, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:21.81 . The recombinant microorganism of embodiment 74 or embodiment 75, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:22.82. The recombinant microorganism of embodiment 74 to 76 and 81 , wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity SEQ ID NO:22.83. The recombinant microorganism of any one of embodiments 74 to 76 and 81 to 82, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:22.84. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:23.85. The recombinant microorganism of embodiment 74 to 76 and 84, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:23.86. The recombinant microorganism of any one of embodiments 74 to 76 and 84 to 85, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:23.87. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:24.88. The recombinant microorganism of embodiment 74 to 76 and 87, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:24.89. The recombinant microorganism of any one of embodiments 74 to 76 and 87 to 88, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:24.90. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:25.91 . The recombinant microorganism of embodiment 74 to 76 and 90, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:25.92. The recombinant microorganism of any one of embodiments 74 to 76 and 90 to 91 , wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:25.93. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:26.94. The recombinant microorganism of embodiment 74 to 76 and 93, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:26.95. The recombinant microorganism of any one of embodiments 74 to 76 and 93 to 94, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:26.96. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:27.97. The recombinant microorganism of embodiment 74 to 76 and 96, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:27.98. The recombinant microorganism of any one of embodiments 74 to 76 and 96 to 97, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:27.99. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:28.100. The recombinant microorganism of embodiment 74 to 76 and 99, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:28.101. The recombinant microorganism of any one of embodiments 74 to 76 and 99 to 100, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:28.102. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:29.103. The recombinant microorganism of embodiment 74 to 76 and 102, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:29.104. The recombinant microorganism of any one of embodiments 74 to 76 and 102 to 103, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:29.105. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NQ:30.106. The recombinant microorganism of embodiment 74 to 76 and 105, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NQ:30.107. The recombinant microorganism of any one of embodiments 74 to 76 and 105 to 106, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:30.108. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:31.109. The recombinant microorganism of embodiment 74 to 76 and 108, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:31.110. The recombinant microorganism of any one of embodiments 74 to 76 and 108 to 109, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:31.111. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:32.112. The recombinant microorganism of embodiment 74 to 76 and 111 , wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:32.113. The recombinant microorganism of any one of embodiments 74 to 76 and 111 to 112, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:32.114. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:33.115. The recombinant microorganism of embodiment 74 to 76 and 114, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:33.116. The recombinant microorganism of any one of embodiments 74 to 76 and 114 to 115, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:33.117. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:34.118. The recombinant microorganism of embodiment 74 to 76 and 117, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:34.119. The recombinant microorganism of any one of embodiments 74 to 76 and 117 to 118, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:34.120. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:35.121. The recombinant microorganism of embodiment 74 to 76 and 120, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:35.122. The recombinant microorganism of any one of embodiments 74 to 76 and 120 to 121 , wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:35.123. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:36.124. The recombinant microorganism of embodiment 74 to 76 and 123, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:36.125. The recombinant microorganism of any one of embodiments 74 to 76 and 123 to 124, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:36.126. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:37.127. The recombinant microorganism of embodiment 74 to 76 and 126, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:37.128. The recombinant microorganism of any one of embodiments 74 to 76 and 126 to 127, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:37.129. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:38.130. The recombinant microorganism of embodiment 74 to 76 and 129, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:38.131. The recombinant microorganism of any one of embodiments 74 to 76 and 129 to 130, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:38.132. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:39.133. The recombinant microorganism of embodiment 74 to 76 and 132, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:39.134. The recombinant microorganism of any one of embodiments 74 to 76 and 132 to 133, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:39.135. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NQ:40.136. The recombinant microorganism of embodiment 74 to 76 and 135, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NQ:40.137. The recombinant microorganism of any one of embodiments 74 to 76 and 135 to 136, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:40.138. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:41.139. The recombinant microorganism of embodiment 74 to 76 and 138, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:41.140. The recombinant microorganism of any one of embodiments 74 to 76 and 138 to 139, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:41.141. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:42.142. The recombinant microorganism of embodiment 74 to 76 and 141 , wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:42.143. The recombinant microorganism of any one of embodiments 74 to 76 and 141 to 142, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:42.144. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:43.145. The recombinant microorganism of embodiment 74 to 76 and 144, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:43.146. The recombinant microorganism of any one of embodiments 74 to 76 and 144 to 145, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:43.147. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:44.148. The recombinant microorganism of embodiment 74 to 76 and 147, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:44.149. The recombinant microorganism of any one of embodiments 74 to 76 and 147 to 148, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:44.150. The recombinant microorganism of any one of embodiments 73 to 149, which further has increased fructose isomerase activity relative to a parental microorganism.151. The recombinant microorganism of embodiment 150, wherein the increased fructose isomerase activity is at a mesophilic temperature.152. The recombinant microorganism of any one of embodiments 1 to 151 , wherein the recombinant microorganism is a Bacillus subtilis.153. The recombinant microorganism of any one of embodiments 1 to 151 , wherein the recombinant microorganism is a Pseudomonas putida.154. The recombinant microorganism of any one of embodiments 1 to 151 , wherein the recombinant microorganism is a Klebsiella oxytoca.155. The recombinant microorganism of any one of embodiments 1 to 151 , wherein the recombinant microorganism is a Pantoea ananatis.156. The recombinant microorganism of any one of embodiments 1 to 151 , wherein the recombinant microorganism is a Tatumella citrea.157. The recombinant microorganism of any one of embodiments 1 to 151 , wherein the recombinant microorganism is a Zymomonas mobilis.158. The recombinant microorganism of any one of embodiments 1 to 151 , wherein the recombinant microorganism is a Corynebacterium glutamicum.159. The recombinant microorganism of any one of embodiments 1 to 151 , wherein the recombinant microorganism is an E. coli.160. The recombinant microorganism of embodiment 159, wherein the E. coli is E. coli K12 or a strain derived therefrom, such as E. coli MG1655; or the E. coli is E. coli W.161. A method for producing 2-keto-3-deoxygluconate (“KDG”), comprising culturing the recombinant microorganism of any one of embodiments 1 to 160 in a production medium comprising sucrose.162. The method of embodiment 161 , wherein the production medium comprises from 0.1 w / v% to 5 w / v% sucrose.163. The method of embodiment 161 or embodiment 162, wherein the recombinant microorganism is capable of non-phosphorylatively transporting sucrose from the production medium into a cell of the recombinant microorganism.164. The method of any one of embodiments 161 to 163, wherein the recombinant microorganism is capable of hydrolyzing sucrose to glucose and fructose.165. The method of any one of embodiments 161 to 164, wherein the method further comprises growing the recombinant microorganism in a growth medium comprising a carbon source other than sucrose, prior to culturing in the production medium.166. The method of embodiment 165, wherein the growth medium comprises glycerol.167. The method of embodiment 165 or embodiment 166, wherein the growth medium comprises from 0.1 w / v% to 5 w / v% glycerol.168. The method of any one of embodiments 101 to 167, wherein the growth medium lacks added sucrose.169. The method of embodiment 165 or embodiment 166, wherein the growth medium comprises no more than 0.1% sucrose.170. The method of any one of embodiments 161 to 169, further comprising use of the KDG.171. The method of embodiment 170, wherein the KDG is used for the production of pyruvate and / or glyceraldehyde.172. The method of embodiment 170, wherein the KDG is used for the production of isopentenyl pyrophosphate (IPP) and / or dimethylallyl pyrophosphate (DMAPP).173. The method of embodiment 170, wherein the KDG is used for the production of terpenoids.174. A method for non-phosphorylatively transporting sucrose, comprising culturing the recombinant microorganism of any one of embodiments 1 to 160 in a production medium comprising sucrose.175. The method of embodiment 174, wherein the production medium comprises from 0.1 w / v% to 5 w / v% sucrose.176. The method of embodiment 174 or embodiment 175, wherein the method further comprises growing the recombinant microorganism in a growth medium comprising a carbon source other than sucrose, prior to culturing in the production medium.177. The method of embodiment 175, wherein the growth medium comprises glycerol.178. The method of embodiment 175 or embodiment 177, wherein the growth medium comprises from 0.1 w / v% to 5 w / v% glycerol.179. The method of any one of embodiments 176 to 178, wherein the growth medium lacks added sucrose.180. The method of any one of embodiments 175 to 178, wherein the growth medium comprises no more than 0.1% sucrose.181. A method for hydrolyzing sucrose to glucose and fructose, comprising culturing the recombinant microorganism of any one of embodiments 1 to 160 in a production medium comprising sucrose.182. The method of embodiment 181 , wherein the production medium comprises from 0.1 w / v% to 5 w / v% sucrose.183. The method of embodiment 181 or embodiment 182, wherein the recombinant microorganism is capable of non-phosphorylatively transporting sucrose from the production medium into a cell of the recombinant microorganism.184. The method of any one of embodiments 181 to 183, wherein the method further comprises growing the recombinant microorganism in a growth medium comprising a carbon source other than sucrose, prior to culturing in the production medium.185. The method of embodiment 184, wherein the growth medium comprises glycerol.186. The method of embodiment 184 or embodiment 185, wherein the growth medium comprises from 0.1 w / v% to 5 w / v% glycerol.187. The method of any one of embodiments 184 to 186, wherein the growth medium lacks added sucrose.188. The method of any one of embodiments 184 to 186, wherein the growth medium comprises no more than 0.1% sucrose.189. A method for isomerizing fructose and glucose, comprising culturing the recombinant microorganism of any one of embodiments 1 to 160 in a production medium comprising sucrose, fructose, and / or glucose, optionally at a mesophilic temperature.190. The method of embodiment 189, wherein the production medium comprises from 0.1 w / v% to 5 w / v% sucrose fructose, and / or glucose.191. The method of embodiment 189 or embodiment 182, wherein the recombinant microorganism is capable of non-phosphorylatively transporting sucrose from the production medium into a cell of the recombinant microorganism.192. The method of any one of embodiments 189 to 191 , wherein the recombinant microorganism is capable of hydrolyzing sucrose to glucose and fructose.193. The method of any one of embodiments 189 to 192, wherein the method further comprises growing the recombinant microorganism in a growth medium comprising a carbon source other than sucrose, fructose, and glucose prior to culturing in the production medium.194. The method of embodiment 193, wherein the growth medium comprises glycerol.195. The method of embodiment 193 or embodiment 194, wherein the growth medium comprises from 0.1 w / v% to 5 w / v% glycerol.196. The method of any one of embodiments 193 to 195, wherein the growth medium lacks added sucrose, fructose, and glucose.197. The method of any one of embodiments 193 to 196, wherein the growth medium comprises no more than 0.1% sucrose, fructose, and / or glucose.198. A method for producing 2-keto-3-deoxygluconate (“KDG”), comprising culturing the recombinant microorganism of any one of embodiments 1 to 160 in a production medium comprising glucose.199. The method of embodiment 198, wherein the production medium comprises from 0.1 w / v% to 5 w / v% glucose.200. The method of embodiment 198 or embodiment 199, wherein the recombinant microorganism is capable of non-phosphorylatively transporting glucose from the production medium into a cell of the recombinant microorganism.201. The method of any one of embodiments 198 to 200, wherein the method further comprises growing the recombinant microorganism in a growth medium comprising a carbon source other than glucose, prior to culturing in the production medium.202. The method of embodiment 201 , wherein the growth medium comprises glycerol.203. The method of embodiment 201 or embodiment 202, wherein the growth medium comprises from 0.1 w / v% to 5 w / v% glycerol.204. The method of any one of embodiments 201 to 203, wherein the growth medium lacks added glucose.205. The method of any one of embodiments 201 to 203, wherein the growth medium comprises no more than 0.1% glucose.206. A recombinant microorganism comprising a nucleic acid or a plurality of nucleic acids encoding means for non-phosphorylatively transporting sucrose, wherein at least one nucleic acid is heterologous to the recombinant microorganism.207. The recombinant microorganism of embodiment 206, wherein the means for non-phosphorylatively transporting sucrose comprise means for passively transporting nonphosphorylated sucrose through a cell membrane of the recombinant microorganism.208. The recombinant microorganism of embodiment 206 or embodiment 207, wherein the means for non-phosphorylatively transporting sucrose comprise means for actively transporting non-phosphorylated sucrose through a cell membrane of the recombinant microorganism.209. A recombinant microorganism, which is optionally the recombinant microorganism of any one of embodiments 206 to 208, comprising a nucleic acid or a plurality of nucleic acids encoding means for hydrolyzing sucrose to glucose and fructose, wherein at least one nucleic acid is heterologous to the recombinant microorganism.210. A recombinant microorganism, which is optionally the recombinant microorganism of any one of embodiments 206 to 209, comprising a nucleic acid or a plurality of nucleic acids encoding means for isomerizing fructose and glucose, optionally at a mesophilic temperature, wherein at least one nucleic acid is heterologous to the recombinant microorganism.211. A recombinant microorganism, which is optionally the recombinant microorganism of any one of embodiments 206 to 210, comprising a nucleic acid or aplurality of nucleic acids encoding means for producing 2-keto-3-deoxygluconic acid (KDG) from glucose, wherein at least one nucleic acid is heterologous to the recombinant microorganism.212. The recombinant microorganism of embodiment 211, wherein the means for producing KDG from glucose comprise means for catalyzing the conversion of glucose to gluconolactone.213. The recombinant microorganism of embodiment 211 or embodiment 212, wherein the means for producing KDG from glucose comprise means for catalyzing the conversion of gluconolactone to gluconate.214. The recombinant microorganism of any one of embodiments 211 to 213, wherein the means for producing KDG from glucose comprise means for catalyzing the conversion of gluconate to KDG,215. The recombinant microorganism of any one of embodiments 206 to 214, wherein the recombinant microorganism lacks means for phosphorylating at least one of sucrose, fructose, or glucose.216. A method of non-phosphorylatively transporting sucrose, comprising: culturing the recombinant microorganism of any one of embodiments 206 to 215, wherein the recombinant microorganism comprises a nucleic acid or a plurality of nucleic acids encoding means for non- phosphorylatively transporting sucrose, in a production medium comprising sucrose.217. A method of isomerizing fructose and glucose, comprising: culturing the recombinant microorganism of any one of embodiments 206 to 215, wherein the recombinant microorganism comprises a nucleic acid or a plurality of nucleic acids encoding means for isomerizing fructose and glucose, in a production medium comprising sucrose, fructose, and / or glucose.218. The method of embodiment 217, wherein the culturing is at a mesophilic temperature.219. A method of producing KDG, comprising: culturing the recombinant microorganism of any one of embodiments206 to 215, wherein the recombinant microorganism comprises anucleic acid or a plurality of nucleic acids encoding means for producing KDG from glucose, in a production medium comprising sucrose or glucose.220. A recombinant microorganism comprising means for non-phosphorylatively transporting sucrose, wherein the means is at least partially heterologous to the recombinant microorganism.221. The recombinant microorganism of embodiment 220, wherein the means for non-phosphorylatively transporting sucrose comprise means for passively transporting nonphosphorylated sucrose through a cell membrane of the recombinant microorganism.222. The recombinant microorganism of embodiment 220 or embodiment 221 , wherein the means for non-phosphorylatively transporting sucrose comprise means for actively transporting non-phosphorylated sucrose through a cell membrane of the recombinant microorganism.223. A recombinant microorganism, which is optionally the recombinant microorganism of any one of embodiments 220 to 222, comprising means for hydrolyzing sucrose to glucose and fructose, wherein the means is at least partially heterologous to the recombinant microorganism.224. A recombinant microorganism, which is optionally the recombinant microorganism of any one of embodiments 220 to 223, comprising means for isomerizing fructose and glucose, optionally at a mesophilic temperature, wherein the means is at least partially heterologous to the recombinant microorganism.225. A recombinant microorganism, which is optionally the recombinant microorganism of any one of embodiments 220 to 224, comprising means for producing 2- keto-3-deoxygluconic acid (KDG) from glucose, wherein the means is at least partially heterologous to the recombinant microorganism.226. The recombinant microorganism of embodiment 225, wherein the means for producing KDG from glucose comprise means for catalyzing the conversion of glucose to gluconolactone.227. The recombinant microorganism of embodiment 225 or embodiment 226, wherein the means for producing KDG from glucose comprise means for catalyzing the conversion of gluconolactone to gluconate.228. The recombinant microorganism of any one of embodiments 225 to 227, wherein the means for producing KDG from glucose comprise means for catalyzing the conversion of gluconate to KDG,229. The recombinant microorganism of any one of embodiments 220 to 228, wherein the recombinant microorganism lacks means for phosphorylating at least one of sucrose, fructose, or glucose.230. A method of non-phosphorylatively transporting sucrose, comprising:(a) culturing the recombinant microorganism of any one of embodiments 220 to 229, wherein the recombinant microorganism comprises means for non-phosphorylatively transporting sucrose, in a production medium comprising sucrose.231. A method of isomerizing fructose and glucose, comprising:(a) culturing the recombinant microorganism of any one of embodiments 220 to 229, wherein the recombinant microorganism comprises means for isomerizing fructose and glucose, in a production medium comprising sucrose, fructose, and / or glucose.232. The method of embodiment 231 , wherein the culturing is at a mesophilic temperature.233. A method of producing KDG, comprising: culturing the recombinant microorganism of any one of embodiments 220 to 229, wherein the recombinant microorganism comprises means for producing KDG from glucose, in a production medium comprising sucrose or glucose.

[0243] Various aspects of the present disclosure are described in the embodiments set forth in the following numbered paragraphs of Group 2.

[0244] Group 2:1. A recombinant microorganism configured to non-phosphorylatively transport sucrose; hydrolyze sucrose to glucose and fructose; isomerize fructose and glucose, optionally at a mesophilic temperature; produce 2-keto-3-deoxygluconic acid (KDG) from glucose; or any two, any three, or all four thereof.2. A recombinant microorganism, which is optionally the recombinant microorganism of embodiment 1 , comprising one or more nucleic acids comprising:(a) a nucleotide sequence encoding a sucrose porin (the “sucrose porin nucleotide sequence”),(b) a nucleotide sequence encoding a sucrose permease (the “sucrose permease nucleotide sequence”), and(c) a nucleotide sequence encoding a sucrose invertase (the “sucrose invertase nucleotide sequence”), wherein at least one of the sucrose porin nucleotide sequence, the sucrose permease nucleotide sequence, and the sucrose invertase nucleotide sequence is heterologous to the microorganism.3. The recombinant microorganism of embodiment 2, wherein the sucrose porin nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:1.4. The recombinant microorganism of embodiment 2 or embodiment 3, wherein the sucrose porin nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:1.5. The recombinant microorganism of any one of embodiments 2 to 4, wherein the sucrose porin nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:1.6. The recombinant microorganism of any one of embodiments 2 to 5, wherein the sucrose permease nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:4.7. The recombinant microorganism of any one of embodiments 2 to 6, wherein the sucrose permease nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:4.8. The recombinant microorganism of any one of embodiments 2 to 7, wherein the sucrose permease nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:4.9. The recombinant microorganism of any one of embodiments 2 to 8, wherein the sucrose invertase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:7.10. The recombinant microorganism of any one of embodiments 2 to 9, wherein the sucrose invertase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:7.11 . The recombinant microorganism of any one of embodiments 2 to 10, wherein the sucrose invertase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:7.12. A recombinant microorganism, which is optionally the recombinant microorganism of any one of embodiments 1 to 11 , comprising one or more nucleic acids comprising:(a) a nucleotide sequence encoding a glucose dehydrogenase (the “glucose dehydrogenase nucleotide sequence”), and(b) a nucleotide sequence encoding a gluconate dehydratase (the “gluconate dehydratase nucleotide sequence”), wherein at least one of the glucose dehydrogenase nucleotide sequence and the gluconate dehydratase nucleotide sequence is heterologous to the microorganism.13. The recombinant microorganism of embodiment 12, wherein the glucose dehydrogenase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to any one of SEQ ID NO: 10- 13.14. The recombinant microorganism of embodiment 12 or embodiment 13, wherein the glucose dehydrogenase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to any one of SEQ ID NQ:10-13.15. The recombinant microorganism of any one of embodiments 12 to 14, wherein the glucose dehydrogenase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to any one of SEQ ID NQ:10-13.16. The recombinant microorganism of any one of embodiments 12 to 15, wherein the gluconate dehydratase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 18.17. The recombinant microorganism of any one of embodiments 12 to 16, wherein the gluconate dehydratase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 18.18. The recombinant microorganism of any one of embodiments 12 to 17, wherein the gluconate dehydratase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 18.19. A recombinant microorganism, which is optionally the recombinant microorganism of any one of embodiments 1 to 18, comprising one or more nucleic acids comprising a nucleotide sequence encoding a gluconolactonase (the “gluconolactonase nucleotide sequence”).20. The recombinant microorganism of embodiment 19, wherein the gluconolactonase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to any one of SEQ ID NO: 14- 17.21 . The recombinant microorganism of embodiment 19 or embodiment 20, wherein the gluconolactonase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to any one of SEQ ID NO:14-17.22. The recombinant microorganism of any one of embodiments 19 to 21 , wherein the gluconolactonase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to any one of SEQ ID NO:14-17.23. The recombinant microorganism of any one of embodiments 1 to 22, which further has reduced glucokinase activity relative to a parental microorganism.24. The recombinant microorganism of any one of embodiments 1 to 23, which further has reduced fructokinase activity relative to a parental microorganism.25. The recombinant microorganism of any one of embodiments 1 to 24, which further has reduced glucose phosphotransferase (PTS) activity relative to a parental microorganism.26. The recombinant microorganism of any one of embodiments 1 to 25, which further has reduced fructose PTS activity relative to a parental microorganism.27. The recombinant microorganism of any one of embodiments 1 to 26, which further has reduced mannose PTS activity relative to a parental microorganism.28. The recombinant microorganism of any one of embodiments 1 to 27, which further has reduced gluconate kinase activity relative to a parental microorganism.29. The recombinant microorganism, which is optionally the recombinant microorganism of any one of embodiments 1 to 28, comprising one or more nucleic acids comprising:a nucleotide sequence encoding a fructose isomerase (the “fructose isomerase nucleotide sequence”).30. The recombinant microorganism of embodiment 29, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to any one of SEQ ID NO:21-44.31 . The recombinant microorganism of embodiment 29 or embodiment 30, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to any one of SEQ ID NO: 21-44.32. The recombinant microorganism of any one of embodiments 29 to 31 , wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to any one of SEQ ID NO: 21-44.33. The recombinant microorganism of any one of embodiments 1 to 32, wherein the recombinant microorganism is an E. coli, a Bacillus subtilis, a Pseudomonas putida, a Klebsiella oxytoca, a Pantoea ananatis, a Tatumella citrea, a Zymomonas mobilis, or a Corynebacterium glutamicum.34. The recombinant microorganism of embodiment 33, wherein the recombinant microorganism is an E. coli.35. The recombinant microorganism of embodiment 34, wherein the E. coli is of strain K12.36. A method for producing 2-keto-3-deoxygluconate (“KDG”), comprising culturing the recombinant microorganism of any one of embodiments 1 to 35 in a production medium comprising sucrose.37. The method of embodiment 36, wherein the recombinant microorganism is capable of non-phosphorylatively transporting sucrose from the production medium into a cell of the recombinant microorganism.38. The method of embodiment 36 or embodiment 37, wherein the method further comprises growing the recombinant microorganism in a growth medium comprising a carbon source other than sucrose, prior to culturing in the production medium.39. The method of embodiment 38, wherein the growth medium comprises glycerol.40. The method of embodiment 38 or embodiment 39, wherein the growth medium lacks added sucrose.41 . The method of embodiment 38 or embodiment 39, wherein the growth medium comprises no more than 0.1% sucrose.42. A recombinant microorganism configured to non-phosphorylatively transport sucrose; and hydrolyze sucrose to glucose and fructose.43. A recombinant microorganism, which is optionally the recombinant microorganism of embodiment 42, comprising one or more nucleic acids comprising:(a) a nucleotide sequence encoding a sucrose porin (the “sucrose porin nucleotide sequence”),(b) a nucleotide sequence encoding a sucrose permease (the “sucrose permease nucleotide sequence”), and(c) a nucleotide sequence encoding a sucrose invertase (the “sucrose invertase nucleotide sequence”), wherein at least one of the sucrose porin nucleotide sequence, the sucrose permease nucleotide sequence, and the sucrose invertase nucleotide sequence is heterologous to the microorganism.44. The recombinant microorganism of embodiment 43, wherein the sucrose porin nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:1.45. The recombinant microorganism of embodiment 43 or embodiment 44, wherein the sucrose porin nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:1 .46. The recombinant microorganism of any one of embodiments 43 to 45, wherein the sucrose porin nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:1 .47. The recombinant microorganism of any one of embodiments 43 to 46, wherein the sucrose permease nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:4.48. The recombinant microorganism of any one of embodiments 43 to 47, wherein the sucrose permease nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:4.49. The recombinant microorganism of any one of embodiments 43 to 48, wherein the sucrose permease nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:4.50. The recombinant microorganism of any one of embodiments 43 to 49, wherein the sucrose invertase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:7.51 . The recombinant microorganism of any one of embodiments 43 to 50, wherein the sucrose invertase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:7.52. The recombinant microorganism of any one of embodiments 43 to 51 , wherein the sucrose invertase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:7.53. A recombinant microorganism configured to isomerize fructose and glucose, optionally at a mesophilic temperature.54. The recombinant microorganism, which is optionally the recombinant microorganism of embodiment 53, comprising one or more nucleic acids comprising: a nucleotide sequence encoding a fructose isomerase (the “fructose isomerase nucleotide sequence”).55. The recombinant microorganism of embodiment 54, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to any one of SEQ ID NO:21-44.56. The recombinant microorganism of embodiment 54 or embodiment 55, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to any one of SEQ ID NO: 21-44.57. The recombinant microorganism of any one of embodiments 54 to 56, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to any one of SEQ ID NO: 21-44.58. A recombinant microorganism configured to produce 2-keto-3-deoxygluconic acid (KDG) from glucose.59. A recombinant microorganism, which is optionally the recombinant microorganism of embodiment 58, comprising one or more nucleic acids comprising:(a) a nucleotide sequence encoding a glucose dehydrogenase (the “glucose dehydrogenase nucleotide sequence”), and(b) a nucleotide sequence encoding a gluconate dehydratase (the “gluconate dehydratase nucleotide sequence”), wherein at least one of the glucose dehydrogenase nucleotide sequence and the gluconate dehydratase nucleotide sequence is heterologous to the microorganism.60. The recombinant microorganism of embodiment 59, wherein the glucose dehydrogenase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to any one of SEQ ID NO: 10- 13.61 . The recombinant microorganism of embodiment 59 or embodiment 60, wherein the glucose dehydrogenase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to any one of SEQ ID NO:10-13.62. The recombinant microorganism of any one of embodiments 59 to 61 , wherein the glucose dehydrogenase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to any one of SEQ ID NQ:10-13.63. The recombinant microorganism of any one of embodiments 59 to 62, wherein the gluconate dehydratase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 18.64. The recombinant microorganism of any one of embodiments 59 to 63, wherein the gluconate dehydratase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 18.65. The recombinant microorganism of any one of embodiments 59 to 64, wherein the gluconate dehydratase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 18.66. A recombinant microorganism, which is optionally the recombinant microorganism of any one of embodiments 53 to 65, comprising one or more nucleic acids comprising a nucleotide sequence encoding a gluconolactonase (the “gluconolactonase nucleotide sequence”).67. The recombinant microorganism of embodiment 66, wherein the gluconolactonase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to any one of SEQ ID NO: 14- 17.68. The recombinant microorganism of embodiment 66 or embodiment 67, wherein the gluconolactonase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to any one of SEQ ID NO:14-17.69. The recombinant microorganism of any one of embodiments 66 to 68, wherein the gluconolactonase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to any one of SEQ ID NO:14-17.70. The recombinant microorganism of any one of embodiments 66 to 69, which further has increased fructose isomerase activity at mesophilic temperatures relative to a parental microorganism.71 . A method for producing 2-keto-3-deoxygluconate (“KDG”), comprising culturing the recombinant microorganism of any one of embodiments 53 to 70 in a production medium comprising glucose.72. The method of embodiment 71, wherein the recombinant microorganism is capable of non-phosphorylatively transporting glucose from the production medium into a cell of the recombinant microorganism.73. The method of embodiment 71 or embodiment 72, wherein the method further comprises growing the recombinant microorganism in a growth medium comprising a carbon source other than glucose, prior to culturing in the production medium.74. The method of embodiment 73, wherein the growth medium comprises glycerol.75. The method of embodiment 73 or embodiment 74, wherein the growth medium lacks added glucose.76. The method of embodiment 73 or embodiment 74, wherein the growth medium comprises no more than 0.1% glucose.77. A method for isomerizing fructose and glucose, comprising culturing the recombinant microorganism of any one of embodiments 53 to 57 in a production medium comprising sucrose, fructose, and / or glucose, optionally at a mesophilic temperature.78. The method of embodiment 77, wherein the recombinant microorganism is capable of non-phosphorylatively transporting sucrose from the production medium into a cell of the recombinant microorganism.79. The method of embodiment 77 or embodiment 78, wherein the recombinant microorganism is capable of hydrolyzing sucrose.80. The method of any one of embodiments 77 to 79, wherein the method further comprises growing the recombinant microorganism in a growth medium comprising a carbon source other than glucose, prior to culturing in the production medium.81 . The method of embodiment 80, wherein the growth medium comprises glycerol.82. The method of embodiment 80 or embodiment 81 , wherein the growth medium lacks added sucrose, fructose, and glucose.83. The method of embodiment 80 or embodiment 81 , wherein the growth medium comprises no more than 0.1% sucrose, fructose, and / or glucose.84. A recombinant microorganism configured to non-phosphorylatively transport sucrose; hydrolyze sucrose to glucose and fructose; isomerize fructose and glucose, optionally at a mesophilic temperature; and produce 2-keto-3-deoxygluconic acid (KDG) from glucose.85. A recombinant microorganism, which is optionally the recombinant microorganism of embodiment 84, comprising one or more nucleic acids comprising:(a) a nucleotide sequence encoding a sucrose porin (the “sucrose porin nucleotide sequence”),(b) a nucleotide sequence encoding a sucrose permease (the “sucrose permease nucleotide sequence”),(c) a nucleotide sequence encoding a sucrose invertase (the “sucrose invertase nucleotide sequence”),(d) a nucleotide sequence encoding a glucose dehydrogenase (the “glucose dehydrogenase nucleotide sequence”),(e) a nucleotide sequence encoding a gluconate dehydratase (the “gluconate dehydratase nucleotide sequence”),(f) a nucleotide sequence encoding a fructose isomerase (the “fructose isomerase nucleotide sequence”), and(g) optionally a nucleotide sequence encoding a gluconolactonase (the “gluconolactonase nucleotide sequence. wherein at least one of the sucrose porin nucleotide sequence, the sucrose permease nucleotide sequence, the sucrose invertase nucleotide sequence, the glucose dehydrogenase nucleotide sequence, the gluconate dehydratase nucleotide sequence, the gluconolactonase nucleotide sequence, and the fructose isomerase nucleotide sequence is heterologous to the recombinant microorganism.86. The recombinant microorganism of embodiment 84 or embodiment 85, wherein the recombinant microorganism further has reduced glucokinase activity, reduced fructokinase activity, reduced glucose phosphotransferase (PTS) activity, reduced fructose PTS activity, reduced mannose PTS activity, or reduced gluconate kinase activity relative to a parental microorganism.87. A recombinant microorganism comprising a nucleic acid or a plurality of nucleic acids encoding means for non-phosphorylatively transporting sucrose; means for hydrolyzing sucrose to glucose and fructose; means for isomerizing fructose and glucose; means for producing 2-keto-3-deoxygluconic acid (KDG) from glucose; or any two, any three, or all four thereof, wherein at least one nucleic acid is heterologous to the recombinant microorganism.88. The recombinant microorganism of embodiment 87, wherein the means for non-phosphorylatively transporting sucrose comprise means for passively transporting nonphosphorylated sucrose through a cell membrane of the recombinant microorganism and / or means for actively transporting non-phosphorylated sucrose through a cell membrane of the recombinant microorganism.89. The recombinant microorganism of embodiment 87 or embodiment 88, wherein the means for producing KDG from glucose comprise means for catalyzing the conversion of glucose to gluconolactone and / or means for catalyzing the conversion of gluconate to KDG, and optionally further comprise means for catalyzing the conversion of gluconolactone to gluconate.90. The recombinant microorganism of any one of embodiments 87 to 89, wherein the recombinant microorganism lacks means for phosphorylating at least one of sucrose, fructose, or glucose.91 . A method of non-phosphorylatively transporting sucrose, comprising:culturing the recombinant microorganism of any one of embodiments 87 to 90, wherein the recombinant microorganism comprises a nucleic acid or a plurality of nucleic acids encoding means for non- phosphorylatively transporting sucrose, in a production medium comprising sucrose.92. A method of isomerizing fructose and glucose, comprising: culturing the recombinant microorganism of any one of embodiments 87 to 90, wherein the recombinant microorganism comprises a nucleic acid or a plurality of nucleic acids encoding means for isomerizing fructose and glucose, in a production medium comprising sucrose, fructose, and / or glucose.93. The method of embodiment 92, wherein the culturing is at a mesophilic temperature.94. A method of producing KDG, comprising: culturing the recombinant microorganism of any one of embodiments 87 to 90, wherein the recombinant microorganism comprises a nucleic acid or a plurality of nucleic acids encoding means for producing KDG from glucose, in a production medium comprising sucrose or glucose.95. A recombinant microorganism comprising means for non-phosphorylatively transporting sucrose; means for hydrolyzing sucrose to glucose and fructose; means for isomerizing fructose and glucose, optionally at a mesophilic temperature; means for producing 2-keto-3-deoxygluconic acid (KDG) from glucose; or any two, any three, or all four thereof, wherein at least one means is at least partially heterologous to the recombinant microorganism.96. The recombinant microorganism of embodiment 95, wherein the means for non-phosphorylatively transporting sucrose comprise means for passively transporting nonphosphorylated sucrose through a cell membrane of the recombinant microorganism and / or means for actively transporting non-phosphorylated sucrose through a cell membrane of the recombinant microorganism.97. The recombinant microorganism of embodiment 95 or embodiment 96, wherein the means for producing KDG from glucose comprise means for catalyzing the conversion of glucose to gluconolactone and / or means for catalyzing the conversion ofgluconate to KDG, and optionally further comprise means for catalyzing the conversion of gluconolactone to gluconate.98. The recombinant microorganism of any one of embodiments 95 to 97, wherein the recombinant microorganism lacks means for phosphorylating at least one of sucrose, fructose, or glucose.99. A method of non-phosphorylatively transporting non-phosphorylated sucrose, comprising: culturing the recombinant microorganism of any one of embodiments 95 to 98, wherein the recombinant microorganism comprises a means for non-phosphorylatively transporting non-phosphorylated sucrose, in a production medium comprising sucrose.100. A method of isomerizing fructose and glucose, comprising: culturing the recombinant microorganism of any one of embodiments 95 to 98, wherein the recombinant microorganism comprises means for isomerizing fructose and glucose, in a production medium comprising sucrose, fructose, and / or glucose.101. The method of embodiment 100, wherein the culturing is at a mesophilic temperature.102. A method of producing KDG, comprising: culturing the recombinant microorganism of any one of embodiments 95 to 98, wherein the recombinant microorganism comprises means for producing KDG from glucose, in a production medium comprising sucrose or glucose.6.9. Examples6.9.1. Example 1 : Growth of modified E. coli strains on sucrose

[0245] E. coli strains MG 1655 and SuA6 lack both a native sucrose invertase and a sucrose permease. Accordingly, both strains are incapable of growth in M9 medium containing 1% sucrose. These strains were used to study whether expression of a sucrose porin, a sucrose permease, and a sucrose invertase could enable growth in M9 medium containing 1 % sucrose.

[0246] Plasmid pTrcHis2b-cscA.cscB.scrY was constructed. The plasmid comprises a coding region encoding a protein having 100% identity to SEQ ID NO:7 (E. coil strain W sucrose invertase), a coding region encoding a protein having 100% identity to SEQ ID NO:4 (E. coli strain W sucrose permease), and a coding region encoding a protein having 100% identity to SEQ ID NO:1 (Salmonella thyphimurium sucrose porin) (FIG. 4), operably linked to the pTrc regulatory region. E. coli strains MG 1655 and SuA6 were transformed with the plasmid. Growth of wild type and transformed strains on plates comprising M9 medium containing 1% sucrose was qualitatively assessed as shown in Table 9.

[0247] These results indicate that the expression of a sucrose porin, a sucrose permease, and a sucrose invertase enabled cell growth in a medium containing sucrose.6.9.2. Example 2: Growth on glucose of E. coli SuA7.1 strains expressing GDH

[0248] E. coli strain SuA7.1 was prepared from SuA6 by knocking out the endogenous glucokinase gene (glk) and restoring gluconokinase activity encoded by gntK. As prepared, SuA7.1 is incapable of growth in M9 medium comprising 1% glucose. This strain was used to study whether expression of a glucose dehydrogenase (GDH) could enable growth of SuA7.1 in M9 medium containing 1% glucose.

[0249] First, coding regions encoding protein sequences having 100% identity to SEQ ID NQ:10-13, corresponding to GDH from Priestia megaterium (previously known as Bacillus megaterium), Bacillus pumullis, Bacillus subtilis, and Lysini bacillus sphaericus, respectively, were inserted into pTrcHis2b plasmids. SuA7.1 was transformed with the plasmids or with vector alone as a negative control, and grown in M9 medium containing 1 % glucose. In addition, SuA7.1 with vector alone was grown in medium containing gluconate as a positive control.

[0250] Cell growth was determined from ODeoo after 3 days. Results are shown in FIG. 7. Bm, SEQ ID NO:10; Bp; SEQ ID NO:11; Bs, SEQ ID NO:12; Ls, SEQ ID NO:13; pTrcHis2b, vector only; pTrcHis2b in gluconate, vector only, medium containing gluconate.

[0251] As can be seen in FIG. 7, cells expressing SEQ ID NO: 11 showed a moderately high amount of cell growth. Cells expressing SEQ ID NO:12 showed cell growth comparable to that observed for pTrcHis2b in gluconate.

[0252] In view of these results, SEQ ID NO: 12 was used in subsequent studies of coexpression of a heterologous GDH and a heterologous gluconolactonase.6.9.3. Example 3: Growth on glucose of E. coli SuA7.1 strains expressing GDH and gluconolactonase

[0253] A group of test plasmids was generated from the pTrc plasmid containing a coding region encoding a protein sequence having 100% identity to SEQ ID NO:12 by adding a coding region encoding a protein sequence having 100% identity to each one of SEQ ID NO:14-17.

[0254] SEQ ID NO:14-17 correspond to gluconolactonase (Gnl) from Zymomonas mobilis, 6-phosphogluconolactonase (Pgl) from E. coli K-12, L-alpha-hydroxyglutaric acid gammalactonase (ppgL / Trol) from Pseudomonas putida, and a putative sugar lactone lactonase (YvrE) from Bacillus subtilis 168. The resulting plasmids generally had the structure of pTrcHis2b_GDH+gluconolactonase shown in FIG. 6.

[0255] SuA7.1 was transformed with the plasmids, with vector alone as a negative control, and grown in M9 medium containing 1% glucose. Cell growth was determined from ODeoo after 2 days. Results are shown in FIG. 8. pTrcHis2B, vector alone; GDH, SEQ ID NO:12 alone; GDH_pgl, SEQ ID NO:12 and SEQ ID NO:15; GDH_yvrE, SEQ ID NO:12 and SEQ ID NO:17; GDH_Trol, SEQ ID NO:12 and SEQ ID NO:16; GDH_gnl, SEQ ID NO:12 and SEQ ID NO:14.

[0256] As can be seen in FIG. 8, expression of both SEQ ID NO: 12 and one of SEQ ID NO:14, 15, 16, or 17 provided little improvement in cell growth compared to SEQ ID NO:12 alone.6.9.4. Example 4: Conversion of glucose to KDG by E. coli strains expressing GDH and GAD

[0257] E. coli strain A5_KmR (Apts, AgntK, AidnK, Aglk, AkdgK::KmR) was transformed with two plasmids: 1) a pTrcHis2b plasmid comprising SEQ ID NO:12 as described in Example 2 (pTrc_GDH) and 2) a pLac plasmid comprising a gluconate dehydratase (GAD) coding region having 100% identity to SEQ ID NO: 18 (pLacAchrol). Selection for cells transformed with both plasmids yielded E. coli strain A5_GDH-GAD. Strain A5_GDH-GAD would be expected to grow in a medium comprising glycerol and produce KDG only if the medium comprised glucose.

[0258] Strain A5_GDH-GAD was grown in flasks comprising Hi-Def media under two carbon source conditions: 1) 0.5% glycerol, and 2) 0.5% glycerol + 1% glucose. Each carbon source condition was assessed in triplicate. After 24 hours, the amount (g / L) of gluconate, glucose, and KDG in the flask was measured. The gluconate, glucose and KDG were secreted, the cells were centrifuged down, and the supernatant underwent quantification. The results are shown in Table 10:

[0259] In media essentially comprising only 0.5% glycerol as the carbon source, as expected, no glucose, gluconate, or KDG was produced. When 1% glucose (10 g / L) was added, the cells absorbed from 1.85 g / L to 2.79 g / L glucose from the medium and produced from 1.13 g / L to 1.24 g / L KDG, representing a yield of KDG from absorbed glucose of 41 %- 65%.

[0260] Further, although the A5_GDH-GAD produced gluconate as an intermediate, essentially no gluconate was detected, which indicates GAD activity was sufficiently high that the conversion of gluconate to KDG was not rate-limiting.6.9.5. Example 5: Growth on glucose of additional E. coli SuA7.1 strains expressing GDH

[0261] Additional plasmids comprising coding regions encoding other putative GDH enzymes and / or engineered variants of one or more of enzymes having at least 90% identity to one or more of SEQ ID NO: 10- 13 are transformed into E. coli strain SuA7.1 and the transformed cells are grown in M9 medium comprising 1% glucose. One or more of the other putative GDH enzymes and / or engineered variants yields growth higher than that provided by SEQ ID NO:12 (BsGdh), pTrcHis2b in gluconate, or both.6.9.6. Example 6: Growth on glucose of additional E. coli SuA7.1 strains expressing GDH and gluconolactonase

[0262] Additional plasmids comprising a coding region encoding a coding region encoding a protein sequence having 100% identity to SEQ ID NO: 12 and a coding region for other putative gluconolactonases than those studied in Example 3 and / or engineered versions of enzymes having at least 90% identity to one or more of SEQ ID NO:14-17 are transformed into E. coli strain SuA7.1. Further plasmids are constructed wherein each of one or more of the putative gluconolactonases studied in Example 3 is paired with a GDH found to have higher growth in the study of Example 5, followed by transformation into E. coli strain SuA7.1.

[0263] The transformed E. coli strains SuA7.1 are grown in M9 medium with 1% glucose. Growth of the cells is measured as in Example 3.

[0264] One or more of the strains expressing both a GDH and a gluconolactonase is found to have higher growth than the strains identified as GDH and / or GDH_yvrE in Example 3 and FIG. 8.6.9.7. Example 7: Conversion of glucose to KDG by additional E. coli strains expressing GDH and GAD; or GDH, GAD, and gluconolactonase

[0265] E. coli strain A5_KmR (Apts, AgntK, AidnK, Aglk, AkdgK::KmR) is transformed with one or more plasmids comprising between them a GDH coding region as described in Example 2 or Example 5 and a gluconate dehydratase (GAD) coding region other than that described in Example 4. A subpopulation of the transformed strains is further transformed with a coding region encoding a gluconolactonase described in Example 3 or Example 6.The gluconolactonase coding region may be present on the same plasmid as the GDH coding region, the GAD coding region, both, or neither.

[0266] Cells transformed as described in this Example are grown in flasks in 1) Hi-Def medium comprising 0.5% glycerol as the primary carbon source and 2) Hi-Def medium comprising 0.5% glycerol and 1 % glucose. After 24 hours, production of gluconate, glucose, and KDG are measured as in Example 4.

[0267] One or more of the cells transformed as described in this Example consumes more than 2.79 g / L of glucose from the medium comprising 1% glucose and / or produces more than 1.24 g / L of KDG and / or has a yield of KDG from glucose that is greater than 65%. The one or more cells do so without appreciable gluconate accumulation.6.9.8. Example 8: Fructose Isomerization by Recombinant Microorganisms at Mesophilic Temperature in vivo

[0268] Nucleic acids comprising fructose isomerases (SEQ ID NO:21-44, three of which had site-specific substitutions as indicated in Table 11) were constructed and E. coli SuA6 cells were engineered therewith. The genetic constructs were made in a pTrcHis2b plasmid containing a fructose isomerase coding region. A genetic construct comprising vector only was used as a negative control. Cells were grown in medium containing fructose for 3 days at 37°C. Cell growth was determined from the optical density at 600 nm (OD6oo).

[0269] Table 11 provides the 3-day OD6oo values of E. coli SuA6 cells expressing the fructose isomerase sequences set forth below.

[0270] OD6OO values greater than the negative control indicate that the enzyme has fructose isomerization activity at mesophilic temperatures in vivo. OD6oo values greater than the negative control were found for enzymes having 100% sequence identity to SEQ ID NO:1- 20. This is believed to be the first showing of any enzyme possessing fructose isomerization activity at mesophilic temperatures in vivo.7. INCORPORATION BY REFERENCE

[0271] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes.

[0272] Any discussion of documents, acts, materials, devices, articles or the like that has been included in this specification is solely for the purpose of providing a context for the present disclosure. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed anywhere before the priority date of this application.

Claims

WHAT IS CLAIMED IS:

1. A recombinant microorganism comprising one or more nucleic acids comprising:(a) a nucleotide sequence encoding a glucose dehydrogenase heterologous to the microorganism (the “glucose dehydrogenase nucleotide sequence”); and(b) a nucleotide sequence encoding a gluconate dehydratase heterologous to the microorganism (the “gluconate dehydratase nucleotide sequence”).

2. The recombinant microorganism of claim 1 , wherein the glucose dehydrogenase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 12.

3. The recombinant microorganism of claim 1 or claim 2, wherein the gluconate dehydratase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 18.

4. The recombinant microorganism of any one of claims 1 to 3, further comprising a nucleotide sequence encoding a sucrose invertase heterologous to the microorganism (the “sucrose invertase nucleotide sequence”).

5. The recombinant microorganism of claim 4, wherein the sucrose invertase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:7.

6. The recombinant microorganism of any one of claims 1 to 5, further comprising a nucleotide sequence encoding a gluconolactonase heterologous to the microorganism (the “gluconolactonase nucleotide sequence”).

7. The recombinant microorganism of claim 6, wherein the gluconolactonase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOS:14-17.

8. The recombinant microorganism of any one of claims 1 to 7, further comprising a nucleotide sequence encoding a fructose isomerase heterologous to the microorganism (the “fructose isomerase nucleotide sequence”).

9. The recombinant microorganism of claim 8, wherein the fructose isomerase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:23, optionally wherein the amino acid sequence comprises a W139F substitution and a V186T substitution relative to the amino acid sequence of SEQ ID NO:23.

10. The recombinant microorganism of any one of claims 1 to 9, which further has reduced glucokinase activity, reduced fructokinase activity, reduced gluconate kinase activity, reduced glucose phosphotransferase (PTS) activity, reduced fructose PTS activity, and / or reduced mannose PTS activity relative to a parental microorganism.11 . The recombinant microorganism of any one of claims 1 to 10, further comprising a nucleotide sequence encoding a sucrose porin heterologous to the microorganism (the “sucrose porin nucleotide sequence”).

12. The recombinant microorganism of claim 11 , wherein the sucrose porin nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:1 .

13. The recombinant microorganism of any one of claims 1 to 12, further comprising a nucleotide sequence encoding a sucrose permease heterologous to the microorganism (the “sucrose permease nucleotide sequence”).

14. The recombinant microorganism of claim 13, wherein the sucrose permease nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO:4.

15. The recombinant microorganism of any one of claims 1 to 14, wherein the recombinant microorganism is an E. coli, a Bacillus subtilis, a Pseudomonas putida, a Klebsiella oxytoca, a Pantoea ananatis, a Tatumella citrea, a Zymomonas mobilis, or a Corynebacterium glutamicum.

16. The recombinant microorganism of claim 15, wherein the recombinant microorganism is an E. coli.

17. The recombinant microorganism of claim 16, wherein the E. coli is of strain K12.

18. A method for producing 2-keto-3-deoxygluconate (“KDG”), comprising culturing the recombinant microorganism any one of claims 1 to 10 in a production medium comprising sucrose, fructose, and / or glucose.

19. A method for producing 2-keto-3-deoxygluconate (“KDG”), comprising culturing the recombinant microorganism any one of claims 11 to 17 in a production medium comprising sucrose.

20. The method of claim 19, wherein the method further comprises growing the recombinant microorganism in a growth medium comprising a carbon source other than sucrose prior to culturing in the production medium.21 . The method of claim 20, wherein the growth medium comprises glycerol.

22. The method of claim 20 or claim 21 , wherein the growth medium lacks added sucrose.

23. The method of claim 20 or claim 21 , wherein the growth medium comprises no more than 0.1 % sucrose.

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