Production of 2-KETO-3-deoxygluconate (KDG) from sucrose using engineered fructose isomerases

Abstract

The disclosure relates to recombinant microorganisms configured to express an engineered fructose isomerase and to non-phosphorylatively transport sucrose, hydrolyze sucrose to glucose and fructose, and / or produce 2-keto-3-deoxygluconic acid (KDG) from glucose. The engineered fructose isomerase may be capable of isomerizing fructose and glucose at a mesophilic temperature. The recombinant microorganisms can be used in methods of isomerizing fructose and glucose, producing KDG from glucose, or non-phosphorylatively transporting sucrose, comprising culturing the recombinant microorganisms in a production medium comprising sucrose, fructose, and / or glucose. The culturing can be at mesophilic temperatures.

Description

Docket no. BPC-028WOPRODUCTION OF 2-KETO-3-DEOXYGLUCONATE (KDG) FROM SUCROSE USING ENGINEERED FRUCTOSE ISOMERASES1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of United States provisional application no.63 / 750,173, filed on January 27, 2025, the contents of which are incorporated herein in its entirety.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 January 20, 2026, is named BPC-028WO_SL.xml and is 56,832 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] Glucose isomerases are enzymes that catalyze the reversible isomerization of glucose to fructose. Such enzymes have been purified from microorganisms and used in the production of high fructose corn syrup (HFCS), which typically comprises 55% fructose instead of the 50% fructose found in hydrolyzed corn syrup. Production of HFCS from corn syrup is an in vitro process, and is typically conducted at temperatures of about 60°C or more.

[0005] 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

[0006] The present disclosure applies synthetic biology to engineer recombinant microorganisms to impart the capability of extracellular sucrose uptake, isomerization of fructose and glucose using engineered fructose isomerases, and / or sucrose utilization for the production of KDG within the cell.

[0007] Generally, the overall pathway beginning with extracellular sucrose and resulting in cellular KDG production has four main components. One component of the overall pathway is 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 (e.g., where the microorganism has an outer membrane) and / or a sucrose permease. Examples of microorganisms configured to non-phosphorylatively transport sucrose into the cell are described in Section 6.3.1, and numbered embodiments 1 to 350 and 396 to 405, 410 to 419, and 424 to 602, and their use is described in Section 6.7.3, and numbered embodiments 351 to 398, 406 to 409, and 603 to 608. Examples of sucrose porin amino acid sequences are described in Section 6.4.1. Examples of sucrose permease amino acid sequences are described in Section 6.4.2.

[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, 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 numbered embodiments 2 to 350 and 424 to 602, and their use is described in Section 6.7.4 and numbered embodiments 351 to 398 and 603 to 608.Examples of sucrose invertase amino acid sequences are described in Section 6.4.3.

[0009] 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 and / or a gluconate dehydratase. Examples of microorganisms configured to convert glucose to KDG are described in Section 6.3.4 and numbered embodiments 26 to 350 and 435 to 602, and their use is described in Section 6.7.7 and numbered embodiments 351 to 398 and 603 to 608. Examples of glucose dehydrogenase amino acid sequences are described in Section 6.4.5. Examples of gluconate dehydratase amino acid sequences are described in Section 6.4.7. This component of the pathway may include the spontaneous conversion of gluconolactone to gluconate. This conversion may be catalyzed by gluconolactonase. In some embodiments, arecombinant microorganism of the disclosure is further engineered to express a gluconolactonase. Examples of gluconolactonase amino acid sequences are described in Section 6.4.6.

[0010] 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 an engineered fructose isomerase. Examples of microorganisms configured to isomerize fructose and glucose are described in Section 6.3.3 and numbered embodiments 1 to 350 and 424 to 602, and their use is described in Section 6.7.5 and numbered embodiments 351 to 398 and 603 to 608.Examples of fructose isomerase amino acid sequences are described in Section 6.4.4.

[0011] 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.

[0012] In some embodiments, a parental microorganism may naturally contain the non-phosphorylatively 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 fructose and glucose and beengineered 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.

[0013] 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.

[0014] KDG produced by the microorganisms of the disclosure can subsequently be utilized in chemical or biochemical processes to produce further products, e.g., terpenoids.Examples of these subsequent processes are described in Section 6.7.8.

[0015] 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 numbered embodiments 67 to 72 and 446 to 451.

[0016] Microorganisms of the disclosure can be engineered from parental microorganisms using known engineering techniques. Examples of parental microorganisms are described in Section 6.6. Examples of engineering techniques are described in Section 6.6.1.5. BRIEF DESCRIPTION OF THE FIGURES

[0017] 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).

[0018] 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).

[0019] 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.

[0020] 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.

[0021] 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.

[0022] FIG. 6 shows a schematic of plasmid pTrcHis2b_GDH+gluconolactonase. Bs_GDH, Bacillus subtilis glucose dehydrogenase.

[0023] FIG. 7 shows cell growth (as optical density at 600 nm, OD600) of E. coli SuA7.1 expressing four different heterologous GDH nucleotide sequences in M9 media with 1% glucose in 3 days.

[0024] FIG. 8 shows cell growth (as GD600) 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.

[0025] FIG. 9A schematically represents the effect of fructose on growth of E. coli strain SuA6. As described in Example 9, because E. coli strain SuA6 lacks fructose isomerase activity, it cannot grow on fructose as its carbon source unless a fructose isomerase activity is engineered into it.

[0026] FIG. 9B schematically represents the effect of fructose on growth of E. coli strain SuA6 engineered to have a fructose isomerase activity.

[0027] FIG. 10 shows a schematic of plasmid pTrcHis2b-xylACT. XylA-CT: C. thermosulfurogenes xylose isomerase.

[0028] FIG. 11 graphically depicts the specific activity in vitro of twelve variants of the C. thermosulfurogenes XylA W139FA / 186T double mutant using cell lysate, normalized relative to the activity of the double mutant.6. DETAILED DESCRIPTION6.1. Definitions

[0029] 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.

[0030] 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.

[0031] 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 othermonosaccharides in addition to fructose and glucose can be a fructose isomerase as described herein, provided it catalyzes the isomerization of fructose and glucose. A number of enzymes are known to catalyze isomerization of substrates other than fructose. Many of these enzymes can or potentially could also catalyze isomerization of fructose and are fructose isomerases as the term is used herein. 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). 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). 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 / SGSC1412 / ATCC 700720) (UniProt identifier Q8ZMP7), Archaeoglobus fulgidus (UniProt identifier 028778), Methanosarcina mazei (UniProt identifier Q8PVJ5), and Pyrococcus furiosus (UniProt identifier P83194). Further examples of such enzymes include mannose-6-phosphate isomerase (ManA), also known as phosphomannose isomerase (PMI), which is known to catalyze the interconversion of fructose 6-phosphate and mannose-6-phosphate, an example of which is ManA from E. coli MG 1655 (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).

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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 computerprogram 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 of amino 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.

[0039] 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

[0040] 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.

[0041] 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 recombinantmicroorganism. Those biochemical processes can include production of KDG from glucose, as well as the production of compounds derived from KDG.

[0042] 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 membrane through 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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. The isomerization of fructose and glucose is independent of the cell’s source or sources of these monosaccharides, and the isomerized fructose or glucose can be used in any intracellular process making use of either monosaccharide. Accordingly, the fructose isomerases described in Section 6.4.4 can be included in a variety of intracellular biochemical pathways. For example, fructose and / or glucose can be non-phosphorylatively transported into a cell from media comprising fructose and / or glucose, and subsequently isomerized. For another example, sucrose can be non-phosphorylatively transported into acell from media comprising sucrose. Sucrose can be hydrolyzed to fructose and glucose, and the monosaccharides can subsequently be isomerized. The isomerization of fructose and glucose can provide fructose or glucose for biochemical processes of a recombinant microorganism. Those biochemical processes can include cellular survival and growth. Those biochemical processes can include production of KDG from glucose. In some embodiments, such as is schematically depicted in FIG. 2, fructose can be isomerized to glucose by the activity of a fructose isomerase (reaction 2). Glucose can be converted to gluconolactone by the activity of a glucose dehydrogenase (GDH) (reaction 3).Gluconolactone can spontaneously convert to gluconic acid, or this conversion can be catalyzed by the activity of a gluconolactonase (reaction 4). The activity of a gluconate dehydratase (GAD) can convert gluconic acid to KDG (reaction 5). In some embodiments, the non-phosphorylative transport of sucrose, the hydrolysis of sucrose, the isomerization of fructose and glucose, and the production of KDG from glucose as precursor can be engineered into recombinant microorganisms.

[0048] 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.

[0049] 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 four thereof 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 6.6, by techniques including those described in Section 6.6.1.

[0050] 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 6.7.8.

[0051] Further details of recombinant microorganisms, polypeptides, pathways, methods, and uses of the disclosure are presented below.6.3. Recombinant Microorganisms

[0052] 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.

[0053] 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).

[0054] 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 6.6, and suitable engineering techniques include those described in Section 6.6.1.

[0055] 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. This can 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.

[0056] 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 W (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, Fructose, and / or Glucose

[0057] The non-phosphorylative transport of sucrose, fructose, and / or glucose 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”), a fructose porin (“fructose porin nucleotide sequence”), a fructose permease (“fructose permease nucleotide sequence”), a glucose porin (“glucose porin nucleotide sequence”), a glucose permease (“glucose permease nucleotide sequence”), or any two, three, four, or five, or all six thereof..

[0058] Features of a parental microorganism that can be considered when engineering a recombinant microorganism to gain or improve the function of a porin or 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.

[0059] 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 recombinantmicroorganism comprises outer and inner membranes, to localize both a sucrose porin and a sucrose permease to the same membrane or membranes, etc.

[0060] Of the exemplary parental microorganisms, parental E. coli\< 2 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 or a strain derived therefrom such as or a strain E. coli MG1655, P putida, orZ. mobilis can be engineered to include nucleic acid(s) encoding a sucrose, fructose, and / or glucose porin; nucleic acid(s) encoding a sucrose, fructose, and / or glucose permease, or nucleic acid(s) encoding both.

[0061] 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, fructose, and / or glucose porin.

[0062] Parental B. 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 to localize a sucrose, fructose, and / or glucose porin to its single membrane, to supplement or replace its endogenous phosphorylation-coupled sucrose permease activity with a sucrose, fructose, and / or glucose permease activity that does not phosphorylate sucrose, or both.

[0063] 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, fructose, and / or glucose porin to its inner membrane, to localize a sucrose, fructose, and / or glucose permease to its outer membrane, to operably link either or both of a sucrose, fructose, and / or glucose porin coding region and a sucrose, fructose, and / or glucose permease coding region to a regulatory region with which it is not operably linked in parental K. oxytoca, or two or more thereof.

[0064] 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 nucleic acid(s) encoding a sucrose, fructose, and / or glucose porin, to supplement or replace its endogenous phosphorylation-coupled permease activity with a sucrose, fructose, and / or glucose permease activity that does not phosphorylate sucrose, or both.6.3.2. Recombinant Microorganisms Configured to Hydrolyze Sucrose to Fructose and Glucose

[0065] 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”).

[0066] 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.

[0067] 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.

[0068] Preferably, a recombinant microorganism has intracellular sucrose invertase activity.

[0069] 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.

[0070] Parental E. coli\N 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.

[0071] 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

[0072] 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 nucleotidesequence”). In some embodiments, the fructose isomerase is a heterologous fructose isomerase described in Section 6.4.4.1. In some embodiments, the fructose isomerase is an engineered fructose isomerase described in Section 6.4.4.2.

[0073] 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).

[0074] Turning to the exemplary parental microorganisms, all of them lack fructose isomerase activity. In some embodiments, a recombinant E. coli K12, E. coli\N, 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

[0075] 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”).

[0076] 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.

[0077] Of the exemplary parental microorganisms, parental E. coli K12, 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.

[0078] 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.

[0079] 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.

[0080] 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. coliVM, B. subtilis, P. putida, K. oxytoca, P. ananatis, T. citrea, Z. mobilis, or C. glutamicum can be engineered to include a gluconate dehydratase activity.

[0081] 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\N, K. oxytoca, P. ananatis, T. citrea, Z. mobilis, or C. glutamicum is engineered to include a gluconolactonase activity.

[0082] 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.

[0083] 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).

[0084] 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

[0085] 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 TCA cycle. 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 theEmbden-Meyerhof-Parnas pathway eventually yields 2-keto-3-deoxy-6-phosphogluconate (KDPG), which is not desirable when the production of KDG is intended.

[0086] 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.

[0087] 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 I IB, 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.

[0088] 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, Fructose, or Glucose Porin

[0089] A sucrose, fructose, or glucose porin is a beta barrel protein which, when present in the outer membrane of Gram-negative bacteria, permits passive diffusion of extracellular sucrose, fructose, and / or glucose into the periplasmic space. A porin that permits the passive diffusion of other molecules as well as sucrose, fructose, and / or glucose is a sucrose, fructose, or glucose porin as described herein, provided it permits the diffusion of sucrose, fructose, or glucose, respectively. Given the similarities in size, shape, composition, and hydrophobicity of sucrose, fructose, and glucose, a porin recognized as acting on one of these saccharides may also have activity on the others. For example, a sucrose porin may also permit the passive diffusion of fructose and / or glucose. For another example, a glucoseporin may also permit the passive diffusion of sucrose and / or fructose. For yet another example, a fructose porin may also permit the passive diffusion of sucrose and / or glucose.

[0090] A porin that permits the passive diffusion of extracellular sucrose, fructose, and / or glucose through the single membrane of a Gram-positive bacterium is also a sucrose, fructose, or glucose porin as described herein.

[0091] In aspects, the present disclosure provides a recombinant microorganism engineered to express a sucrose porin. The activity of a sucrose porin 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.

[0092] 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.

[0093] 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. coll 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.

[0094] 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.

[0095] Exemplary sucrose porin amino acid sequences are provided in Table 2:6.4.2. Sucrose, Fructose, or Glucose Permease

[0096] A sucrose, fructose, or glucose permease is a member of major facilitator superfamily (MFS) and is a membrane protein which actively transports sucrose, fructose, and / or glucose across 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 Gram-positive bacteria and other microorganisms. A permease that actively transports other molecules as well as sucrose, fructose, and / or glucose is a sucrose, fructose, or glucose permease as described herein, provided it actively transports sucrose,fructose, or glucose, respectively. Given the similarities in size, shape, composition, and hydrophobicity of sucrose, fructose, and glucose, a permease recognized as acting on one of these saccharides may also have activity on the others. For example, a sucrose permease may also actively transport fructose and / or glucose. For another example, a glucose permease may also actively transport sucrose and / or fructose. For yet another example, a fructose permease may also actively transport sucrose and / or glucose.

[0097] In aspects, the present disclosure provides a recombinant microorganism engineered to express a sucrose permease. The activity of a sucrose permease 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.

[0098] 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.

[0099] A different mechanism to transport sucrose is carried out by sucrose permeases which transport sucrose into the cells without modifying the sucrose molecule.

[0100] 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.

[0101] 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).

[0102] 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 O17:K52:H18 (strain UMN0261 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).

[0103] Known sucrose permeases typically comprise twelve transmembrane helices formed by N and C terminal domains connected by a cytoplasmic loop. Accordingly, in some embodiments, 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.

[0104] The replacement of native Cys residues of E. coll 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.

[0105] Exemplary sucrose permease amino acid sequences are provided in Table 3:6.4.3. Sucrose Invertase

[0106] 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.

[0107] In some embodiments, a sucrose invertase of the present disclosure has an activity identified by EC number 3.2.1.26.

[0108] 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% sequence identity, 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 I Classical Ogawa 395 / 0395) sucrose invertase (cscA) having the UniProt identifier A5EZZ8 (SEQ ID NO:8), orStaphylococcus aureus (strain COL) sucrose invertase (cscA) having the UniProt identifier A0A0H2WWU4 (SEQ ID NO:9).

[0109] 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.

[0110] Exemplary sucrose invertase amino acid sequences are provided in Table 4:6.4.4. Fructose Isomerase

[0111] 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.

[0112] 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.

[0113] 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 identifiedby EC number 5.3.1.5. In some embodiments, a fructose isomerase of the present disclosure is heterologous to a parental microorganism. In some embodiments, the fructose isomerase is an engineered fructose isomerase having one or more amino acid substitutions relative to a wild-type version.6.4.4.1. Heterologous Fructose Isomerases

[0114] Examples of fructose isomerases of the present disclosure include xylose isomerases (which are 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), S. 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).

[0115] 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).

[0116] 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 MG 1655 (UniProt identifier P0A6T1), Salmonella enterica serovar typhimurium (strain LT2 / SGSC1412 / ATCC 700720) (UniProt identifier Q8ZMP7), Archaeoglobus fulgidus (UniProt identifier 028778), Methanosarcina mazei (UniProt identifier Q8PVJ5), and Pyrococcus furiosus (UniProt identifier P83194).

[0117] Further examples of such enzymes include mannose-6-phosphate isomerase (ManA), also known as phosphomannose isomerase (PMI), which is known to catalyze the interconversion of fructose 6-phosphate and mannose-6-phosphate, an example of which is ManA 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 MG 1655 (UniProt identifier Q46938).

[0118] 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.

[0119] 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 olivochromogenes 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) putative glucose-6-phosphate isomerase (pgiA2) having UniProt identifier Q92UI1 (SEQ ID NO:33), E. coli MG 1655 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 Q75WH8 (SEQ ID NO:35), E. coli MG1655 glucose-6-phosphate isomerase (PGI) having UniProt identifier P0A6T 1 (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 MG16555-dehydro-4-deoxy-glucuronate isomerase (Kdul) having UniProt identifier Q46938 (SEQ ID NO:40), Archaeoglobus fulgidus glucose-6-phosphate isomerase (PG I) having UniProt identifier 028778 (SEQ ID N0:41), 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).

[0120] 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.

[0121] In some embodiments, a fructose isomerase of the present disclosure comprises the amino acid sequence of SEQ ID NO:21.

[0122] The active site of E. coll 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.

[0123] In some embodiments, a fructose isomerase of the present disclosure comprises the amino acid sequence of SEQ ID NO:22.

[0124] 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 ID NO:23 and / or is identical to SEQ ID NO:23 at positions 101 , 104, 232, 268, 271, 296, 307, 309, and 339.

[0125] 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%, atleast 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.

[0126] In some embodiments, a fructose isomerase of the present disclosure comprises the amino acid sequence of SEQ ID NO:24.

[0127] The active site of Bacillus licheniformis xy\ / 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.

[0128] In some embodiments, a fructose isomerase of the present disclosure comprises the amino acid sequence of SEQ ID NO:25.

[0129] 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.

[0130] In some embodiments, a fructose isomerase of the present disclosure comprises the amino acid sequence of SEQ ID NO:26.

[0131] 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.

[0132] In some embodiments, a fructose isomerase of the present disclosure comprises the amino acid sequence of SEQ ID NO:27.

[0133] 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.

[0134] In some embodiments, a fructose isomerase of the present disclosure comprises the amino acid sequence of SEQ ID NO:28.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] In some embodiments, a fructose isomerase of the present disclosure comprises the amino acid sequence of SEQ ID NO:32.

[0141] 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 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:33 or SEQ ID NO:38 and is identical to SEQ ID NO:33 orSEQ ID NO:38 at positions 92, 94, 101, and 140.

[0142] 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.The binding site of E. coli manAforZn2+ (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 forZn2+ (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 fructoseisomerase 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 I 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 5:6.4.4.2. Engineered Fructose Isomerases

[0153] In one aspect, the present disclosure provides engineered fructose isomerases.

[0154] In some embodiments, engineered fructose isomerases of the present disclosure can isomerize fructose to glucose in vivo at mesophilic temperatures. Mesophilic temperatures are 20°C-45°C and includes 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.

[0155] In some embodiments, an engineered 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.

[0156] In some embodiments, when engineered fructose isomerases are heterologous to and are expressed by recombinant organisms that otherwise lack the ability to metabolize fructose, a recombinant organism expressing an engineered fructose isomerase has greater growth than a non-expressing 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.

[0157] In some embodiments, engineered fructose isomerases of the present disclosure comprises an amino acid sequence having at least one amino acid substitution relative to a corresponding fructose isomerase wild-type sequence and at least 90% sequence identity, such as at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% 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 UniProtidentifier 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 olivochromogenes 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) putative glucose-6-phosphate isomerase (pgiA2) having UniProt identifier Q92UI1 (SEQ ID NO:33), E. coll MG 1655 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 Q75WH8 (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 MG16555-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).

[0158] In some embodiments, fructose isomerases of the present disclosure differ from any one of SEQ ID NO:21-44 by (a) at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acid substitutions, deletions, or insertions and / or (b) up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, or up to 20 substitutions, deletions, or insertions. For example, the fructose isomerase can comprise an amino acid sequence having 3-10, 3-12, 3-15, 3-20, 4-10, 4-12, 4-15, or 4-20 amino acid substitutions as compared to the amino acid sequence of any one of SEQ ID NOS:21-44.ln particular embodiments, fructose isomerases can comprise an amino acid sequence having at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:23, with particularamino acid substitutions engineered in view of the information set forth below. Optionally, the amino acid sequences of the fructose isomerases can comprise at least one, at least two, or at least three of the amino acid substitutions N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, W139F, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, V186T, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, and R424K as compared to the amino acid sequence of SEQ ID NO:23. In some embodiments, the amino acid substitutions do not consist of W139F and V186T.

[0159] 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. The substitutions W139F and V186T enhance activity. Unexpectedly, and as described in Example 1, the inventors discovered that adding to or modifying the W139F / V186T double mutant with one or more of the substitutions N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, or R424K further enhanced activity over the W139F / V186T double mutant.

[0160] 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises both the substitutions W139F and / or V186T and optionally one or more substitutions N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, or R424K as compared to the amino acid sequence of SEQ ID NO:23.

[0161] In some embodiments, polypeptides of the present disclosure comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:23 and one or more of the amino acid substitutions:a) N19E or N19T;b) Y22F;c) A47S;d) Q59M;e) C99S orC99T;f) D128E;g) T141C orT141S;h) A142N, A142S, orA142T;i) L144C, L144K, or L144M;j) F150Y;k) S155A;l) V186C;m) F187L, F187Q, F187W, orF187Y;n) E203Q;o) N249H or N249S;p) F276Q;q) T299E;r) F363Y;s) K410E;t) R415K;u) Q417E or Q417K; andv) R424K;as compared to the amino acid sequence of SEQ ID NO:23.

[0162] In some embodiments, polypeptides of the present disclosure comprise at least two of the substitutions (a)-(v).

[0163] In some embodiments, polypeptides of the present disclosure comprise at least three of the substitutions (a)-(v).

[0164] In some embodiments, polypeptides of the present disclosure comprise at least four of the substitutions (a)-(v).

[0165] In some embodiments, polypeptides of the present disclosure comprise at least five of the substitutions (a)-(v).

[0166] In some embodiments, polypeptides of the present disclosure comprise up to six of the substitutions (a)-(v).

[0167] In some embodiments, polypeptides of the present disclosure comprise up to seven of the substitutions (a)-(v).

[0168] In some embodiments, polypeptides of the present disclosure comprise up to eight of the substitutions (a)-(v).

[0169] In some embodiments, polypeptides of the present disclosure comprise up to nine of the substitutions (a)-(v).

[0170] In some embodiments, polypeptides of the present disclosure comprise up to ten of the substitutions (a)-(v).

[0171] In some embodiments, polypeptides of the present disclosure further comprise one or both of the amino acid substitutions W139F and V186T.

[0172] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, A142N, and V186T as compared to the amino acid sequence of SEQ ID NO:23.

[0173] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, A142S, and V186T as compared to the amino acid sequence of SEQ ID NO:23.

[0174] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions A47S, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:23.

[0175] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions C99S, W139F, T141S, A142S, and V186C as compared to the amino acid sequence of SEQ ID NO:23.

[0176] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions C99S, W139F, T141S, A142T, and V186C as compared to the amino acid sequence of SEQ ID NO:23.

[0177] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions C99T, W139F, T141 S, A142S, V186T, and F 187W as compared to the amino acid sequence of SEQ ID NO:23.

[0178] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions C99T, W139F, T141 S, A142T, V186T, and F 187W as compared to the amino acid sequence of SEQ ID NO:23.

[0179] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions C99T, W139F, L144M, and V186C as compared to the amino acid sequence of SEQ ID NO:23.

[0180] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions C99T, W139F, and V186C as compared to the amino acid sequence of SEQ ID NO:23.

[0181] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions C99T, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:23.

[0182] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions D128E, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:23.

[0183] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and E203Q as compared to the amino acid sequence of SEQ ID NO:23.

[0184] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, F150Y, and V186T as compared to the amino acid sequence of SEQ ID NO:23.

[0185] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and F187L as compared to the amino acid sequence of SEQ ID NO:23.

[0186] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and F187Q as compared to the amino acid sequence of SEQ ID NO:23.

[0187] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and F187W as compared to the amino acid sequence of SEQ ID NO:23.

[0188] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and F187Y as compared to the amino acid sequence of SEQ ID NO:23.

[0189] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and F276Q as compared to the amino acid sequence of SEQ ID NO:23.

[0190] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and F363Y as compared to the amino acid sequence of SEQ ID NO:23.

[0191] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and K410E as compared to the amino acid sequence of SEQ ID NO:23.

[0192] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, L144C, and V186T as compared to the amino acid sequence of SEQ ID NO:23.

[0193] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, L144K, and V186T as compared to the amino acid sequence of SEQ ID NO:23.

[0194] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, L144M, and V186T as compared to the amino acid sequence of SEQ ID NO:23.

[0195] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions N19E, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:23.

[0196] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions N19T, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:23.

[0197] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and N249H as compared to the amino acid sequence of SEQ ID NO:23.

[0198] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and N249S as compared to the amino acid sequence of SEQ ID NO:23.

[0199] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and Q417E as compared to the amino acid sequence of SEQ ID NO:23.

[0200] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and Q417K as compared to the amino acid sequence of SEQ ID NO:23.

[0201] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions Q59M, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:23.

[0202] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and R415K as compared to the amino acid sequence of SEQ ID NO:23.

[0203] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and R424K as compared to the amino acid sequence of SEQ ID NO:23.

[0204] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, S155A, and V186T as compared to the amino acid sequence of SEQ ID NO:23.

[0205] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, T141C, and V186T as compared to the amino acid sequence of SEQ ID NO:23.

[0206] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, T141S, A142T, and V186T as compared to the amino acid sequence of SEQ ID NO:23.

[0207] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, T141S, L144M, V186T, and F187W as compared to the amino acid sequence of SEQ ID NO:23.

[0208] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, T141S, and V186T as compared to the amino acid sequence of SEQ ID NO:23.

[0209] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F and V186C as compared to the amino acid sequence of SEQ ID NO:23.

[0210] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions W139F, V186T, and T299E as compared to the amino acid sequence of SEQ ID NO:23.

[0211] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions Y22F, W139F, and V186T as compared to the amino acid sequence of SEQ ID NO:23.- M -

[0212] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions Q59M, W139F, V186T, N249H, and R424K as compared to the amino acid sequence of SEQ ID NO:23.

[0213] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions Q59M, W139F, V186T, T299E, and R424K as compared to the amino acid sequence of SEQ ID NO:23.

[0214] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions D128E, W139F, F150Y, V186T, and F363Y as compared to the amino acid sequence of SEQ ID NO:23.

[0215] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions Q59M, W139F, V186T, N249H, and F363Y as compared to the amino acid sequence of SEQ ID NO:23.

[0216] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions N19E, D128E, W139F, V186T, F276Q, and R415K as compared to the amino acid sequence of SEQ ID NO:23.

[0217] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions N19E, A47S, D128E, W139F, V186T, and F276Q as compared to the amino acid sequence of SEQ ID NO:23.

[0218] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions N19E, Q59M, W139F, V186T, N249H, and R424K as compared to the amino acid sequence of SEQ ID NO:23.

[0219] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions A47S, W139F, V186T, N249H, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:23.

[0220] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions Q59M, W139F, V186T, N249H, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:23.

[0221] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions A47S, W139F, V186T, T299E, F363Y, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:23.

[0222] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions D128E, W139F, V186T, N249H, F363Y, F276Q, and K410E as compared to the amino acid sequence of SEQ ID NO:23.

[0223] In some embodiments, polypeptides of the present disclosure comprise amino acid sequences comprising the substitutions Q59M, D128E, W139F, V186T, N249H, F276Q, and R424K as compared to the amino acid sequence of SEQ ID NO:23.

[0224] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and A142N. as compared to the amino acid sequence of SEQ ID NO:23.

[0225] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and A142S as compared to the amino acid sequence of SEQ ID NO:23.

[0226] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and A47S as compared to the amino acid sequence of SEQ ID NO:23.

[0227] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, C99S, T141S, A142S, and V186C as compared to the amino acid sequence of SEQ ID NO:23.

[0228] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, C99S, T141 S, A142T, and V186C as compared to the amino acid sequence of SEQ ID NO:23.

[0229] In particular 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%, atleast 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, C99T, T141S, A142S, and F187W as compared to the amino acid sequence of SEQ ID NO:23.

[0230] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, C99T, T141S, A142T, and F187W as compared to the amino acid sequence of SEQ ID NO:23.

[0231] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, C99T, L144M, and V186C as compared to the amino acid sequence of SEQ ID NO:23.

[0232] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, C99T, and V186C as compared to the amino acid sequence of SEQ ID NO:23.

[0233] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and C99T as compared to the amino acid sequence of SEQ ID NO:23.

[0234] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and D128E as compared to the amino acid sequence of SEQ ID NO:23.

[0235] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and E203Q as compared to the amino acid sequence of SEQ ID NO:23.

[0236] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and F150Y as compared to the amino acid sequence of SEQ ID NO:23.

[0237] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and F187L as compared to the amino acid sequence of SEQ ID NO:23.

[0238] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and F187Q as compared to the amino acid sequence of SEQ ID NO:23.

[0239] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and F187W as compared to the amino acid sequence of SEQ ID NO:23.

[0240] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and F187Y.

[0241] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and F276Q as compared to the amino acid sequence of SEQ ID NO:23.

[0242] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and F363Y as compared to the amino acid sequence of SEQ ID NO:23.

[0243] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and K410E as compared to the amino acid sequence of SEQ ID NO:23.

[0244] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and L144C as compared to the amino acid sequence of SEQ ID NO:23.

[0245] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and L144K as compared to the amino acid sequence of SEQ ID NO:23.

[0246] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprisesthe substitutions W139F, V186T, and L144M as compared to the amino acid sequence of SEQ ID NO:23.

[0247] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and N19E as compared to the amino acid sequence of SEQ ID NO:23.

[0248] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and N19T as compared to the amino acid sequence of SEQ ID NO:23.

[0249] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and N249H as compared to the amino acid sequence of SEQ ID NO:23.

[0250] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and N249S as compared to the amino acid sequence of SEQ ID NO:23.

[0251] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and Q417E as compared to the amino acid sequence of SEQ ID NO:23.

[0252] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical toSEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and Q417K as compared to the amino acid sequence of SEQ ID NO:23.

[0253] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and Q59M.

[0254] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and R415K as compared to the amino acid sequence of SEQ ID NO:23.

[0255] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and R424K as compared to the amino acid sequence of SEQ ID NO:23.

[0256] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and S155A as compared to the amino acid sequence of SEQ ID NO:23.

[0257] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and T141C as compared to the amino acid sequence of SEQ ID NO:23.

[0258] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical toSEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, T141S, and A142T as compared to the amino acid sequence of SEQ ID NO:23.

[0259] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, T141S, L144M, and F187W as compared to the amino acid sequence of SEQ ID NO:23.

[0260] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and T141S as compared to the amino acid sequence of SEQ ID NO:23.

[0261] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F and V186C as compared to the amino acid sequence of SEQ ID NO:23.

[0262] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and T299E as compared to the amino acid sequence of SEQ ID NO:23.

[0263] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, and Y22F as compared to the amino acid sequence of SEQ ID NO:23.

[0264] In particular 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%, atleast 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, Q59M, N249H, and R424K as compared to the amino acid sequence of SEQ ID NO:23.

[0265] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, Q59M, T299E, and R424K as compared to the amino acid sequence of SEQ ID NO:23.

[0266] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, D128E, F150Y, and F363Y as compared to the amino acid sequence of SEQ ID NO:23.

[0267] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, Q59M, N249H, and F363Y as compared to the amino acid sequence of SEQ ID NO:23.

[0268] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, N19E, D128E, F276Q, and R415K as compared to the amino acid sequence of SEQ ID NO:23.

[0269] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, N19E, A47S, D128E, and F276Q as compared to the amino acid sequence of SEQ ID NO:23.

[0270] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, N19E, Q59M, N249H, and R424K as compared to the amino acid sequence of SEQ ID NO:23.

[0271] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, A47S, N249H, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:23.

[0272] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, Q59M, N249H, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:23.

[0273] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, A47S, T299E, F363Y, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:23.

[0274] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprises the substitutions W139F, V186T, D128E, N249H, F363Y, F276Q, and K410E as compared to the amino acid sequence of SEQ ID NO:23.

[0275] In particular 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%, or at least 99% sequence identity to SEQ ID NO:23; is identical to SEQ ID NO:23 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339; and comprisesthe substitutions W139F, V186T, Q59M, D128E, N249H, F276Q, and R424K as compared to the amino acid sequence of SEQ ID NO:23.

[0276] 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, an engineered Thermotoga neapolitana xylA comprises one, two, or three amino acid substitutions V186T, L283P, and / or F187S as compared to the amino acid sequence of SEQ ID NO:29. In some embodiments, an engineered Thermotoga neapolitana xylA 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%, or at least 99% sequence identity to SEQ ID NO:29. In some embodiments, an engineered Thermotoga neapolitana xylA is identical (i.e., does not have a substitution relative) to SEQ ID NO:29 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339 of SEQ ID NO:29. In some particular embodiments, an engineered Thermotoga neapolitana xylA (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:29; (b) is identical (i.e., does not have a substitution relative) to SEQ ID NO:29 at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339, and / or (c) comprises the amino acid substitutions V186T, L283P, and F187S as compared to SEQ ID NO:29.

[0277] Alignment of the primary amino acid sequences or the tertiary structures of xylAs (e.g., SEQ ID NQ:21-30, 32, or 44) can identify residues of any xylA which are aligned with residues of SEQ ID NO:23 and / or SEQ ID NO:29 where the substitutions increased fructose isomerase activity.

[0278] In some embodiments, the aligned residues of any xylA can thus be substituted with the same amino acids of the substitutions of SEQ ID NO:23 and / or SEQ ID NO:29 set forth in particular embodiments above.

[0279] The active site of Actinoplanes missouriensis xy\A 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, an engineered Actinoplanes missouriensis xylA (a) comprises an amino acid sequence having at least 90% (e g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:21 and (b) is identical (i.e., does not have a substitution relative) to SEQ ID NO:21 at positions 54, 57, 181, 217, 220, 245, 255, 257, and 292. In further embodiments, a fructose isomerase according to this paragraph can further comprise one or more the substitutions at positions aligned with W139F, V186T, N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y,S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K41 OE, R415K, Q417E, Q417K, or R424K of SEQ ID NO:23 or V186T, L283P, or F187S of SEQ ID NO:29.

[0280] The active site of E. co / / 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, an engineered E. coli xylA (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:22 and (b is identical (i.e., does not have a substitution relative) to SEQ ID NO:22 at positions 101 , 104, 232, 268, 271, 296, 307, 309, and 339. In further embodiments, a fructose isomerase according to this paragraph can further comprise one or more the substitutions at positions aligned with W139F, V186T, N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, or R424K of SEQ ID NO:23 orV186T, L283P, or F187S of SEQ ID NO:29.

[0281] 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, an engineered Anoxybacillus kamchatkensis xylA (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:24 and (b) is identical (i.e., does not have a substitution relative) to SEQ ID NO:24 at positions 99, 102, 230, 266, 269, 294, 305, 307, and 337. In further embodiments, a fructose isomerase according to this paragraph can further comprise one or more the substitutions at positions aligned with W139F, V186T, N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, or R424K of SEQ ID NO:23 or V186T, L283P, or F187S of SEQ ID NO:29.

[0282] 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, an engineered Bacillus licheniformis xylA (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:25 and (b) is identical (i.e., does not have a substitution relative) to SEQ ID NO:25 at positions 99, 102, 230, 266, 269, 294, 305, 307, and 337. In further embodiments, a fructose isomerase according to this paragraph can further comprise one or more the substitutions at positionsaligned with W139F, V186T, N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, or R424K of SEQ ID NO:23 or V186T, L283P, or F187S of SEQ ID NO:29.

[0283] 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, an engineered Bacillus coagulans xylA (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:26 and (b) is identical (i.e., does not have a substitution relative) to SEQ ID NO:26 at positions 100, 103, 231, 267, 270, 295, 306, 308, and 338. In further embodiments, a fructose isomerase according to this paragraph can further comprise one or more the substitutions at positions aligned with W139F, V186T, N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, or R424K of SEQ ID NO:23 or V186T, L283P, or F187S of SEQ ID NO:29.

[0284] 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, an engineered Streptomyces rubiginosus xylA (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:27 and (b is identical (i.e., does not have a substitution relative) to SEQ ID NO:27 at positions 54, 57, 181, 217, 220, 245, 255, 257, and 287. In further embodiments, a fructose isomerase according to this paragraph can further comprise one or more the substitutions at positions aligned with W139F, V186T, N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, or R424K of SEQ ID NO:23 or V186T, L283P, or F187S of SEQ ID NO:29.

[0285] 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, an engineered Streptomyces olivochromogenes xylA (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:28 and (b) is identical (i.e., does not have a substitution relative) to SEQ ID NO:28 at positions 54, 57, 181, 217, 220, 245, 255, 257, and 287. In furtherembodiments, a fructose isomerase according to this paragraph can further comprise one or more the substitutions at positions aligned with W139F, V186T, N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, or R424K of SEQ ID NO:23 or V186T, L283P, or F187S of SEQ ID NO:29.

[0286] 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, an engineered l-rhamnose isomerase of Pseudomonas stutzeri (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:31 or SEQ ID NO:35 and (b) is identical (i.e., does not have a substitution relative) to SEQ ID NO:31 or SEQ ID NO:35 at positions 219, 254, 257, 281 , 289, 291 , 298, and 327.

[0287] 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, an engineered Arthrobacter sp. (strain NRRL B3728) xylA (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:32 and (b) is identical (i.e., does not have a substitution relative) to SEQ ID NO:32 at positions 54, 57, 181, 217, 220, 245, 255, 257, and 293. In further embodiments, a fructose isomerase according to this paragraph can further comprise one or more the substitutions at positions aligned with W139F, V186T, N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, or R424K of SEQ ID NO:23 or V186T, L283P, or F187S of SEQ ID NO:29.

[0288] The binding site of Pyrococcus furiosus PGI for Fe cation (cofactor) comprises residues 88, 90, 97, and 146. The substitution T85Q may impart higher fructose isomerase activity. In some embodiments, an engineered Pyrococcus furiosus PGI (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:43; (b) is identical (i.e., does not have a substitution relative) to SEQ ID NO:43 at positions 88, 90, 97, and 146; and / or comprises the substitution T85Q relative to SEQ ID NO:43.

[0289] Alignment of the primary amino acid sequences or the tertiary structures of PGIs (e.g., SEQ ID NO:33, 36, 38, 39, 41 , 42, or 43) can identify a residue of any PGI which is aligned with position 85 of SEQ ID NO:43.

[0290] In some embodiments, the residue of any PGI aligned with T85 of SEQ ID NO:43 can thus be substituted with Gin.

[0291] The binding sites of both Rhizobium meliloti pgiA1 and pgiA2 for Fe cation (cofactor) comprise residues 92, 94, 101, and 140. In some embodiments, an engineered Rhizobium meliloti pgiA1 or pgiA2 (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:33 or SEQ ID NO:38 and (b) is identical (i.e., does not have a substitution relative) to SEQ ID NO:33 or SEQ ID NO:38 at positions 92, 94, 101, and 140.

[0292] The binding site of E. coli manAfor 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, an engineered E. coli manA(a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:34 and (b) is identical (i.e., does not have a substitution relative) to SEQ ID NO:34 at positions 97, 99, 134, 255, and 274.

[0293] The active site of E. coli PGI comprises residues 355, 386, and 514. In some embodiments, an engineered E. coli PGI (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NO:36 and (b) is identical (i.e., does not have a substitution relative) to SEQ ID NO:36 at positions 355, 386, and 514.

[0294] The binding site of E. coli Kdul forZn2+ (cofactor) comprises residues 196, 198, 203, and 245. In some embodiments, an engineered E. coli Kdul (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity to SEQ ID NQ:40 and (b) is identical (i.e., does not have a substitution relative) to SEQ ID NQ:40 at positions 196, 198, 203, and 245.

[0295] The active site of Actinoplanes sp. (strain ATCC 31351 I 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, an engineered Actinoplanes sp. (strain ATCC 31351 / 3876) xylA (a) comprises an amino acid sequence having at least 90% (e.g., at least 90%, at least 92.5%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99%) sequence identity to SEQ ID NO:44 and (b) is identical (i.e., does not have a substitution relative) to SEQ ID NO:44 at positions 54, 57, 181, 217, 220, 245, 255, 257, and 292.6.4.5. Glucose Dehydrogenase

[0296] 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, ?-D-glucose:oxygen 1-oxidoreductase, and pyranose:oxygen 2-oxidoreductase. A dehydrogenase that has oxidoreductase activity and can act on CH-OH moieties of molecules other than glucose is a glucose dehydrogenase as described herein, provided it catalyzes the conversion of glucose to gluconolactone.

[0297] In some embodiments, a glucose dehydrogenase of the present disclosure has an activity identified by EC number 1.1.1.47.

[0298] 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 (gdhIV) having the UniProt identifier P39485 (SEQ ID NQ: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).

[0299] 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.

[0300] Exemplary glucose dehydrogenase amino acid sequences are provided in Table 6:6.4.6. Gluconolactonase

[0301] 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.

[0302] 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.-.

[0303] 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 least95%, 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 Zymomonas mobilis gluconolactonase (gn / ) having the UniProt identifier Q01578 (SEQ ID NO:14); E.coli K12 gluconolactonase (pgl) 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).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.

[0304] 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 etal., 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.

[0305] 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.

[0306] 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.

[0307] Exemplary gluconolactonase amino acid sequences are provided in Table 7:6.4.7. Gluconate Dehydratase

[0308] 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.

[0309] 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.-.

[0310] 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 (HvD_1) having the UniProt identifier A0A0M7KL98 (SEQ ID NO: 19), or Achromobacter veterisilvae gluconate dehydratase ( / 7vD_3) having the UniProt identifier A0A446D025 (SEQ ID NQ:20).

[0311] 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.

[0312] 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 NO:20 and is identical to SEQ ID NO:20 at positions 81 , 123, 124, 494, and 520.

[0313] Exemplary gluconate dehydratase amino acid sequences are provided in Table 8:6.5. Nucleic acids

[0314] 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.

[0315] 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.

[0316] 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.

[0317] 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 a peptide 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.

[0318] 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.

[0319] 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 thepolypeptide 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.

[0320] 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 an inducing agent, such as a sugar (e.g., lactose) or an organic acid or a salt thereof (e.g., gluconate).

[0321] 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.

[0322] 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.

[0323] 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.

[0324] A nucleotide sequence encoding an amino acid sequence of any heterologous polypeptide can be codon optimized fora 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).

[0325] 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.

[0326] 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).

[0327] A particular plasmid, pTrcHis2b-XylACT, has the schematic structure shown in FIG.10, wherein xylA-CT is a coding region encoding a protein differing from SEQ ID NO:23 (C. thermosulfurogenes xylA) by including the substitutions W139F and V186T as compared to the amino acid sequence of SEQ ID NO:23.6.6. Parental Microorganisms

[0328] 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.

[0329] In some embodiments, the microorganism is E. coli. In particular aspects, the E. coli is E. CO / / -K12 or a strain derived therefrom, such as E. coli MG 1655. In other aspects, the E. coli is E. coli W.

[0330] In some embodiments, the microorganism is Bacillus subtilis.

[0331] In some embodiments, the microorganism is Pseudomonas putida.

[0332] In some embodiments, the microorganism is Klebsiella oxytoca.

[0333] In some embodiments, the microorganism is Pantoea ananatis.

[0334] In some embodiments, the microorganism is Tatumella citrea.

[0335] In some embodiments, the microorganism is Zymomonas mobilis.

[0336] In some embodiments, the microorganism is Corynebacterium glutamicum.

[0337] In some embodiments, the microorganism is E. coll SuA6 (parent strain K12 MG1655, APTS fruBKA, APTS manXYZ, APTS Hlcrr, Amak, AgntK, AidnK).

[0338] In some embodiments, the microorganism is E. coll SuA7.1 (parent strain K12 MG1655, APTS fruBKA, APTS manXYZ, APTS Hlcrr, Amak, AidnK, Aglk).

[0339] In some embodiments, the microorganism is E. coli SuA5_KmR (Apts, AgntK, AidnK, Aglk, AkdgK::KmR).6.6.1. Engineering Methods

[0340] 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.

[0341] 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.

[0342] 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.”

[0343] 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 orto 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).

[0344] 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

[0345] 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, the isomerization of fructose and glucose, and the production of KDG from glucose.6.7.1. Culture media

[0346] 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.

[0347] 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).

[0348] 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).

[0349] 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).

[0350] 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).

[0351] 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.”

[0352] 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.

[0353] 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 from 0.1 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).

[0354] 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.

[0355] 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).

[0356] 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.

[0357] 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.

[0358] 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 the person of ordinary skill in the art having the benefit of the present disclosure as a routine matter.

[0359] 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

[0360] 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.

[0361] The fermentation process is typically run at a temperature that is optimal for growth of a recombinant microorganism. Fermentation for a mesophilic microorganism is typically carried 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.

[0362] 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

[0363] 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.

[0364] 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.

[0365] 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.

[0366] 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

[0367] 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.

[0368] 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.

[0369] 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.

[0370] 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

[0371] 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.

[0372] 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 asucrose invertase nucleotide sequence. Such a recombinant microorganism may be capable of isomerizing fructose and glucose in embodiments wherein a production medium comprises sucrose.

[0373] 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.

[0374] The isomerization of fructose and glucose can enhance a recombinant microorganism’s ability to survive or grow in media comprising fructose relative to a parental microorganism that lacks this ability. Alternatively, the isomerization of fructose and glucose can enhance a recombinant microorganism’s ability to survive or grow in media comprising glucose relative to a parental microorganism that lacks this ability.

[0375] The isomerization of fructose and glucose can be useful in vitro. For example, a fructose isomerase can be purified from cells in which it is translated, and the purified fructose isomerase can be used to isomerize fructose and glucose in the production of high fructose corn syrup, other sweetening agents, or other products of processes to which fructose or glucose can be a feedstock.6.7.6. Methods of Using Fructose and / or Glucose

[0376] Fructose or glucose produced by the methods described in Section 6.7.5 can be used for any desired purpose. In some embodiments, fructose and / or glucose can be purified from the recombinant microorganisms or from fructose isomerases used in vitro and used either individually or together in any proportion as a sweetening agent in foodstuffs. In some embodiments, fructose or glucose can be supplied to biochemical processes for the production of further products. The biochemical processes can be intracellular to the recombinant microorganisms or can be performed in other organisms. In some embodiments, the biochemical processes are intracellular to the recombinantmicroorganisms.

[0377] For example, recombinant microorganisms can be further configured to convert glucose to 2-keto-3-deoxygluconate (“KDG”), such as by being engineered to comprise nucleic acids comprising nucleotide sequences encoding glucose dehydrogenase, gluconate dehydratase, and optionally gluconolactonase. The action of these enzymes on glucose and products thereof increases the ratio of fructose to glucose, and thus the fructose isomerases of the recombinant microorganisms would be expected to convert fructose to glucose more than the reverse. Thus, the yield of KDG from fructose can be increased.6.7.7. Methods Of Producing 2-Keto-3-Deoxygluconate

[0378] 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.

[0379] 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.

[0380] 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.

[0381] 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.

[0382] 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.

[0383] Producing methods of the disclosure can further comprise growing a recombinant microorganism in a growth medium comprising a carbon source other than glucose orsucrose, 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.

[0384] 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.6.7.8. Methods of Using KDG

[0385] KDG produced by the methods described in Section 6.7.7can 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.

[0386] 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 WO2021016220.7. SPECIFIC EMBODIMENTS

[0387] The present disclosure is exemplified by the specific embodiments below.1. A recombinant microorganism configured to non-phosphorylatively transport sucrose and to express an engineered fructose isomerase.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.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.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.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.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.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.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.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.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.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.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.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.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 NQ: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.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.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.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.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. 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.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.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. 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.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.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. 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.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.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.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.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.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. 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:20.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 NO:20.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. 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”).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.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.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.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.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.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.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.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.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. 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.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.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. 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.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.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. The recombinant microorganism of any one of embodiments 1 to 66, which further has reduced glucokinase activity relative to a parental microorganism.The recombinant microorganism of any one of embodiments 1 to 67, which further has reduced fructokinase activity relative to a parental microorganism.The recombinant microorganism of any one of embodiments 1 to 68, which further has reduced glucose phosphotransferase (PTS) activity relative to a parental microorganism. The recombinant microorganism of any one of embodiments 1 to 69, which further has reduced fructose PTS activity relative to a parental microorganism.The recombinant microorganism of any one of embodiments 1 to 70, which further has reduced mannose PTS activity relative to a parental microorganism.The recombinant microorganism of any one of embodiments 1 to 71 , which further has reduced gluconate kinase activity relative to a parental microorganism.A recombinant microorganism, which is optionally the recombinant microorganism of any one of embodiments 1 to 72, configured to isomerize fructose and glucose at a mesophilic temperature.The recombinant microorganism of any one of embodiments 1 to 73 , wherein the recombinant microorganism comprises one or more nucleic acids comprising a nucleotide sequence encoding the engineered fructose isomerase (the “engineered fructose isomerase nucleotide sequence”).The recombinant microorganism of embodiment 74, wherein the engineered fructose isomerase nucleotide sequence encodes at least one amino acid substitution as compared to a “reference” fructose isomerase of any one of SEQ ID NOS:21-44, optionally wherein(a) the engineered fructose isomerase has at least 90% sequence identity to the reference fructose isomerase of any one of SEQ ID NOS:21-44; and / or(b) the engineered fructose isomerase has improved fructose isomerase activity as compared to the reference fructose isomerase having the amino acid sequence of any one of SEQ ID NOS:21-44.The recombinant microorganism of embodiment 75, wherein the engineered 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.The recombinant microorganism of embodiment 75 or embodiment 76, wherein the engineered 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. The recombinant microorganism of any one of embodiments 75 to 77, wherein the engineered 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. The recombinant microorganism of any one of embodiments 75 to 78, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:23, optionally wherein the amino acid sequencecomprises at least one, at least two, or at least three of the amino acid substitutions N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, W139F, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, V186T, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, and R424K as compared to the amino acid sequence of SEQ ID NO:23. The recombinant microorganism of any one of embodiments 75 to 78, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:23, optionally wherein the amino acid sequence comprises an A47S substitution as compared to the amino acid sequence of SEQ ID NO:23 and at least one, at least two, or at least three of the amino acid substitutions N19E, N19T, Y22F, Q59M, C99S, C99T, D128E, W139F, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, V186T, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, and R424K as compared to the amino acid sequence of SEQ ID NO:23. The recombinant microorganism of any one of embodiments 74 to 80, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:23.The recombinant microorganism of any one of embodiments 74 to 81 , wherein the engineered fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:23.The recombinant microorganism of any one of embodiments 74 to 82, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:23.The recombinant microorganism of any one of embodiments 74 to 83, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:23.The recombinant microorganism of any one of embodiments 74 to 84, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:23.The recombinant microorganism of any one of embodiments 74 to 85, wherein the engineered fructose isomerase comprises an amino acid sequence having amino acid substitutions W139F and / or V186T, and optionally one or more of the amino acid substitutions N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, and R424K as compared to the amino acid sequence of SEQ ID NO:23.The recombinant microorganism of any one of embodiments 74 to 85, wherein the engineered fructose isomerase comprises an amino acid sequence having amino acid substitutions A47S, W139F, and / or V186T, and optionally one or more of the amino acid substitutions N19E, N19T, Y22F, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, and R424K as compared to the amino acid sequence of SEQ ID NO:23.The recombinant microorganism of any one of embodiments 74 to 87, wherein the engineered fructose isomerase comprises an amino acid sequence identical to (i.e., does not have a substitution) at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339 as compared to the amino acid sequence of SEQ ID NO:23.The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and A142N as compared to the amino acid sequence of SEQ ID NO:23.The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and A142S as compared to the amino acid sequence of SEQ ID NO:23.The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and A47S as compared to the amino acid sequence of SEQ ID NO:23.The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, C99S, T141S, A142S, and V186C as compared to the amino acid sequence of SEQ ID NO:23.The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, C99S, T141S, A142T, and V186C as compared to the amino acid sequence of SEQ ID NO:23.The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, C99T, T141S, A142S, and F187Was compared to the amino acid sequence of SEQ ID NO:23.The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising thesubstitutions W139F, V186T, C99T, T141S, A142T, and F187Was compared to the amino acid sequence of SEQ ID NO:23.The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, C99T, L144M, and V186C as compared to the amino acid sequence of SEQ ID NO:23.The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, C99T, and V186C as compared to the amino acid sequence of SEQ ID NO:23.The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and C99T as compared to the amino acid sequence of SEQ ID NO:23.The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and D128E as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and E203Q as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and F150Y as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and F187L as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and F187Q as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising thesubstitutions W139F, V186T, and F187W as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and F187Y as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and F276Q as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and F363Y as compared to the amino acid sequence of SEQ ID NO:23.. Th The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and K410E as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and L144C as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and L144K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and L144M as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and N19E as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising thesubstitutions W139F, V186T, and N19T as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and N249H as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and N249S as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and Q417E as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and Q417K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and Q59M as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and R415K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and R424K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and S155A as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising thesubstitutions W139F, V186T, and T141C as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, T141S, and A142T as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, T141S, L144M, and F187W as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and T141S as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F and V186C as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and T299E as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and Y22F as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, Q59M, N249H, and R424K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, Q59M, T299E, and R424K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising thesubstitutions W139F, V186T, D128E, F150Y, and F363Y as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, Q59M, N249H, and F363Y as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, N19E, D128E, F276Q, and R415K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, N19E, A47S, D128E, and F276Q as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, N19E, Q59M, N249H, and R424K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, A47S, N249H, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, Q59M, N249H, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, A47S, T299E, F363Y, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, D128E, N249H, F363Y, F276Q, and K410E as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising thesubstitutions W139F, V186T, Q59M, D128E, N249H, F276Q, and R424K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, and A142N as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, and A142S as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, C99S, T141S, A142S, and V186C as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, C99S, T141S, A142T, and V186C as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, C99T, T141S, A142S, and F187W as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, C99T, T141S, A142T, and F187W as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, C99T, L144M, and V186C as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, C99T, and V186C as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising thesubstitutions A47S, W139F, V186T, and C99T as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, and D128E as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, and E203Q as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, and F150Y as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, and F187L as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, and F187Q as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, and F187W as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, and F187Y as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, and F276Q as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising thesubstitutions A47S, W139F, V186T, and F363Y as compared to the amino acid sequence of SEQ ID NO:23.. Th The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, and K410E as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, and L144C as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, and L144K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, and L144M as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, and N19E as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, and N19T as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, and N249H as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, and N249S as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising thesubstitutions A47S, W139F, V186T, and Q417E as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, and Q417K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, and Q59M as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, and R415K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, and R424K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, and S155A as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, and T141C as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, T141S, and A142T as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, T141S, L144M, and F187Was compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising thesubstitutions A47S, W139F, V186T, and T141S as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F and V186C as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, and T299E as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, and Y22F as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, Q59M, N249H, and R424K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, Q59M, T299E, and R424K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, D128E, F150Y, and F363Y as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, Q59M, N249H, and F363Y as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, N19E, D128E, F276Q, and R415K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising thesubstitutions A47S, W139F, V186T, N19E, Q59M, N249H, and R424K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, Q59M, N249H, K410E, and R424K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, D128E, N249H, F363Y, F276Q, and K410E as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 74 to 88, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions A47S, W139F, V186T, Q59M, D128E, N249H, F276Q, and R424K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:21.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:21.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:21.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:21.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:21.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:21.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase an amino acid sequence having at least 90% sequence identity to SEQ ID NO:22.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:22.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:22.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:22.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:22.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:22.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:24.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase an amino acid sequence having at least 95% sequence identity to SEQ ID NO:24.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:24.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:24.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:24.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:24.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:25.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:25.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:25.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:25.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:25.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:25.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:26.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:26.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:26.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:26.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:26.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:26.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:27.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:27.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:27.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:27.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:27.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:27.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:28.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:28.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:28.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:28.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:28.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:28.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:29.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:29.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:29.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:29.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:29.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:29.. The recombinant microorganism of any one of embodiments 74, 75, or 231 to 236, wherein the engineered fructose isomerase, comprising at least one amino acid substitution V186T, L283P, or F187S.. The recombinant microorganism of embodiment 237, comprising at least two amino acid substitutions V186T, L283P, or F187S.. The recombinant microorganism of embodiment 237 or embodiment 238, comprising amino acid substitutions V186T, L283P, and F187S.. The recombinant microorganism of any one of embodiments 74, 75, or 231 to 239, comprising at least one amino acid substitution corresponding to an amino acid substitution W139F, V186T, N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, or R424K in the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NQ:30.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NQ:30.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:30.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:30.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:30.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NQ:30.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:31.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:31.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:31.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:31.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:31.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:31.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:32.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:32.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:32.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:32.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:32.. The recombinant microorganism of any one of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:32.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:33.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:33.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:33.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:33.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:33.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:33.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:34.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:34.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:34.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:34.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:34.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:34.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:35.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:35.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:35.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:35.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:35.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:35.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:36.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:36.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:36.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:36.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:36.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:36.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:37.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:37.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:37.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:37.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:37.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:37.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:38.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:38.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:38.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:38.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:38.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:38.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:39.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:39.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:39.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:39.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:39.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:39.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NQ:40.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NQ:40.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:40.- HO -. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:40.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:40.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NQ:40.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:41.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:41.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:41.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:41.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:41.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:41.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:42.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:42.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:42.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:42.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:42.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:42.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:43.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:43.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:43.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:43.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:43.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:43.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:43.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:44.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:44.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:44.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:44.. The recombinant microorganism of embodiment 74 or embodiment 75, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:44.. The recombinant microorganism of any one of embodiments 74, 75, or 189 to 330, wherein the engineered fructose isomerase comprises at least one amino acid substitution corresponding to an amino acid substitution W139F, V186T, N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, or R424K in the amino acid sequence of SEQ ID NO:3.. The recombinant microorganism of any one of embodiments 74 to 331 , wherein the engineered fructose isomerase comprises 3-10 amino acid substitutions as compared to the reference amino acid sequence.. The recombinant microorganism of any one of embodiments 74 to 331 , wherein the engineered fructose isomerase comprises 3-12 amino acid substitutions as compared to the reference amino acid sequence.. The recombinant microorganism of any one of embodiments 74 to 331 , wherein the engineered fructose isomerase comprises 3-15 amino acid substitutions as compared to the reference amino acid sequence.. The recombinant microorganism of any one of embodiments 74 to 331 , wherein the engineered fructose isomerase comprises 3-20 amino acid substitutions as compared to the reference amino acid sequence.. The recombinant microorganism of any one of embodiments 74 to 331 , wherein the engineered fructose isomerase comprises 4-10 amino acid substitutions as compared to the reference amino acid sequence.. The recombinant microorganism of any one of embodiments 74 to 331 , wherein the engineered fructose isomerase comprises 4-12 amino acid substitutions as compared to the reference amino acid sequence.. The recombinant microorganism of any one of embodiments 74 to 331 , wherein the engineered fructose isomerase comprises 4-15 amino acid substitutions as compared to the reference amino acid sequence.- US -. The recombinant microorganism of any one of embodiments 74 to 331 , wherein the engineered fructose isomerase comprises 4-20 amino acid substitutions as compared to the reference amino acid sequence.. The recombinant microorganism of any one of embodiments 74 to 339, which further has increased fructose isomerase activity relative to a parental microorganism.. The recombinant microorganism of embodiment 340, wherein the increased fructose isomerase activity is at a mesophilic temperature.. The recombinant microorganism of any one of embodiments 1 to 341 , wherein the recombinant microorganism is a Bacillus subtilis.. The recombinant microorganism of any one of embodiments 1 to 341 , wherein the recombinant microorganism is a Pseudomonas putida.. The recombinant microorganism of any one of embodiments 1 to 341 , wherein the recombinant microorganism is a Klebsiella oxytoca.. The recombinant microorganism of any one of embodiments 1 to 341 , wherein the recombinant microorganism is a Pantoea ananatis.. The recombinant microorganism of any one of embodiments 1 to 341 , wherein the recombinant microorganism is a Tatumella citrea.. The recombinant microorganism of any one of embodiments 1 to 341 , wherein the recombinant microorganism is a Zymomonas mobilis.. The recombinant microorganism of any one of embodiments 1 to 341 , wherein the recombinant microorganism is a Corynebacterium glutamicum.. The recombinant microorganism of any one of embodiments 1 to 341 , wherein the recombinant microorganism is an E. coli.. The recombinant microorganism of embodiment 349, 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. . A method for producing 2-keto-3-deoxygluconate (“KDG”), comprising culturing the recombinant microorganism of any one of embodiments 1 to 350 in a production medium comprising sucrose.. The method of embodiment 351, wherein the production medium comprises from 0.1 w / v% to 5 w / v% sucrose.. The method of embodiment 351 or embodiment 352, wherein the recombinant microorganism is capable of non-phosphorylatively transporting sucrose from the production medium into a cell of the recombinant microorganism.. The method of any one of embodiments 351 to 353, wherein the recombinant microorganism is capable of hydrolyzing sucrose to glucose and fructose.. The method of any one of embodiments 351 to 354, 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.. The method of embodiment 355, wherein the growth medium comprises glycerol. . The method of embodiment 355 or embodiment 356, wherein the growth medium comprises from 0.1 w / v% to 5 w / v% glycerol.. The method of any one of embodiments 355 to 357, wherein the growth medium lacks added sucrose.. The method of embodiment 355 or embodiment 356, wherein the growth medium comprises no more than 0.1% sucrose.. The method of any one of embodiments 351 to 359, further comprising use of the KDG.. The method of embodiment 360, wherein the KDG is used for the production of pyruvate and / or glyceraldehyde.. The method of embodiment 360, wherein the KDG is used for the production of isopentenyl pyrophosphate (IPP) and / or dimethylallyl pyrophosphate (DMAPP).. The method of embodiment 360, wherein the KDG is used for the production of terpenoids.. A method for non-phosphorylatively transporting sucrose, comprising culturing the recombinant microorganism of any one of embodiments 1 to 350 in a production medium comprising sucrose.. The method of embodiment 360, wherein the production medium comprises from 0.1 w / v% to 5 w / v% sucrose.. The method of embodiment 364 or embodiment 365, 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.. The method of embodiment 365, wherein the growth medium comprises glycerol. . The method of embodiment 365 or embodiment 367, wherein the growth medium comprises from 0.1 w / v% to 5 w / v% glycerol.. The method of any one of embodiments 366 to 368, wherein the growth medium lacks added sucrose.. The method of any one of embodiments 365 to 368, wherein the growth medium comprises no more than 0.1% sucrose.. A method for hydrolyzing sucrose to glucose and fructose, comprising culturing the recombinant microorganism of any one of embodiments 1 to 350 in a production medium comprising sucrose.. The method of embodiment 371 , wherein the production medium comprises from 0.1 w / v% to 5 w / v% sucrose.. The method of embodiment 371 or embodiment 372, wherein the recombinant microorganism is capable of non-phosphorylatively transporting sucrose from the production medium into a cell of the recombinant microorganism.. The method of any one of embodiments 371 to 373, 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.. The method of embodiment 374, wherein the growth medium comprises glycerol. . The method of embodiment 374 or embodiment 375, wherein the growth medium comprises from 0.1 w / v% to 5 w / v% glycerol.. The method of any one of embodiments 374 to 376, wherein the growth medium lacks added sucrose.. The method of any one of embodiments 374 to 376, wherein the growth medium comprises no more than 0.1% sucrose.. A method for isomerizing fructose and glucose, comprising culturing the recombinant microorganism of any one of embodiments 1 to 350 in a production medium comprising sucrose, fructose, and / or glucose, optionally at a mesophilic temperature.. The method of embodiment 379, wherein the production medium comprises from 0.1 w / v% to 5 w / v% sucrose fructose, and / or glucose.. The method of embodiment 379 or embodiment 372, wherein the recombinant microorganism is capable of non-phosphorylatively transporting sucrose from the production medium into a cell of the recombinant microorganism.. The method of any one of embodiments 379 to 381 , wherein the recombinant microorganism is capable of hydrolyzing sucrose to glucose and fructose.. The method of any one of embodiments 379 to 382, 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.. The method of embodiment 383, wherein the growth medium comprises glycerol. . The method of embodiment 383 or embodiment 384, wherein the growth medium comprises from 0.1 w / v% to 5 w / v% glycerol.. The method of any one of embodiments 383 to 385, wherein the growth medium lacks added sucrose, fructose, and glucose.. The method of any one of embodiments 383 to 386, wherein the growth medium comprises no more than 0.1% sucrose, fructose, and / or glucose.. A method for producing 2-keto-3-deoxygluconate (“KDG”), comprising culturing the recombinant microorganism of any one of embodiments 1 to 350 in a production medium comprising glucose.. The method of embodiment 388, wherein the production medium comprises from 0.1 w / v% to 5 w / v% glucose.. The method of embodiment 379 or embodiment 389, wherein the recombinant microorganism is capable of non-phosphorylatively transporting glucose from the production medium into a cell of the recombinant microorganism.. The method of any one of embodiments 379 to 390, 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.. The method of embodiment 391 , wherein the growth medium comprises glycerol. . The method of embodiment 391 or embodiment 392, wherein the growth medium comprises from 0.1 w / v% to 5 w / v% glycerol.. The method of any one of embodiments 391 to 393, wherein the growth medium lacks added glucose.. The method of any one of embodiments 391 to 393, wherein the growth medium comprises no more than 0.1% glucose.. 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.. The recombinant microorganism of embodiment 396, wherein the means for non-phosphorylatively transporting sucrose comprise means for passively transporting nonphosphorylated sucrose through a cell membrane of the recombinant microorganism. . The recombinant microorganism of embodiment 396 or embodiment 397, wherein the means for non-phosphorylatively transporting sucrose comprise means for actively transporting non-phosphorylated sucrose through a cell membrane of the recombinant microorganism.. A recombinant microorganism, which is optionally the recombinant microorganism of any one of embodiments 396 to 398, 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.. A recombinant microorganism, which is optionally the recombinant microorganism of any one of embodiments 396 to 399, 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.. A recombinant microorganism, which is optionally the recombinant microorganism of any one of embodiments 396 to 400, comprising a nucleic acid or a plurality 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. . The recombinant microorganism of embodiment 400, wherein the means for producing KDG from glucose comprise means for catalyzing the conversion of glucose to gluconolactone.. The recombinant microorganism of embodiment 401 or embodiment 402, wherein the means for producing KDG from glucose comprise means for catalyzing the conversion of gluconolactone to gluconate.. The recombinant microorganism of any one of embodiments 400 to 403, wherein the means for producing KDG from glucose comprise means for catalyzing the conversion of gluconate to KDG.. The recombinant microorganism of any one of embodiments 396 to 404, wherein the recombinant microorganism lacks means for phosphorylating at least one of sucrose, fructose, or glucose.. A method of non-phosphorylatively transporting sucrose, comprising:culturing the recombinant microorganism of any one of embodiments 396 to 405, 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.. A method of isomerizing fructose and glucose, comprising:culturing the recombinant microorganism of any one of embodiments 396 to 405, 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.. The method of embodiment 407, wherein the culturing is at a mesophilictemperature.. A method of producing KDG, comprising:culturing the recombinant microorganism of any one of embodiments 396 to 405, wherein the recombinant microorganism comprises a nucleic acid or a plurality of nucleic acids encoding means for- I -producing KDG from glucose, in a production medium comprising sucrose or glucose.. A recombinant microorganism comprising means for non-phosphorylatively transporting sucrose, wherein the means is at least partially heterologous to the recombinant microorganism.. The recombinant microorganism of embodiment 410, wherein the means for non-phosphorylatively transporting sucrose comprise means for passively transporting nonphosphorylated sucrose through a cell membrane of the recombinant microorganism. . The recombinant microorganism of embodiment 410 or embodiment 411, wherein the means for non-phosphorylatively transporting sucrose comprise means for actively transporting non-phosphorylated sucrose through a cell membrane of the recombinant microorganism.. A recombinant microorganism, which is optionally the recombinant microorganism of any one of embodiments 410 to 412, comprising means for hydrolyzing sucrose to glucose and fructose, wherein the means is at least partially heterologous to the recombinant microorganism.. A recombinant microorganism, which is optionally the recombinant microorganism of any one of embodiments 410 to 413, comprising means for isomerizing fructose and glucose, optionally at a mesophilic temperature, wherein the means is at least partially heterologous to the recombinant microorganism.. A recombinant microorganism, which is optionally the recombinant microorganism of any one of embodiments 410 to 414, comprising means for producing 2-keto-3-deoxygluconic acid (KDG) from glucose, wherein the means is at least partially heterologous to the recombinant microorganism.. The recombinant microorganism of embodiment 415, wherein the means for producing KDG from glucose comprise means for catalyzing the conversion of glucose to gluconolactone.. The recombinant microorganism of embodiment 415 or embodiment 416, wherein the means for producing KDG from glucose comprise means for catalyzing the conversion of gluconolactone to gluconate.. The recombinant microorganism of any one of embodiments 415 to 417, wherein the means for producing KDG from glucose comprise means for catalyzing the conversion of gluconate to KDG,. The recombinant microorganism of any one of embodiments 410 to 418, wherein the recombinant microorganism lacks means for phosphorylating at least one of sucrose, fructose, or glucose.. A method of non-phosphorylatively transporting sucrose, comprising:(a) culturing the recombinant microorganism of any one of embodiments 410 to 419, wherein the recombinant microorganism comprises means for non-phosphorylatively transporting sucrose, in a production medium comprising sucrose.421. A method of isomerizing fructose and glucose, comprising:(a) culturing the recombinant microorganism of any one of embodiments 410 to 419, wherein the recombinant microorganism comprises means for isomerizing fructose and glucose, in a production medium comprising sucrose, fructose, and / or glucose.422. The method of embodiment 421 , wherein the culturing is at a mesophilic temperature.423. A method of producing KDG, comprising:(a) culturing the recombinant microorganism of any one of embodiments 410 to 419, wherein the recombinant microorganism comprises means for producing KDG from glucose, in a production medium comprising sucrose or glucose.424. 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; wherein the recombinant microorganism comprises one or more nucleic acids comprising a nucleotide sequence encoding an engineered fructose isomerase (the “engineered fructose isomerase nucleotide sequence”).425. A recombinant microorganism, which is optionally the recombinant microorganism of embodiment 424, 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.426. The recombinant microorganism of embodiment 425, wherein the sucrose porin nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:1.427. The recombinant microorganism of embodiment 425 or embodiment 426, wherein the sucrose porin nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:1.428. The recombinant microorganism of any one of embodiments 425 to 427, wherein the sucrose porin nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:1.429. The recombinant microorganism of any one of embodiments 425 to 428, wherein the sucrose permease nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:4.430. The recombinant microorganism of any one of embodiments 425 to 429, wherein the sucrose permease nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:4.431. The recombinant microorganism of any one of embodiments 425 to 430, wherein the sucrose permease nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:4.432. The recombinant microorganism of any one of embodiments 425 to 431 , wherein the sucrose invertase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO:7.433. The recombinant microorganism of any one of embodiments 425 to 432, wherein the sucrose invertase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:7.434. The recombinant microorganism of any one of embodiments 425 to 433, wherein the sucrose invertase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO:7.435. A recombinant microorganism, which is optionally the recombinant microorganism of any one of embodiments 424 to 434, 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.. The recombinant microorganism of embodiment 435, 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.. The recombinant microorganism of embodiment 435 or embodiment 436, 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. . The recombinant microorganism of any one of embodiments 435 to 437, 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. . The recombinant microorganism of any one of embodiments 435 to 438, wherein the gluconate dehydratase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 18.. The recombinant microorganism of any one of embodiments 435 to 439, wherein the gluconate dehydratase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 18.. The recombinant microorganism of any one of embodiments 435 to 440, wherein the gluconate dehydratase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 99% identity to SEQ ID NO: 18.. A recombinant microorganism, which is optionally the recombinant microorganism of any one of embodiments 424 to 442, comprising one or more nucleic acids comprising a nucleotide sequence encoding a gluconolactonase (the “gluconolactonase nucleotide sequence”).. The recombinant microorganism of embodiment 442, 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.. The recombinant microorganism of embodiment 442 or embodiment 443, 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.. The recombinant microorganism of any one of embodiments 442 to 444, 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.. The recombinant microorganism of any one of embodiments 424 to 445, which further has reduced glucokinase activity relative to a parental microorganism.. The recombinant microorganism of any one of embodiments 424 to 446, which further has reduced fructokinase activity relative to a parental microorganism.. The recombinant microorganism of any one of embodiments 424 to 447, which further has reduced glucose phosphotransferase (PTS) activity relative to a parental microorganism.. The recombinant microorganism of any one of embodiments 424 to 448, which further has reduced fructose PTS activity relative to a parental microorganism.. The recombinant microorganism of any one of embodiments 424 to 449, which further has reduced mannose PTS activity relative to a parental microorganism.. The recombinant microorganism of any one of embodiments 424 to 450, which further has reduced gluconate kinase activity relative to a parental microorganism.. The recombinant microorganism of any one of embodiments 424 to 451 , wherein the engineered fructose isomerase nucleotide sequence encodes at least one amino acid substitution as compared to a “reference” fructose isomerase of any one of SEQ ID NOS:21-44, optionally wherein(a) the engineered fructose isomerase has at least 90% sequence identity to the reference fructose isomerase of any one of SEQ ID NOS:21-44; and / or(b) the engineered fructose isomerase has improved fructose isomerase activity as compared to the reference fructose isomerase having the amino acid sequence of any one of SEQ ID NOS:21-44.. The recombinant microorganism of embodiment 452, wherein the engineered 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.. The recombinant microorganism of embodiment 452 or embodiment 453, wherein the engineered 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.. The recombinant microorganism of any one of embodiments 452 to 454, wherein the engineered 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. . The recombinant microorganism of any one of embodiments 452 to 455, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:23, optionally wherein the amino acid sequence comprises at least one, at least two, or at least three of the amino acid substitutions N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, W139F, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, V186T, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, and R424K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 456, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:23, optionally wherein the amino acid sequence comprises an A47S substitution as compared to the amino acid sequence of SEQ ID NO:23 and at least one, at least two, or at least three of the amino acid substitutions N19E, N19T, Y22F, Q59M, C99S, C99T, D128E, W139F, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, V186T, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, and R424K as compared to the amino acid sequence of SEQ ID NO:23. . The recombinant microorganism of any one of embodiments 452 to 457, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 458, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 459, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 460, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 461 wherein the engineered fructose isomerase comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 462, wherein the engineered fructose isomerase comprises an amino acid sequence having amino acid substitutions W139F and / or V186T, and optionally one or more of the amino acid substitutions N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, and R424K as compared to the amino acid sequence of SEQ ID NO:23. . The recombinant microorganism of any one of embodiments 452 to 463, wherein the engineered fructose isomerase comprises an amino acid sequence having amino acid substitutions A47S, W139F, and / or V186T, and optionally one or more of the amino acid substitutions N19E, N19T, Y22F, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W,F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, and R424K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 464, wherein the engineered fructose isomerase comprises an amino acid sequence identical to (i.e., does not have a substitution) at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339 as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and A142N as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and A142S as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and A47S as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, C99S, T141S, A142S, and V186C as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, C99S, T141S, A142T, and V186C as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, C99T, T141S, A142S, and F187Was compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, C99T, T141S, A142T, and F187Was compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising thesubstitutions W139F, C99T, L144M, and V186C as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, C99T, and V186C as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and C99T as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and D128E as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and E203Q as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and F150Y as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and F187L as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and F187Q as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and F187W as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising thesubstitutions W139F, V186T, and F187Y as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and F276Q as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and F363Y as compared to the amino acid sequence of SEQ ID NO:23.. Th The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and K410E as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and L144C as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and L144K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and L144M as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and N19E as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and N19T as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising thesubstitutions W139F, V186T, and N249H as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and N249S as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and Q417E as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and Q417K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and Q59M as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and R415K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and R424K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and S155A as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and T141 C as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising thesubstitutions W139F, V186T, T141S, and A142T as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, T141S, L144M, and F187W as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and T141 S as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F and V186C as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and T299E as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and Y22F as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, Q59M, N249H, and R424K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, Q59M, T299E, and R424K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, D128E, F150Y, and F363Y as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising thesubstitutions W139F, V186T, Q59M, N249H, and F363Y as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, N19E, D128E, F276Q, and R415K as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, N19E, A47S, D128E, and F276Q as compared to the amino acid sequence of SEQ ID NO:23.. The recombinant microorganism of any one of embodiments 452 to 465, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, N19E, Q59M, N249H, and R424K as compared to th...

Claims

1. WHAT IS CLAIMED IS:

1. A recombinant microorganism configured to non-phosphorylatively transport sucrose and to express an engineered fructose isomerase.

2. A recombinant microorganism, which is optionally the recombinant microorganism of claim 1 , configured to hydrolyze sucrose to glucose and fructose.

3. A recombinant microorganism, which is optionally the recombinant microorganism of claim 1 or claim 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 claims 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, the sucrose invertase nucleotide sequence, the glucose dehydrogenase nucleotide sequence, the gluconate dehydratase nucleotide sequence, the gluconolactonase nucleotide sequence, and the engineered fructose isomerase nucleotide sequence is heterologous to the microorganism.

5. A recombinant microorganism, which is optionally the recombinant microorganism of any one of claims 1 to 4, comprising one or more nucleic acids comprising:(a) a nucleotide sequence encoding a glucose dehydrogenase (the “glucose dehydrogenase nucleotide sequence”),(b) a nucleotide sequence encoding a gluconate dehydratase (the “gluconate dehydratase nucleotide sequence”),(c) a nucleotide sequence encoding a gluconolactonase (the “gluconolactonase nucleotide sequence”), and(d) a nucleotide sequence encoding the engineered fructose isomerase (the “engineered fructose isomerase nucleotide sequence”),wherein at least one of the glucose dehydrogenase nucleotide sequence, the gluconate dehydratase nucleotide sequence, and the gluconolactonase nucleotide sequence is heterologous to the microorganism.

6. The recombinant microorganism of claim 4 or 5, wherein the sucrose porin nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90%, 95%, 99%, or 100% identity to any one of SEQ ID NOS:1-7.

7. The recombinant microorganism of claim 5 or 6, wherein the glucose dehydrogenase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90%, 95%, 99%, or 100% identity to any one of SEQ ID NQS:10-13.

8. The recombinant microorganism of any one of claims 4 to 7, wherein the gluconate dehydratase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90%, 95%, 99%, or 100% identity to any one of SEQ ID NQS:18-20.

9. The recombinant microorganism of any one of claims 4 to 8, wherein the gluconolactonase nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 90%, 95%, 99%, or 100% identity to any one of SEQ ID NOS:14-17.

10. The recombinant microorganism of any one of claims 1 to 9, which further has at least one of reduced glucokinase activity, reduced fructokinase activity, reduced glucose phosphotransferase (PTS) activity, reduced fructose PTS activity, reduced mannose PST activity, or reduced gluconate kinase activity relative to a parental microorganism.

11. A recombinant microorganism, which is optionally the recombinant microorganism of any one of claims 1 to 10, configured to isomerize fructose and glucose at a mesophilic temperature.

12. The recombinant microorganism of any one of claims 1 to 11 , wherein the engineered fructose isomerase nucleotide sequence encodes at least one amino acid substitution as compared to a “reference” fructose isomerase of any one of SEQ ID NOS:21-44, optionally wherein(a) the engineered fructose isomerase has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference fructose isomerase of any one of SEQ ID NOS:21-44; and / or(b) the engineered fructose isomerase has improved fructose isomerase activity as compared to the reference fructose isomerase having the amino acid sequence of any one of SEQ ID NOS:21-44.

13. The recombinant microorganism of claim 12, wherein the engineered fructose isomerase comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:23, optionally wherein the amino acid sequencecomprises at least one, at least two, or at least three of the amino acid substitutions N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, W139F, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, V186T, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, and R424K as compared to the amino acid sequence of SEQ ID NO:23.

14. The recombinant microorganism of claim 13, wherein the engineered fructose isomerase comprises an amino acid sequence having amino acid substitutions W139F and / or V186T, and optionally one or more of the amino acid substitutions N19E, N19T, Y22F, A47S, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, and R424K as compared to the amino acid sequence of SEQ ID NO:23.

15. The recombinant microorganism of claim 14, wherein the engineered fructose isomerase comprises an amino acid sequence having amino acid substitutions A47S, W139F, and / or V186T, and optionally one or more of the amino acid substitutions N19E, N19T, Y22F, Q59M, C99S, C99T, D128E, T141C, T141S, A142N, A142S, A142T, L144C, L144K, L144M, F150Y, S155A, V186C, F187L, F187Q, F187W, F187Y, E203Q, N249H, N249S, F276Q, T299E, F363Y, K410E, R415K, Q417E, Q417K, and R424K as compared to the amino acid sequence of SEQ ID NO:23.

16. The recombinant microorganism of any one of claims 13 to 15, wherein the engineered fructose isomerase comprises an amino acid sequence identical to (i.e., does not have a substitution) at positions 101, 104, 232, 268, 271, 296, 307, 309, and 339 as compared to the amino acid sequence of SEQ ID NO:23.

17. The recombinant microorganism of any one of claims 13 to 16, wherein the engineered fructose isomerase comprises an amino acid sequence comprising the substitutions W139F, V186T, and A47S as compared to the amino acid sequence of SEQ ID NO:23.

18. The recombinant microorganism of any one of claims 1 to 17, wherein the engineered fructose isomerase comprises 3-20 amino acid substitutions as compared to the reference amino acid sequence.

19. The recombinant microorganism of any one of claims 14 to 18, which further has increased fructose isomerase activity relative to a parental microorganism; and optionally wherein the increase fructose isomerase activity is at a mesophilic temperature.

20. The recombinant microorganism of any one of claims 1 to 19, wherein the recombinant microorganism is a Bacillus subtilis, Pseudomonas putida, Klebsiella oxytoca, Pantoeaananatis, Tatumella citrea, Zymomonas mobilis, Corynebacterium glutamicum, or an E. coli.

21. The recombinant microorganism of claim 20, wherein the E. coli is E. coli K12 or a strain derived therefrom, such as E. coli MG 1655; or the E. coli is E. coli W.

22. A method for producing 2-keto-3-deoxygluconate (“KDG”), comprising culturing the recombinant microorganism of any one of claims 1 to 21 in a production medium comprising sucrose; and optionally wherein,(a) the production medium comprises from 0.1 w / v% to 5 w / v% sucrose; and / or (b) the recombinant microorganism is capable of non-phosphorylatively transporting sucrose from the production medium into a cell of the recombinant microorganism; and / or(c) the recombinant microorganism is capable of hydrolyzing sucrose to glucose and fructose; and / or(d) 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.

23. The method of claim 22, wherein the growth medium comprises glycerol.

24. The method of claim 22 or claim 23, wherein the growth medium lacks added sucrose or comprises no more than 0.1% sucrose.

25. The method of any one of claims 22 to 24, further comprising use of the KDG, wherein the KDG is optionally used for the production of pyruvate and / or glyceraldehyde, the production of isopentenyl pyrophosphate (IPP) and / or dimethylallyl pyrophosphate (DMAPP), or the production of terpenoids.

26. A method for non-phosphorylatively transporting sucrose, comprising culturing the recombinant microorganism of any one of claims 1 to 21 in a production medium comprising sucrose, 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.

27. The method of claim 26, wherein the growth medium comprises glycerol.

28. The method of claim 26 or claim 27, wherein the growth medium lacks added sucrose or comprises no more than 0.1% sucrose.

29. A method for hydrolyzing sucrose to glucose and fructose, comprising culturing the recombinant microorganism of any one of claims 1 to 21 in a production mediumcomprising sucrose, wherein the recombinant microorganism is capable of non- phosphorylatively transporting sucrose from the production medium into a cell of the recombinant microorganism, wherein the method further comprises growing the recombinant microorganism in a growth medium comprising a carbon source other than sucrose, such as glycerol, prior to culturing in the production medium.

30. The method of any one of claim 29, wherein the growth medium lacks added sucrose or comprises no more than 0.1% sucrose.

31. A method for isomerizing fructose and glucose, comprising culturing the recombinant microorganism of any one of claims 1 to 21 in a production medium comprising sucrose, fructose, and / or glucose, optionally at a mesophilic temperature, wherein the recombinant microorganism is capable of non-phosphorylatively transporting sucrose or glucose from the production medium into a cell of the recombinant microorganism and / or hydrolyzing sucrose to glucose and fructose.

32. The method of any one of claim 31 , 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.

33. The method of claim 32, wherein the growth medium comprises glycerol.

34. The method of any one of claim 32 or claim 33, wherein the growth medium lacks added sucrose, fructose, and glucose or comprises no more than 0.1% sucrose, fructose, and / or glucose.

35. A method for producing 2-keto-3-deoxygluconate (“KDG”), comprising culturing the recombinant microorganism of any one of claims 1 to 21 in a production medium comprising glucose, wherein the recombinant microorganism is capable of non- phosphorylatively transporting glucose from the production medium into a cell of the recombinant microorganism.

36. The method of any one of claims 31 to 35, 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, optionally wherein the growth medium comprises glycerol.

37. The method of any one of claim 36, wherein the growth medium lacks added glucose or comprises no more than 0.1% glucose.

38. A recombinant microorganism comprising a nucleic acid or a plurality of nucleic acids encoding means for non-phosphorylatively transporting sucrose, wherein at least onenucleic acid is heterologous to the recombinant microorganism, in a production medium comprising sucrose or glucose.

39. The recombinant microorganism of claim 38, wherein the means for non- phosphorylatively transporting sucrose comprise means for passively or actively transporting non-phosphorylated sucrose through a cell membrane of the recombinant microorganism.

40. A recombinant microorganism, which is optionally the recombinant microorganism of claim 38 or claim 39 comprising a nucleic acid or a plurality of nucleic acids encoding means for,(a) hydrolyzing sucrose to glucose and fructose,(b) isomerizing fructose and glucose, optionally at a mesophilic temperature, and / or (c) producing 2-keto-3-deoxygluconic acid (KDG) from glucose, wherein at least one nucleic acid is heterologous to the recombinant microorganism.

41. A recombinant microorganism, which is optionally the recombinant microorganism of any one of claims 38 to 40, 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.

42. A recombinant microorganism, which is optionally the recombinant microorganism of any one of claims 38 to 41 , comprising a nucleic acid or a plurality 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.

43. The recombinant microorganism of claim 42, wherein the means for producing KDG from glucose comprise at least one of the:(a) means for catalyzing the conversion of glucose to gluconolactone,(b) means for catalyzing the conversion of gluconolactone to gluconate, and / or (c) means for catalyzing the conversion of gluconate to KDG.

44. The recombinant microorganism of any one of claims 38 to 43, wherein the recombinant microorganism lacks means for phosphorylating at least one of sucrose, fructose, or glucose.

45. A method of isomerizing fructose and glucose, comprising culturing the recombinant microorganism of any one of claims 38 to 44, wherein the recombinant microorganism comprises a nucleic acid or a plurality of nucleic acids encoding means for isomerizingfructose and glucose, in a production medium comprising sucrose, fructose, and / or glucose, optionally wherein the culturing is at a mesophilic temperature.

46. A recombinant microorganism comprising means for non-phosphorylatively transporting sucrose, wherein the means is at least partially heterologous to the recombinant microorganism, wherein the means for non-phosphorylatively transporting sucrose comprise means for passively or actively transporting non-phosphorylated sucrose through a cell membrane of the recombinant microorganism, optionally wherein the recombinant microorganism further comprises means for hydrolyzing sucrose to glucose and fructose, wherein the means is at least partially heterologous to the recombinant microorganism, optionally wherein the recombinant microorganism lacks means for phosphorylating at least one of sucrose, fructose, or glucose.

47. A recombinant microorganism, which is optionally the recombinant microorganism of claim 46, comprising means for producing 2-keto-3-deoxygluconic acid (KDG) from glucose, wherein the means is at least partially heterologous to the recombinant microorganism.

48. The recombinant microorganism of claim 47, wherein the means for producing KDG from glucose comprises(a) means for catalyzing the conversion of glucose to gluconolactone;(b) means for producing KDG from glucose comprise means for catalyzing the conversion of gluconolactone to gluconate;(c) means for producing KDG from glucose comprise means for catalyzing the conversion of gluconate to KDG; and optionally wherein the recombinant microorganism lacks means for phosphorylating at least one of sucrose, fructose, or glucose.

49. A method of non-phosphorylatively transporting sucrose, comprising culturing the recombinant microorganism of any one of claims 46 to 48, wherein the recombinant microorganism comprises means for non-phosphorylatively transporting sucrose, in a production medium comprising sucrose.

50. A method of producing KDG, comprising culturing the recombinant microorganism of any one of claims 46 to 48, wherein the recombinant microorganism comprises means for producing KDG from glucose, in a production medium comprising sucrose or glucose.

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