Compositions and methods for producing dihydrofuran from ketose

Abstract

The present disclosure provides compositions and methods for the production of dihydrofuran by a glycosyl hydrolase enzyme capable of dehydrating 2-keto-3-deoxygluconic acid (KDG) to K4. Also provided are compositions and methods for further processing K4 to produce HMFA (5-hydroxymethyl-2-furoic acid) and / or FDCA (2, 5-furandicarboxylic acid).

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 352,145, filed on June 14, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] Reference to an electronic sequence listing

[0004] The contents of the electronic sequence listing (ARZE_035_03WO_SeqList_ST26.xml; size: 144,098 bytes; and creation date: June 12, 2023) are incorporated herein by reference in their entirety. Background Art

[0005] Furandicarboxylic acid (FDCA) is one of the main building blocks of polyethylene 2,5-furandicarboxylate (PEF), a plant-based polymer that has a 50-70% smaller carbon footprint than its petroleum-based competitor, polyethylene terephthalate (PET). Currently, PET dominates the global packaging market because it is strong, lightweight and does not break easily. Most of PET is used to make synthetic fibers, while the remaining 30% is used to produce bottles. However, PEF has several advantages over PET. When used in combination with PET, it can be reused up to 5 times more times than PET alone, it degrades much faster than PET, and can replace currently non-recyclable film packaging.

[0006] Previous methods for producing FDCA or producing more basic and natural carbohydrate starting materials such as dihydrofuran have focused on chemical catalytic methods in industry. However, these methods involve the use of high-cost catalysts, which also present significant limitations and process drawbacks. Currently, no biocatalytic process used by the industry can produce FDCA yields comparable to those of the chemical catalytic route.

[0007] Therefore, there is a need to provide new enzymes and methods for producing dihydrofurans from ketoses via a biocatalytic route under thermochemically favorable conditions. Summary of the invention

[0008] Provided herein are methods for producing dihydrofurans from ketoses.

[0009] In one embodiment, the present disclosure relates to a biocatalytic method for producing dihydrofuran, the method comprising contacting 2-keto-3-deoxygluconic acid (KDG) with a glycoside hydrolase, thereby producing dihydrofuran, wherein the contacting comprises: a. a pH of about 4 to about 7 as measured by a pH meter; b. a temperature of 45°C to 74°C; or both a. and b., thereby producing dihydrofuran. In one embodiment, the method comprises a., wherein the pH is about 4 to 5. In one embodiment, the method comprises b., wherein the temperature is 70°C to 74°C. In one embodiment, the method comprises a. and b., wherein the pH is about 4 to 5 and the temperature is about 62°C to 72°C. In one embodiment, the method comprises a. and b., wherein the pH and temperature are selected from the group consisting of: a. pH about 4 and temperature about 63°C; b. pH about 4.5 and temperature about 69°C; and c. pH about 5 and temperature about 72°C. In one embodiment, the method comprises c. In one embodiment, KDG is 180mM to 300mM. In one embodiment, KDG is 180mM to 220mM. In one embodiment, the glycoside hydrolase comprises a protein having at least 80% sequence identity with a sequence selected from SEQ ID NOs: 1-116. In one embodiment, the sequence identity is at least 85%, 90%, 95%, 98%, 99% or 100%. In one embodiment, the glycoside hydrolase comprises a first motif that binds to KDG, and a second motif that comprises a catalytic residue, the catalytic residue comprises aspartic acid, the first motif comprises at least two residues, the first residue comprises arginine, the second residue comprises tryptophan, phenylalanine or tyrosine, and the glycoside hydrolase is determined by SWISS-MODEL modeling to be a homolog of SEQ ID NO: 1 or SEQ ID NO: 19. In one embodiment, the glycoside hydrolase comprises a protein having 100% sequence identity with SEQ ID NO: 27. In one embodiment, the contact time is 0.5 hours to 24 hours. In one embodiment, the contact time is 0.5 hours to 5 hours. In one embodiment, the contact time is about 3 hours. In one embodiment, the method further comprises dehydrating dihydrofuran to generate 5-hydroxymethyl-2-furoic acid (HMFA), wherein after dehydration, a HMFA yield of at least 40% is observed. In one embodiment, dehydration comprises contacting dihydrofuran with an acid selected from the group consisting of formic acid, hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, hydrobromic acid, and C-6 carboxylic acids. In one embodiment, the acid comprises formic acid. In one embodiment, the method further comprises oxidizing HMFA to generate 2,5-furandicarboxylic acid (FDCA). In one embodiment, oxidation comprises a chemical oxidation reaction. In one embodiment, oxidation comprises an enzymatic oxidation reaction.

[0010] In one embodiment, the disclosure relates to an isolated polypeptide having at least 85% identity to any one of SEQ ID NO: 35 to SEQ ID NO: 116. In an embodiment, the disclosure also relates to a biocatalytic method for producing dihydrofuran, the method comprising contacting 2-keto-3-deoxygluconic acid (KDG) with an isolated polypeptide to produce dihydrofuran. In one embodiment, the contacting comprises: a. a pH of about 4 to about 7 as measured by a pH meter; b. a temperature of 45°C to 74°C; or both a. and b., thereby producing dihydrofuran. In one embodiment, the method comprises a., wherein the pH is about 4 to 5. In one embodiment, the method comprises b., wherein the temperature is 70°C to 74°C. In one embodiment, the method comprises a. and b., wherein the pH is about 4 to 5 and the temperature is about 62°C to 72°C. In one embodiment, the method includes a. and b., wherein the pH and temperature are selected from the group consisting of: a. pH about 4 and temperature about 63°C; b. pH about 4.5 and temperature about 69°C; and c. pH about 5 and temperature about 72°C. In one embodiment, the method includes c. In one embodiment, KDG is about 180mM to 300mM. In one embodiment, KDG is about 180mM to 220mM. In one embodiment, the contact time is 0.5 hours to 24 hours. In one embodiment, the contact time is up to 5 hours. In one embodiment, the contact time is about 3 hours. In one embodiment, the method also includes dehydrating dihydrofuran to produce 5-hydroxymethyl-2-furoic acid (HMFA), wherein a HMFA yield of at least 40% is observed after dehydration. In one embodiment, dehydration includes contacting dihydrofuran with an acid selected from the group consisting of: formic acid, hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, hydrobromic acid and Ci-6 carboxylic acid. In one embodiment, the acid includes formic acid. In one embodiment, the method further comprises oxidizing HMFA to generate 2,5-furandicarboxylic acid (FDCA). In one embodiment, the oxidation comprises a chemical oxidation reaction. In one embodiment, the oxidation comprises an enzymatic oxidation reaction. In one embodiment, the disclosure relates to a composition comprising an isolated polypeptide.

[0011] In one embodiment, the present disclosure relates to a modified microorganism comprising an exogenous glycoside hydrolase, wherein the exogenous glycoside hydrolase comprises a sequence having at least 85% sequence identity with a sequence selected from SEQ ID NOs: 1-116. In one embodiment, the exogenous glycoside hydrolase comprises a sequence having at least 90%, 95%, 97%, 98%, 99% or 100% sequence identity with a sequence selected from SEQ ID NOs: 1-116. In one embodiment, the exogenous glycoside hydrolase comprises SEQ ID NO: 27. In one embodiment, the modified microorganism is a bacterium. In one embodiment, the bacterium is selected from the group consisting of E. coli, Saccharomyces sp., Aspergillus sp., Pichia sp., Pseudomonas sp. and Bacillus sp. In one embodiment, the bacterium is E. coli. In one embodiment, the present disclosure relates to a composition comprising a modified microorganism. In one embodiment, the present disclosure is directed to a method of producing polyethylene 2,5-furandicarboxylate (PEF) comprising a biocatalytic process.

[0012] In one embodiment, the present disclosure relates to an isolated non-natural glycoside hydrolase comprising: a first motif that binds to 2-keto-3-deoxygluconic acid, the first motif comprising at least two residues, the first residue being arginine and the second residue being tryptophan, phenylalanine or tyrosine; and a second motif comprising a catalytic residue, the catalytic residue being aspartic acid or glutamic acid, wherein the isolated non-natural glycoside hydrolase has at least 20% identity to SEQ ID NO: 1 or SEQ ID NO: 19. In one embodiment, the isolated non-natural glycoside hydrolase has at least 25%, 45%, 65%, 85%, 95% or 99% identity to SEQ ID NO: 1 or SEQ ID NO: 19. In one embodiment, the second of the at least two residues of the first motif is tryptophan and / or the catalytic residue of the second motif is aspartic acid. In one embodiment, the first motif has a sequence of Rx QTW, wherein x is serine or an aliphatic amino acid. In one embodiment, the first motif has the sequence Rx1QTW(2x2)Yx2Y, wherein x1 is serine and x2 is an aliphatic amino acid. In one embodiment, the second motif has the sequence xD, wherein x is an aliphatic amino acid. In one embodiment, the second motif has the sequence (2x)KSE(3x)DT(2M)xSxPFx, wherein x is an aliphatic amino acid. In one embodiment, the arginine of the first motif and the catalytic residue of the second motif are separated by about 70 residues.

[0013] In one embodiment, the present disclosure relates to an isolated glycoside hydrolase comprising: a sequence of Formula 1: P(19x)LPP(4x)HYHQGVxLxG(4x)(W / Y)(10x)Y(3x)(Y / W)x(D / E)(6x)G(9x)D(2x)Q(P / A)Gx(L / I)L(2x)L(7x)(R / K)Y(2x)(A / G)(3x)(L / I)(9x)(T / N)xE(G / Q)G(F / Y)(W / F)H(K / N)(3x)Px(Q / E)(M / Q)WLDGLYMxG(5x)Y(A / G)(9x)D(4x)Q(6x)(H / K)(T / M)(R / K)(3x)TG L(2x)H(A / G)(W / F)(D / S)(2x)(R / K)(3x)W(A / S)(D / N)(2x)(T / S)Gx(S / A)PE xW(G / A)R(S / A)xGW(9x)(D / E)x(I / L)P(2x)H(20x)Q(4x)GxWxQ(V / I)x(D / N) (K / R)(G / V)(4x)NW(L / P)ExSx(S / T)xL(6x)K(G / A)(15x)(K / Q)(A / G)(F / Y)x G(18x)(I / V)C(I / V)GT(S / G)xGxY(5x)R(5x)D(L / M)HG(V / A)GA(F / L); and with SEQ ID NO: 1, wherein x is any amino acid. In one embodiment, the isolated glycoside hydrolase has at least 55%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or 100% homology to SEQ ID NO: 1. In one embodiment, the isolated glycoside hydrolase further comprises a Y at residue 41; a D at residue 88; an H at residue 132; a W at residue 141; a D at residue 143; an M at residue 147; an H at residue 189; a W at residue 211; an A at residue 212; an R at residue 213; a W at residue 217; an S at residue 278; an L or M at residue 282; a C at residue 330; and an H or K at residue 352, wherein the residues are numbered according to SEQ ID NO: 1. In one embodiment, the isolated glycoside hydrolase further comprises a loop region having at least 21 residues. In one embodiment, the isolated glycoside hydrolase further comprises a loop region having at least one modification selected from the group consisting of 331 to 336 of SEQ ID NO:1.In one embodiment, at least one modification selected from the group consisting of 331 to 336 of SEQ ID NO: 1 includes one or more of the following: V331K, G332E, G332M, G332V, S334A, S334C, S334D, S334E, S334G, S334I, S334K, S334M, S334N, S334Q, S334R, S334T, S334V, A335V, A335P, A335L and A335C. In one embodiment, the isolated glycoside hydrolase further comprises two amino acids appended at the n-terminus of the sequence and five amino acids appended at the c-terminus of the sequence.

[0014] In one embodiment, the present disclosure relates to a biocatalytic method for producing dihydrofuran, comprising: contacting 2-keto-3-deoxygluconic acid (KDG) with a glycoside hydrolase, thereby producing dihydrofuran, the contacting comprising a. a pH of about 4 to about 7 as measured by a pH meter; b. a temperature of 45°C to 74°C; or a and b. both, thereby producing dihydrofuran, wherein the glycoside hydrolase has at least 50% homology to SEQ ID NO: 1, and wherein the glycoside hydrolase comprises a D at residue 143, an R at residue 213, and a W at residue 217 relative to SEQ ID NO: 1. In one embodiment, residue 143 of the glycoside hydrolase is a catalytic residue, and residues 213 and 217 are substrate binding residues.

[0015] In one embodiment, the present disclosure relates to an isolated glycoside hydrolase comprising: a sequence of Formula 2: (F / Y)P(8x)(W / Y)(7x)W(T / M)(2x)F(2x)G(2x)(W / Y)(2x)Y(11x)(A / G)(10x)(L / I)(8x)(H / F)D(L / I)GF(4x)(S / T)(4x)(W / Y)(15x)(A / G)(13x)(L / I)(16x)IDx(L / M) (L / M)(N / S)(22x)H(3x)(T / S)(5x)RxDxS(S / T)(6x)(D / N)(10x)TxQG(4x)SxW(A / S / T)RG(Q / L)(A / T)W(2x)YG(28x)PxD(4x)(Y / W)D(F / L)(12x)S(6x)(S / C)(33x)Y(30x)(W / F / Y)(G / A)DY(Y / F)(2x)ExL; and having at least 50% homology to SEQ ID NO: 19, wherein x is any amino acid. In one embodiment, the isolated glycoside hydrolase has at least 55%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or 100% homology to SEQ ID NO: 19. In one embodiment, the isolated glycoside hydrolase further comprises I or L at residue 26; H at residue 41; W at residue 42; M at residue 43; H at residue 87; D at residue 88; G at residue 134; D at residue 149; T at residue 150; M at residue 152; Q at residue 193; W at residue 219; R at residue 221; W at residue 225; S at residue 280; I at residue 284; and F at residue 352, wherein the residues are numbered according to SEQ ID NO: 19. In one embodiment, the isolated glycoside hydrolase comprises a sequence selected from SEQ ID NOs: 24 to 27. In one embodiment, the isolated glycoside hydrolase further comprises three amino acids appended at the n-terminus of the sequence and five amino acids appended at the c-terminus of the sequence.

[0016] These and other embodiments are described below. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings, which are incorporated herein and form a part of the specification, illustrate some, but not the only or exclusive, example embodiments and / or features. It is intended that the embodiments and drawings disclosed herein be considered illustrative rather than restrictive.

[0018] Figure 1AThe conversion (dehydration) of 2-keto-3-deoxygluconic acid (KDG) (1) to 4,5-dihydro-4-hydroxy-5-hydroxymethyl-2-furancarboxylic acid (K4) (2) is shown, and then K4 is further converted (dehydrated) to 5-hydroxymethyl-2-furoic acid (HMFA) (3).

[0019] Figure 1B It is a two-dimensional diagram of KDG.

[0020] Figure 2A A schematic representation of SEQ ID NO: 1 (a glycoside hydrolase, bound to KDG) is shown. The catalytic residues arginine, tryptophan and aspartate are conserved in both glycosyl hydrolase family 88 and 105 enzymes.

[0021] Figure 2B A representative active site of SEQ ID NO: 1 (a glycoside hydrolase, bound to KDG) is shown. The catalytic residues arginine (1), tryptophan (2), and aspartate (3) are conserved in both glycosyl hydrolase family 88 and 105 enzymes.

[0022] Figure 3 A graphic representation of the geometric parameters of the active site of the glycosyl hydrolase of SEQ ID NO: 1 is shown. Eight geometric parameters (d for distance and θ for bond angle) are specified to describe the spatial position of the functional groups relative to KDG.

[0023] Figure 4 Shown is the detection of K4 determined by liquid chromatography / mass spectrometry (LC / MS) tracking the glycoside hydrolase of SEQ ID NO: 27, which converts KDG to K4 under favorable thermochemical conditions (sodium acetate, pH 4-5, 150 mM NaCl, 63-74°C, 3 hours, 1 uM enzyme and 180 mM KDG substrate), ultimately producing HMFA. The glycoside hydrolase, SEQ ID NO: 27, is used to dehydrate KDG to K4, which is then further dehydrated to HMFA spontaneously and irreversibly.

[0024] Figure 5 Shown is the detection of K4 determined by LC / MS tracking of the glycoside hydrolase of SEQ ID NO: 1, which converts KDG to K4 under favorable thermochemical conditions (sodium acetate, pH 5, 25 mM KDG, 45°C, 3 hours), ultimately producing HMFA. The glycoside hydrolase, SEQ ID NO: 1, is used to dehydrate KDG to K4, which is a detectable intermediate.

[0025] Figure 6 An alignment of the sequences disclosed herein (SEQ ID No: 24 to 27) is shown. The catalytic aspartate residue (D) is indicated by "*", as are the substrate binding residues arginine (R) and tryptophan (W).

[0026] Figure 7 An alignment of functional key residues is shown with reference to SEQ ID NO: 19. Corresponding residues from other sequences are provided. Note that despite the large spatial distance between the amino acids, the degree of correspondence is still high. For SEQ ID Nos: 23-27, additional common residues are shown in bold. DETAILED DESCRIPTION

[0027] The following description includes information that may be useful in understanding the present disclosure. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed disclosure, or that any publication explicitly or implicitly referenced is prior art.

[0028] definition

[0029] While it is believed that the following terms are well understood by those of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the subject matter disclosed herein.

[0030] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. Reference to the techniques employed herein is intended to refer to techniques commonly understood in the art, including modifications of those techniques and / or substitutions of equivalent techniques that are obvious to those skilled in the art.

[0031] As used herein, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise.

[0032] The term "about" or "approximately" means a range (e.g., the value plus or minus 10%) when it is immediately before a numerical value. For example, "about 50" may mean 45 to 55, "about 25,000" may mean 22,500 to 27,500, etc., unless otherwise specified in the context of the present disclosure, or inconsistent with such an explanation. For example, in a numerical list such as "about 49, about 50, about 55...", "about 50" means extending to a range less than half of the interval between the previous value and the next value, for example, greater than 49.5 to less than 52.5. In addition, the phrase "less than about" a value or "greater than about" a value should be understood according to the definition of the term "about" provided herein. Similarly, the term "about" when before a series of numerical values ​​or value ranges (e.g., "about 10, 20, 30" or "about 10-30"), refers to all values ​​in this series or the endpoints of this range, respectively.

[0033] As used herein, the term "microorganism" or "microbe" should be understood in a broad sense. These terms are used interchangeably and include, but are not limited to, two prokaryotic domains, namely bacteria and archaea, as well as certain eukaryotic fungi and protists. In some embodiments, the present disclosure relates to "microorganisms" in the lists and diagrams present in the present disclosure. This characterization can refer not only to identified taxonomic genera, but also to identified taxonomic species, as well as various novel and newly identified or designed strains of any organism in the table or diagram. The same characterization applies to other parts of the specification, such as the description of these terms in the examples.

[0034] When referring to nucleic acid sequences or protein sequences, the term "identity" is used to indicate the similarity between two sequences. Sequence similarity or identity can be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith and Waterman, Adv. Appl. Math. 2, 482 (1981), by the sequence identity alignment algorithm of Needleman and Wunsch, J Mol. Biol. 48, 443 (1970), by the similarity search method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85, 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA by Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), the best fit sequence programs described by Devereux et al., Nucl. Ac id Res. 12, 387-395 (1984), or by inspection. Another suitable algorithm is the BLAST algorithm, described in Altschul et al., J Mol. Biol. 215, 403-410, (1990) and Karlin et al., Proc. Natl. Acad. Sci. USA 90, 5873-5787 (1993). A particularly useful BLAST program is the WU-BLAST-2 program, available from Altschul et al., Methods in Enzymology, 266, 460-480 (1996); blast.wustl / edu / blast / README.html. WU-BLAST-2 uses several search parameters, which are optionally set to default values. The parameters are dynamic values ​​and are established by the program itself based on the composition of the sequence and the composition of the particular database being searched for the sequence of interest; however, these values ​​can be adjusted to increase sensitivity. In addition, another useful algorithm is Gapped BLAST, as reported in Altschul et al., (1997) Nucleic Acids Res. 25, 3389-3402. Unless otherwise stated, percent identity herein is determined using an algorithm available on the Internet at blast.ncbi.nlm.nih.gov / Blast.cgi.

[0035] As used herein, an "isolated" or "purified" polynucleotide or polypeptide, or a biologically active portion thereof, is substantially or essentially free of components that normally accompany or interact with the polynucleotide or polypeptide as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or polypeptide is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Optimally, an "isolated" polynucleotide is free of sequences (optimally protein coding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various embodiments, an isolated polynucleotide may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. Polypeptides that are substantially free of cellular material include polypeptide preparations having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating proteins.

[0036] As used herein, the term "nucleic acid" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or their analogs. The term refers to the primary structure of the molecule and thus includes double-stranded and single-stranded DNA, and double-stranded and single-stranded RNA. It also includes modified nucleic acids, such as methylated nucleic acids and / or capped nucleic acids, nucleic acids containing modified bases, backbone modifications, and the like.

[0037] As used herein, "SWISS-MODEL" refers to a fully automated protein structure homology modeling server for 3D protein structure homology modeling, which can be accessed through the Expasy web server or the DeepView (Swiss Pdb-Viewer) program. SWISS-MODEL consists of three integrated components: (1) SWISS-MODEL pipeline - a set of software tools and databases for automated protein structure modeling; (2) SWISS-MODEL workspace - a web-based graphical user workbench; and (3) SWISS-MODEL repository - a continuously updated homology model database for a set of model organism proteomes of high biomedical interest. Using the SWISS-MODEL pipeline includes four major steps that involve building a homology model of a given protein structure: (1) Identification of structural templates. BLAST and HHblits are used to identify templates. Templates are stored in the SWISS-MODEL Template Library (SMTL) derived from the PDB. (2) Alignment of target and template sequences. (3) Model building and energy minimization. SWISS-MODEL implements a rigid fragment assembly method for modeling. (4) The quality of the model was evaluated using QMEAN (statistical potential of mean force).

[0038] The present disclosure provides enzymes and biocatalytic methods for producing dihydrofuran and downstream products such as 5-hydroxymethyl-2-furoic acid (HMFA), 2,5-furandicarboxylic acid (FDCA), methyl furandicarboxylate (FDME), and polyethylene 2,5-furandicarboxylate (PEF). FDME is the methyl ester of FDCA and is a derivative that can be polymerized with ethylene glycol to produce PEF. A method comprising a biocatalytic process is also provided for producing dihydrofuran by contacting a substrate ketose with a glycoside hydrolase to produce dihydrofuran. Dihydrofuran can be further processed to produce HMFA. In some embodiments, HMFA can also be further processed by chemical or biocatalytic means to produce FDCA. FDCA can be used to produce PEF.

[0039] Also provided are isolated and / or modified glycoside hydrolases that can be used to effect the dehydration of ketoses, such as 2-keto-3-deoxygluconic acid (KDG).

[0040] Glycoside hydrolase

[0041] One or more glycoside hydrolases or their motifs are provided herein. Glycoside hydrolases are enzymes that can hydrolyze glycosidic bonds between carbohydrates or between carbohydrates and non-carbohydrate moieties. In some embodiments, glycoside hydrolases can be used in biocatalytic reactions to dehydrate substrates (e.g., KDG).

[0042] Glycosyl hydrolases can be divided into different families based on sequence similarity. This classification can be found on the CAZy (CArbohydrate-Active EnZymes) website. Because the folding of a protein is more conservative than its sequence, some families can be classified as "clans." In some embodiments, a glycoside hydrolase can be part of any of the 128 glycoside hydrolase families and / or part of any identified clan.

[0043] In some embodiments, the glycoside hydrolase is from the GH88 and / or GH105 family. In some embodiments, the glycoside hydrolase has a classification of E.C3.2.1.179 and / or E.C3.2.1.172. In some embodiments, the glycoside hydrolase comprises Figure 2A , Figure 2B and / or Figure 3 The geometry and / or active sites provided.

[0044] In some embodiments, computational methods can be used to identify and / or design the geometry of glycoside hydrolases capable of undergoing a dehydration reaction with a substrate (eg, KDG). Figure 1B A linear representation of KDG is shown. Figure 1A An example of such a reaction is shown. The biodehydration reaction of ketose (e.g., KDG 1) to dihydrofuran (e.g., FDCA 2) can be carried out by glycosyl hydrolases (further dehydration to HMFA 3). In one example, the hydroxyl group of KDG1 is protonated by aspartate to fully activate it and cause it to leave. Reference Figure 2A and Figure 2B , partially showing that the overall structure of the glycosyl hydrolase is folded into an (alpha / alpha)6 fold. In addition, Figure 2B Glycosyl hydrolase residues that are critical to the reaction are shown. For example, aspartic acid 3 can act as a general acid / base. In the first step, aspartic acid 3 acts as an acid to provide a proton to the leaving hydroxyl group at the anomeric carbon C1 of the substrate (e.g., KDG). Aspartic acid 3 (i.e., the catalytic residue) is located about 100 μg / cm2 from the anomeric carbon C1 of the substrate. In the second step, aspartate 3 acts as a base to abstract a proton from the anomeric carbon C2, thereby promoting the dehydration reaction. Substrate binding occurs through the interaction of the ketocarboxylic acid group on C1 with the arginine 1 and tryptophan 2 residues in the active site.

[0045] In some embodiments, the glycoside hydrolase comprises an active site geometry listed in Table 1. Figure 3Table 1 provides exemplary active site / residue geometries of glycoside hydrolase polypeptides that can be used for KDG dehydration. For example, Table 1 describes the distances (e.g., d1, d2, d3, d4) and angles (e.g., θ1, θ2, θ3, θ4) at each key residue, such as Figure 3 shown.

[0046] In some embodiments, residue #1 and residue #2 in Table 1 coordinate to the carboxylate group at the anomeric carbon C1 of KDG within the active site. In some embodiments, residue #1 and residue #2 are arginine (Arg) and tryptophan (Trp), respectively. In some embodiments, residue #3 has a catalytic effect and acts as a proton donor, adding a proton to the leaving hydroxyl group at the anomeric carbon C1 of KDG. As shown above, catalytic residue #3 is about 100 μg / cm2 away from the anomeric carbon C1 with the leaving hydroxyl group. In some embodiments, residue #1, residue #2, and residue #3 perform glycoside hydrolase activity in the KDG dehydration reaction.

[0047] Table 1 - Exemplary geometries of glycoside hydrolases for KDG dehydration

[0048]

[0049] In some embodiments, glycoside hydrolases comprising substantially similar geometries as provided in Table 1 are also contemplated. For example, the glycoside hydrolase may further comprise residues, such as residue #1, residue #2, and residue #3, whose values ​​are within about 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 angstroms of the values ​​shown in Table 1. In some embodiments, the glycoside hydrolase comprises residues, such as residue #1, residue #2, and residue #3, whose values ​​are within at least 0.5 angstroms of the values ​​shown in Table 1. Similarly, for example, the glycoside hydrolase may comprise residues, such as residue #1, residue #2, and residue #3, whose values ​​are within about 1.0, 2.5, 5.0, 10, 15, 30, 45, 60, 90, 120, 150, and 180 degrees of the values ​​shown in Table 1.

[0050] In embodiments, provided are glycoside hydrolases comprising the geometry shown in Table 1. In embodiments, provided are isolated polypeptides encoding glycoside hydrolases comprising the geometry shown in Table 1.

[0051] Glycoside hydrolase motif

[0052] Also provided is the motif of the glycoside hydrolase. "Motif" is intended to refer to a part of a polynucleotide or a part of an amino acid sequence. The motif may retain the activity of KDG dehydration. Therefore, the motif range of the polynucleotide sequence can vary from at least about 2 nucleotides, about 10 nucleotides, about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, to the full-length polynucleotide sequence corresponding to the glycoside hydrolase. In some embodiments, the motif of a glycoside hydrolase is at least about 2, 5, 8, 10, 35, 60, 85, 110, 135, 160, 185, 210, 235, 260, 285, 310, 335, 360, 385, 410, 435, 460, 485, 510, 535, 560, 585, 610, 635, 660, 685, 710, 735, 760, 785, 810, 835, 860, 885, 910, 935, 960, 985, or up to about 1000 amino acid residues, or up to about the total number of amino acid residues present in a full-length glycoside hydrolase, such as any one of SEQ ID NOs: 1-116.

[0053] In some embodiments, the motif of the glycoside hydrolase comprises a biologically active portion of a glycoside hydrolase capable of at least partially dehydrating KDG. In some embodiments, the motif comprises a residue geometry as shown in any one of Table 1 or has a substantially similar residue geometry.

[0054] In some embodiments, the motif of the glycoside hydrolase can be prepared by isolating a portion of one of the polynucleotides encoding a polypeptide capable of KDG dehydratation, expressing the encoding portion of the polypeptide capable of KDG dehydratation (e.g., by in vitro recombinant expression), and assaying for KDG dehydratase activity.

[0055] In some embodiments, glycoside hydrolase is provided in any form. In some embodiments, glycoside hydrolase is present in DNA, RNA, protein or a combination thereof. In some embodiments, glycoside hydrolase is provided in an isolated form. In some embodiments, glycoside hydrolase is provided as a whole cell system.

[0056] In some embodiments, the present invention provides a recombinant glycoside hydrolase polypeptide comprising an amino acid sequence that is at least 10% to at least 99.73% identical to an amino acid sequence selected from SEQ ID NO: 1-116. In some embodiments, the recombinant glycoside hydrolase polypeptide comprises an amino acid sequence selected from SEQ ID NO: 1-116. In some embodiments, the glycoside hydrolase polypeptide further comprises a tag amino acid sequence. In some embodiments, the tag amino acid sequence is His6.

[0057] In some embodiments, the composition comprising the glycoside hydrolase is at least partially pure. In some embodiments, the composition comprising the glycoside hydrolase is pure basically. The purity of the glycoside hydrolase can change, for example, it can be provided as a rough, semi-purified or purified enzyme. In some embodiments, the glycoside hydrolase polypeptide does not contain impurities.

[0058] Modified glycoside hydrolases

[0059] The present disclosure also provides modified glycoside hydrolases. In some embodiments, the glycoside hydrolases provided herein include one or more modifications. The modification may occur in any region of the glycoside hydrolase. In some embodiments, the modification is within the active site. Modifications to the active site can enhance the binding and / or catalytic efficiency of the glycoside hydrolase to a substrate (e.g., KDG). In some embodiments, the modified glycoside hydrolase provided herein includes modifications around the catalytic residue to recognize, bind to KDG and / or be more catalytically efficient to KDG. In some embodiments, the glycoside hydrolase is modified to include the geometry shown in Table 1 or a geometry substantially similar thereto. In some embodiments, the glycoside hydrolase is modified to improve the geometry listed in Table 1, thereby improving the recognition, binding and / or catalytic efficiency of the substrate (e.g., KDG). Such modifications can be learned from the experimental results shown in Table 3. Different reaction conditions were evaluated for each of SEQ ID NOs: 1-116, and the reaction results were evaluated.

[0060] Any method can be used to generate modified glycoside hydrolases. In some embodiments, the nucleotide sequence or amino acid sequence is modified to produce a recombinant glycoside hydrolase. In some embodiments, the amino acid sequence is modified. Modifications include one or more substitutions, deletions, insertions and any combination thereof. Modifications can include use of natural amino acid residues, synthetic amino acid residues or a combination thereof. In some embodiments, modifications include substitutions.

[0061] In some embodiments, the polynucleotide encoding the glycoside hydrolase polypeptide is modified. The modified polynucleotide may include a deletion. In some cases, the deletion is a base truncation at the 5' end and / or the 3' end and / or one or more nucleotides are deleted at one or more internal sites in the natural polynucleotide. In some cases, the modification includes inserting one or more bases at any position in the 5' end, 3' end and / or one or more internal sites of the polynucleotide. In some embodiments, the modification includes replacing one or more nucleotides at one or more sites in the polynucleotide. With respect to polynucleotides, the modification may include conservative modifications. Conservative modifications may include amino acid substitutions in proteins, changing a given amino acid to a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, and / or size). In some embodiments, conservative modifications include sequences encoding the amino acid sequence of any polypeptide capable of KDG dehydration due to the degeneracy of the genetic code.

[0062] In some embodiments, modified glycoside hydrolase refers to a modified sequence encoding a polypeptide or protein. Protein modification can include deletion, truncation, addition, substitution or a combination thereof. In some embodiments, the glycoside hydrolase protein is modified by truncating at the 5' end and / or 3' end. In certain embodiments, the glycoside hydrolase coding sequence is modified by adding, deleting or adding and deleting one or more residues at the 5' end, 3' end and / or internal region. Modified glycoside hydrolase can retain biological activity. In some embodiments, compared with unmodified glycoside hydrolase, modified glycoside hydrolase retains considerable biological activity. In some cases, biological activity may be reduced. In some cases, biological activity can be increased by modification.

[0063] In some embodiments, the modified glycoside hydrolase comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from SEQ ID NOs: 1-116, an active variant thereof, a fragment thereof, a modified version thereof. In some embodiments, the modified glycoside hydrolase comprises an amino acid sequence that is at least 10% to at least 99.73% identical to an amino acid sequence selected from SEQ ID NOs: 1-116. In some embodiments, the modified glycoside hydrolase comprises an amino acid sequence that is at least 82% identical to SEQ ID NO: 1. In some embodiments, the modified glycoside hydrolase comprises an amino acid sequence that is at least 88% identical to SEQ ID NO: 19. In some embodiments, the modified glycoside hydrolase comprises an amino acid sequence that is at least 85%, 87%, 89%, 91%, 93%, 95%, 97%, 99% or 100% identical to SEQ ID NO: 27. In some embodiments, the computationally designed glycoside hydrolases comprise SEQ ID NOs: 35-116.

[0064] In some embodiments, polynucleotides encoding modified glycoside hydrolases are also provided.In some embodiments, polynucleotides encoding modified glycoside hydrolases comprising any one of SEQ ID NOs: 1-116 are provided.

[0065] In some embodiments, the glycoside hydrolase or motif thereof comprises KDG dehydratability activity and comprises an active site having a catalytic residue geometry as shown in Table 1, or having a substantially similar catalytic residue geometry. In some embodiments, the glycoside hydrolase or motif thereof comprising KDG dehydratability activity and comprising an active site having a catalytic residue geometry as shown in Table 1 further comprises an amino acid sequence having at least 10%, 20%, 30%, 40%, 75%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% percent identity to any one of SEQ ID NOs: 1-116.

[0066] In some embodiments, the glycoside hydrolase comprises an active site having a catalytic residue geometry as shown in any one of Table 1, or having a substantially similar catalytic residue geometry, and further comprises: (a) an amino acid sequence having at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-34, wherein (i) the encoded polypeptide corresponds to SEQ ID NOs: The amino acid residue at amino acid position 40 of SEQ ID NO:1 comprises histidine or glycine or cysteine ​​or serine or tyrosine or phenylalanine or isoleucine or asparagine or glutamine or aspartic acid or glutamic acid; (ii) the amino acid residue at amino acid position 41 of SEQ ID NO:1 in the encoded polypeptide comprises tyrosine or tryptophan; (iii) the amino acid residue at amino acid position 42 of SEQ ID NO:1 in the encoded polypeptide comprises histidine or threonine or methionine or proline or glycine or glutamic acid or glutamine or asparagine or isoleucine or leucine or valine or serine or tryptophan; (iv) the amino acid residue at amino acid position 88 of SEQ ID NO:1 in the encoded polypeptide comprises aspartic acid or leucine or isoleucine or phenylalanine or asparagine or lysine; (v) the amino acid residue at amino acid position 41 of SEQ ID NO:1 in the encoded polypeptide comprises tyrosine or tryptophan; The amino acid residue at amino acid position 132 of SEQ ID NO:1 comprises histidine or alanine or leucine or valine or serine or cysteine ​​or proline or aspartic acid or glutamic acid or asparagine or arginine or glycine or glutamine; (vi) the amino acid residue at amino acid position 146 of SEQ ID NO:1 in the encoded polypeptide comprises tyrosine or phenylalanine or methionine; (vii) the amino acid residue at amino acid position 147 of SEQ ID NO:1 in the encoded polypeptide comprises methionine or alanine or leucine or phenylalanine; (viii) the amino acid residue at amino acid position 189 of SEQ ID NO:1 in the encoded polypeptide comprises histidine or arginine or glutamine or valine or alanine; (ix) the amino acid residue at amino acid position 211 of SEQ ID NO:1 in the encoded polypeptide comprises tryptophan; (x) the amino acid residue at amino acid position 214 of SEQ ID NO:1 in the encoded polypeptide comprises serine or alanine or glycine; (xi) the amino acid residue at amino acid position 216 of SEQ ID NO:1 in the encoded polypeptide comprises serine or alanine or glycine; The amino acid residue at amino acid position 220 of NO: 1 comprises methionine or tyrosine or valine or leucine or alanine or glycine or phenylalanine;(xii) the amino acid residue at amino acid position 278 of SEQ ID NO: 1 in the encoded polypeptide comprises serine; (xiii) the amino acid residue at amino acid position 282 of SEQ ID NO: 1 in the encoded polypeptide comprises leucine or methionine or isoleucine or valine or phenylalanine or threonine or glycine or glutamine; (xiv) the amino acid residue at amino acid position 352 of SEQ ID NO: 1 in the encoded polypeptide comprises histidine or tyrosine or tryptophan or phenylalanine or lysine or valine or arginine; (xv) the amino acid residues at amino acid positions 211-217 of SEQ ID NO: 1 in the encoded polypeptide comprise a fragment of W(A / G / S / T)R(G / A / S)(N / Q / I / L / M)(G / A / T)W; (xvi) the amino acid residue at amino acid position 282 of SEQ ID NO: 1 in the encoded polypeptide comprises leucine or methionine or isoleucine or valine or phenylalanine or threonine or glycine or glutamine; The amino acid residues at amino acid positions 141-145 of SEQ ID NO: 1 include a fragment of (W / I)(L / I / C / A / V / S)D(G / D / A / C / T / V / I / N)(L / M / I / V); and / or (xvii) amino acid residues in the encoded protein corresponding to the amino acid positions of SEQ ID NO: 1 shown in Table 1 and corresponding to the specific amino acid substitutions shown in (d)(i)-(d)(xvii) above, or any combination of these residues.;

[0067] In some embodiments, the glycoside hydrolase comprises a point mutation in SEQ ID NO: 1 selected from the group consisting of: H42I, H42T, H42V, H42W, D88C, D88N, H132E, K133M, W141Y, H189A, H189V, V331K, G332E, G332M, G332V, S334A, S334C, S334D, S334E, S334G, S334I, S334K, S334M, S334N, S334Q, S334R, S334T, S334V, A335V, A335P, A335L, A335C, H352R, and H352V. In some embodiments, the glycoside hydrolase comprises a modification of a residue selected from 332, 334, and 335 of SEQ ID NO: 1. In some embodiments, the glycoside hydrolase comprises a modification in a loop region. In some embodiments, the modification in the loop region comprises any one of the residues at amino acid position numbers 331-336 of SEQ ID NO: 1. In some embodiments, the glycoside hydrolase comprises a point mutation in SEQ ID NO: 19 selected from the group consisting of D41A, D41C, D41E, D41N, D41S, D41T, H87A, H87C, H87E, H87G, H87Q, H87R, H87S, L152M, L152N, W225Y, S337A, Y338V, H339A, H339N, W352F, Y356A, Y356C, Y356F, and Y356H. In embodiments, the point mutation confers increased activity compared to an otherwise comparable glycoside hydrolase lacking the point mutation.

[0068] In some embodiments, the glycoside hydrolases provided herein may not comprise the catalytic residue geometries as shown in any one of Table 1, but retain KDG dehydration activity.

[0069] In some embodiments, a separated polypeptide is provided, which comprises a sequence encoding any provided glycoside hydrolase. In embodiments, the separated polypeptide comprises: a first motif that binds to 2-keto-3-deoxygluconic acid and a second motif that comprises a catalytic residue. In embodiments, the first motif comprises at least two residues, wherein the first residue comprises arginine, and wherein the second residue comprises tryptophan, phenylalanine or tyrosine. In embodiments, the second residue comprises tryptophan. In embodiments, the second residue comprises phenylalanine. In embodiments, the second residue comprises tyrosine. In some embodiments, the catalytic residue comprises aspartic acid. In some embodiments, the separated polypeptide is determined to be a homologue of SEQ ID NO: 1 or SEQ ID NO: 19 by SWISS-MODEL homology modeling. In some embodiments, the separated polypeptide has at least about 25%, 35%, 45%, 55%, 65%, 75%, 85%, 95%, 97% or 100% identity with SEQ ID NO: 1 or SEQ ID NO: 19.

[0070] In embodiments, a separated polypeptide is provided, which comprises a sequence encoding any provided glycoside hydrolase. In embodiments, the separated polypeptide comprises: a first motif that binds to 2-keto-3-deoxygluconic acid and a second motif that comprises a catalytic residue (or vice versa). In embodiments, the first motif comprises at least two residues. The at least two residues can be one of arginine and tryptophan, phenylalanine and tyrosine. In embodiments, the second residue of the first motif can be tryptophan. In embodiments, the second residue of the first motif can be phenylalanine. In embodiments, the second residue of the first motif can be tyrosine. In some embodiments, the catalytic residue of the second motif comprises aspartic acid or glutamic acid. In embodiments, the catalytic residue of the second motif is aspartic acid. In some embodiments, the separated polypeptide is determined to be a homologue of SEQ ID NO: 1 or SEQ ID NO: 19 by SWISS-MODEL homology modeling. In some embodiments, the isolated polypeptide is at least about 25%, 35%, 45%, 55%, 65%, 75%, 85%, 95%, 97% or 100% identical to SEQ ID NO:1 or SEQ ID NO:19.

[0071] In embodiments, alanine scanning, which is interchangeable with other site-directed mutagenesis techniques, can be used to determine the contribution of specific residues to the stability and function of the modified glycoside hydrolase. The results of these analyses are reflected throughout the document.

[0072] In embodiments, the glycoside hydrolase does not comprise a modification to SE Q ID NO: 1 selected from the group consisting of D88 (G / I / L / N / Q / A / R / V / W / Y), H132 (M / P / R / T / W / K / Y / D / C / G), H189 (F / I / L / N / Y / T / V / S), H352 (W / G / E / K / Q), H40 (I / K / L / P / R / W), H42(C / K / Q / S), M147(A / D / F / K / N / H / S / V / E / G / L / P / I / Q / T / Y / C / W), W141(A / D / H / K / M / P / R / T / Y / E / I / N / Q / S / V / C / F), W211(E / L / V / A / F / M / R / G / I / K / N / Q) and Y41(A / D / F / M / R / G / C / E / H / K / N / P / Q / V / I / T).

[0073] According to one embodiment, the isolated polypeptide comprises a first motif that binds to KDG and a second motif that comprises a catalytic residue. In one embodiment, the first motif and the second motif are separated by about 70 residues. In one embodiment, the arginine of the first motif and the catalytic residue of the second motif are separated by 70 residues. In one embodiment, the arginine of the first motif and the catalytic residue of the second motif are separated by 72 residues.

[0074] In one embodiment, the first motif may be at least 5 residues, at least 10 residues, at least 15 residues, at least 20 residues, at least 30 residues, at least 50 residues, at least 100 residues, etc. In one embodiment, the first motif may be 5 residues and have a sequence of RxQTW, wherein R is arginine, x is serine or an aliphatic amino acid, such as glycine, alanine, valine, leucine, isoleucine, and proline, Q is glutamine, T is threonine, W is tryptophan, and R and W are, independently or in combination, substrate binding residues. In one embodiment, the first motif may be 10 residues and have a sequence of Rx1QTW(2x2)Yx2Y, wherein R is arginine, x1 is serine, x2 is an aliphatic amino acid, such as glycine, alanine, valine, leucine, isoleucine, and proline, Q is glutamine, T is threonine, W is tryptophan, Y is tyrosine, and R and W are, independently or in combination, substrate binding residues.

[0075] In one embodiment, the first motif may be at least 5 residues, at least 10 residues, at least 15 residues, at least 20 residues, at least 30 residues, at least 50 residues, at least 100 residues, etc. In one embodiment, the second motif may be 2 residues and have the sequence xD, wherein x is an aliphatic amino acid, such as glycine, alanine, valine, leucine, isoleucine and proline, and D is aspartic acid, and D is a catalytic residue. In one embodiment, the second motif may be 18 residues and have the sequence (2x)KSE(3x)DT(2M)xSxPFx, wherein x is an aliphatic amino acid, such as glycine, alanine, valine, leucine, isoleucine and proline, K is lysine, S is serine, E is glutamic acid, D is aspartic acid, T is threonine, M is methionine, P is proline, F is phenylalanine, and D is a catalytic residue.

[0076] In one embodiment, the modified glycoside hydrolase comprises a sequence of Formula 1: P(19x)LPP(4x)HYHQGVxLxG(4x)(W / Y)(10x)Y(3x)(Y / W)x(D / E)(6x)G(9x)D(2x)Q(P / A)Gx(L / I)L(2x)L(7x)(R / K)Y(2x)(A / G)(3x)(L / I)(9x)(T / N)xE(G / Q)G(F / Y)(W / F)H(K / N)(3x)Px(Q / E)(M / Q)WLDGLYMxG(5x)Y(A / G)(9x)D(4x)Q(6x)(H / K)(T / M)(R / K)(3x)TGL(2x) H(A / G)(W / F)(D / S)(2x)(R / K)(3x)W(A / S)(D / N)(2x)(T / S)Gx(S / A)PExW(G / A)R(S / A)xGW(9x)(D / E)x(I / L)P(2x)H(20x)Q(4x)GxWxQ(V / I)x(D / N)(K / R)(G / V)(4x)NW(L / P)ExSx(S / T)xL(6x)K(G / A)(15x)(K / Q)(A / G)(F / Y)xG(18x)(I / V)C(I / V)GT(S / G)xGxY(5x)R(5x)D(L / M)HG(V / A)GA(F / L), wherein Formula 1 has at least 50% homology with SEQ ID NO: 1, and x can be any amino acid. In one embodiment, the modified glycoside hydrolase has at least 55%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or 100% homology to SEQ ID NO: 1. In one embodiment, the modified glycoside hydrolase comprises Y at residue 41, D at residue 8, H at residue 132, W at residue 141, D at residue 143, M at residue 147, H at residue 189, W at residue 211, G at residue 212, R at residue 213, W at residue 217, S at residue 278, L or M at residue 282, C at residue 330, and H or K at residue 352, wherein the residues are numbered according to SEQ ID NO: 1. In one embodiment, the modified glycoside hydrolase comprises a loop region having at least 21 residues. In one embodiment, the modified glycoside hydrolase comprises a loop region having at least one modification selected from the group consisting of 331 to 336 of SEQ ID NO:1.In one embodiment, the modified glycoside hydrolase comprises at least one modification selected from the group consisting of 331 to 336 of SEQ ID NO: 1, including one or more of the following: V331K, G332E, G332M, G332V, S334A, S334C, S334D, S334E, S334G, S334I, S334K, S334M, S334N, S334Q, S334R, S334T, S334V, A335V, A335P, A335L and A335C.

[0077] In one embodiment, the modified glycoside hydrolase comprises a sequence of Formula 2: (F / Y)P(8x)(W / Y)(7x)W(T / M)(2x)F(2x)G(2x)(W / Y)(2x)Y(11x)(A / G)(10x)(L / I)(8x)(H / F)D(L / I)GF(4x)(S / T)(4x)(W / Y)(15x)(A / G)(13x)(L / I)(16x)IDx(L / M)(L / M) (N / S)(22x)H(3x)(T / S)(5x)RxDxS(S / T)(6x)(D / N)(10x)TxQG(4x)SxW(A / S / T)RG(Q / L)(A / T)W(2x)YG(28x)PxD(4x)(Y / W)D(F / L)(12x)S(6x)(S / C)(33x)Y(30x)(W / F / Y)(G / A)DY(Y / F)(2x)ExL, wherein Formula 2 has at least 50% homology to SEQ ID NO: 19, and x can be any amino acid. In one embodiment, the modified glycoside hydrolase has at least 55%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or 100% homology to SEQ ID NO: 19. In one embodiment, the modified glycoside hydrolase comprises I or L at residue 26, H at residue 41, W at residue 42, M at residue 43, H at residue 87, D at residue 88, G at residue 134, D at residue 149, T at residue 150, M at residue 152, Q at residue 193, W at residue 219, R at residue 221, W at residue 225, S at residue 280, I at residue 284, and F at residue 352, wherein the residues are numbered according to SEQ ID NO: 19. In one embodiment, the modified glycoside hydrolase comprises a sequence selected from SEQ ID NOs: 24 to 27, or a sequence homologous thereto. Figure 6 An alignment of SEQ ID NOs: 24 to 27 is shown, wherein residues marked with * correspond to catalytic residues and substrate binding residues, as described above. Figure 7The same content is given in and further describes other sequences having the same residues at similar positions relative to SEQ ID NO: 19.

[0078] In one embodiment, the modified glycoside hydrolase comprises a sequence of Formula 3, (W / Y)7x(A / G)(92x)WxD(35x)(L / M)(9x)(H / R)(22x)W(A / G / S)R(2x)(G / S)W(8x)(L / I)(27x)Q(3x)(G / K)xW(3x)(I / L)(9x)ExSx(S / T)(9x)(A / G)(52x)Gx(G / A), wherein Formula 3 is at least 50% homologous to SEQ ID NO: 2. In one embodiment, the modified glycoside hydrolase is at least 55%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or 100% homologous to SEQ ID NO: 2.

[0079] Modification method

[0080] In some embodiments, one or more methods can be used to modify the glycoside hydrolase polypeptide and / or its motif. In some embodiments, modifications are identified by rational design modeling. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants and fragments of KDG dehydration polypeptides can be prepared by mutations of polynucleotide sequences. Methods for mutagenesis and polynucleotide alterations are well known in the art. For example, see Kunkel (1985) Proc. Natl. Acad. Sci. USA 82: 488-492; Kunkel et al. (1987) Methods in Enzymol. 154: 367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, ed. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and references cited therein. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the target protein can be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC), the entire contents of which are incorporated herein by reference. Conservative substitutions are also considered, such as exchanging one amino acid with another amino acid having similar properties. In some embodiments, the modification (e.g., mutation) may not cause the sequence to be out of the reading frame. In some embodiments, the modification does not produce a complementary region that can produce a secondary mRNA structure, see European Patent Application Publication No. 75,444.

[0081] In some embodiments, suitable modifications can be identified using well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques and sequencing techniques. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated by using site-directed mutagenesis or gene synthesis but still encoding a polypeptide capable of KDG dehydration or polynucleotides generated by computational modeling.

[0082] In an embodiment, SWISS-MODEL homology modeling can be used to perform homology modeling on the designed and / or generated sequences. In an embodiment, homology modeling is performed using the target glycoside hydrolase sequence as a template. In an embodiment, homology modeling includes: (a) performing a template search to find related homologues; (b) sorting the templates identified from (a) according to global model quality estimation (GMQE) and / or quaternary structure quality estimation (QSQE); (c) determining whether the top-ranked templates cover different regions of the target protein (e.g., glycoside hydrolase) and / or determining whether the top-ranked templates represent different conformational states; and (d) selecting templates. In an embodiment, the top-ranked templates may include the top 1-10, 1-30, 1-50, or 1-100 templates. In embodiments, the selected template may have about 1-5%, 1-10%, 1-20%, 1-30%, 10-30%, 20-40%, 20-60%, 25-65%, 30-70%, 50-80%, 60-95%, or 25-85% identity to the target glycoside hydrolase sequence. In embodiments, the selected template may have at least about 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, or 100% identity to the target glycoside hydrolase sequence. In embodiments, the selected template may have at least about 27% identity to the target glycoside hydrolase.

[0083] Method for preparing dihydrofuran

[0084] A biocatalytic method for producing dihydrofuran from a composition comprising a substrate ketose is also provided. As used herein, "biocatalysis" or "biocatalytic" refers to the use of natural catalysts, such as proteases, to chemically transform organic compounds. Biocatalysis is alternatively referred to as biotransformation or biosynthesis. The biocatalyst protease can be a naturally occurring or recombinant protein. In some embodiments, a method for preparing dihydrofuran by dehydrating ketose is also provided herein. In some embodiments, a glycoside hydrolase polypeptide is capable of converting a ketose moiety into dihydrofuran. The provided method can include any of the described glycoside hydrolases, modified glycoside hydrolases, and portions thereof, including but not limited to glycoside hydrolases comprising a sequence selected from SEQ ID NO: 1-116.

[0085] The method can be biocatalytic, i.e., utilize a biocatalyst. In some embodiments, the biocatalyst is a protease. In some embodiments, the biocatalyst is a glycoside hydrolase polypeptide. In some embodiments, the substrate is a ketose, which is dehydrated by the glycoside hydrolase to generate dihydrofuran.

[0086] Glycoside hydrolases can be provided in free form or fixed form. In some embodiments, glycoside hydrolase preparations can be rough, semi-purified and / or purified. In some embodiments, glycoside hydrolases are provided as a full cell system, for example live or non-live microbial cells, or whole microbial cells, cell lysates and / or any other form known in the art.

[0087] In some embodiments, provided herein is a method for producing a dihydrofuran composition, comprising the steps of: (a) providing a substrate ketose, such as KDG; (b) contacting the ketose with a glycoside hydrolase polypeptide; (c) producing a composition comprising dihydrofuran; and (d) dehydrating the dihydrofuran at an elevated temperature.

[0088] In some embodiments, provided herein is a method for producing a dihydrofuran composition comprising the steps of: (a) providing a composition comprising greater than about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.6% by weight of a substrate ketose on an anhydrous basis; (b) contacting the composition with a glycoside hydrolase polypeptide; (c) producing a composition comprising dihydrofuran; and (d) dehydrating the dihydrofuran at an elevated temperature.

[0089] Substrate

[0090] In some embodiments, the methods provided herein include contacting a substrate (eg, a ketose) with a glycoside hydrolase or a motif thereof.

[0091] In some embodiments, the method provided herein includes contacting a substrate with a glycoside hydrolase or its motif. The substrate may include ketose. In some embodiments, the substrate is a ketose 2-keto-3-deoxygluconic acid, wherein the 2-keto-3-deoxygluconic acid is used as a substrate for glycoside hydrolase biotransformation. Ketose can be synthetic, purified (partially or entirely), commercially available or prepared. An example of a useful composition in the disclosed method is chemically synthesized 2-keto-3-deoxygluconic acid, which is dissolved with a solvent. Another example of a substrate is enzymatically synthesized 2-keto-3-deoxygluconic acid in water. Another example of a substrate is 2-keto-3-deoxygluconic acid fermented in a fermentation broth.

[0092] In some embodiments, the composition comprises purified substrate ketose. For example, the composition can comprise greater than about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.6% substrate ketose by weight on an anhydrous basis.

[0093] In some embodiments, the composition comprises partially purified substrate ketose. For example, the composition contains greater than about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, or about 50% substrate ketose by weight on an anhydrous basis.

[0094] In some embodiments, the composition includes a substrate ketose (e.g., KDG). For example, the composition may include about 250 μM to about 2M, about 500 μM to about 1.5M, about 1mM to about 1M, about 25mM to about 750mM, about 250mM to about 500mM, and about 300mM to about 400mM of substrate ketose. In one embodiment, the composition may include about 180mM of substrate ketose. In one embodiment, the composition may include about 750mM of substrate ketose. In one embodiment, the composition may include about 1.7M of substrate ketose.

[0095] In some embodiments, the composition comprises purified KDG. In some embodiments, the composition contains greater than about 99% KDG by weight on an anhydrous basis. In some embodiments, the composition comprises partially purified KDG. In some embodiments, the composition contains greater than about 50%, about 60%, about 70%, about 80% or about 90% KDG by weight on an anhydrous basis.

[0096] In some embodiments, the present invention provides a method for producing a dihydrofuran composition, comprising the following steps: (a) providing a composition comprising a substrate ketose (e.g., KDG); (b) contacting the ketose with a glycoside hydrolase polypeptide; and (c) producing dihydrofuran. In some embodiments, the composition comprises enzymatically produced ketose. In some embodiments, the glycoside hydrolase utilized in the methods herein is expressed in a modified microorganism.

[0097] In some embodiments, provided herein is a method for producing a dihydrofuran composition, comprising the following steps: (a) providing a composition comprising greater than about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.6% of a substrate ketose by weight on an anhydrous basis; (b) contacting the composition comprising the substrate with a glycoside hydrolase polypeptide; and (c) producing dihydrofuran. In some embodiments, the composition comprises enzymatically produced ketose. In some embodiments, the glycoside hydrolase utilized in the methods herein is expressed in a modified microorganism.

[0098] In some embodiments, the composition comprising KDG is contacted with a glycoside hydrolase to catalyze the reaction of KDG (2-keto-3-deoxygluconic acid) to produce dihydrofuran. In some embodiments, the composition comprises partially purified KDG. In some embodiments, the composition comprises purified KDG. In some embodiments, the composition comprises at least about> 95% KDG. In some embodiments, the composition comprises at least about 95%, 96%, 97%, 98%, 99% or 100% KDG. In some embodiments, the composition comprises greater than 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.6% KDG.

[0099] The present invention also provides a method for producing a dihydrofuran composition, comprising the steps of: (a) providing a composition comprising greater than about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.6% by weight of a substrate 2-keto-3-deoxygluconic acid (KDG) on an anhydrous basis; (b) contacting the composition with a glycoside hydrolase; and (c) producing a composition comprising dihydrofuran. In some embodiments, the composition comprises greater than about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.6% by weight of dihydrofuran 4,5-dihydro-4-hydroxy-5-hydroxymethyl-2-furancarboxylic acid (K4) on an anhydrous basis.

[0100] In some embodiments, K4 can be further converted to 5-hydroxymethyl-2-furoic acid (HM FA) as described in WO 2021016220. In some embodiments, HMFA can be further oxidized to FDCA using chemical oxidation as described in WO 2021016220 or using enzymatic oxidation, such as described in Dijkman et al., 2014, "Discovery and Characterization of a 5-Hydroxymethylfurfural Oxidase from Methylovorus sp. St rain MP688", which is incorporated herein by reference.

[0101] Table 2 - Substrates and products of ketose biocatalytic dehydration and further oxidation

[0102]

[0103] In some embodiments, the method produces a composition comprising greater than about 80% by weight HMFA. In some embodiments, the purification produces a composition comprising greater than about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.6% HMFA by weight. In some embodiments, the composition comprises greater than about 95% HMFA by weight.

[0104] In some embodiments, the step of contacting the composition with a glycoside hydrolase polypeptide and a ketose is performed at a temperature between about 0.5°C and about 75°C. In some embodiments, the temperature is about: 0.5°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, or 70°C, up to about 75°C. In some embodiments, the temperature is about: -0.5°C-5°C, 10°C-15°C, 20°C-30°C, 35°C-45°C, 35°C-50°C, 35°C-55°C, 30°C-60°C, or 30°C, up to about 75°C. In some embodiments, the step of contacting the composition with a glycoside hydrolase polypeptide and a ketose is performed at a temperature of about 45°C.

[0105] In some embodiments, the step of contacting the composition with a glycoside hydrolase polypeptide and a ketose is performed at a pH of about 3 to about 8. In some embodiments, the pH is between about 0.5 to about 1, about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5 to about 8. In some embodiments, the pH is between about 3 to about 6. In some embodiments, the pH is between about 3 to about 5. In some embodiments, the pH is between about 3 to about 4. In some embodiments, the pH is between about 3 to about 3.5. In some embodiments, lowering the pH results in a reduced demand for NaOH during the contacting process. In some embodiments, the step of contacting the composition with a glycoside hydrolase and a ketose is performed at about pH 5.

[0106] In some embodiments, the duration of the step of contacting the composition with a glycoside hydrolase polypeptide and ketose is between 1 hour and 14 days. In some embodiments, the contact duration is about 1 hour, about 3 hours, about 5 hours, about 7 hours, about 9 hours, about 11 hours, about 13 hours, about 15 hours, about 17 hours, about 19 hours, about 21 hours, about 23 hours, about 25 hours, about 27 hours, about 29 hours, about 31 hours, about 33 hours, about 35 hours, about 37 hours, about 39 hours, about 41 hours, about 43 hours, about 45 hours, about 48 hours, about 49 hours, about 51 hours, about 53 hours, about 55 hours, about 57 hours, about 59 hours, about 61 hours, about 63 hours, about 65 hours, about 67 hours, about 69 hours, about 71 hours or about 73 hours, up to about 75 hours. In some embodiments, the duration of the step of contacting the composition with a glycoside hydrolase is about 48 hours. In some embodiments, the duration of contact is about 1 day to about 14 days. In some embodiments, the duration of contact is about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days or about 13 days, up to about 15 days. In some embodiments, the duration of contact is about 4 days to about 10 days. In some embodiments, the duration of the step of contacting the composition with a glycoside hydrolase is about 6 days.

[0107] In some embodiments, the resulting composition comprising K4 comprises greater than 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.6% K4 by weight on a dry basis.

[0108] In some embodiments, at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of the substrate KDG in the composition is converted to K4.

[0109] The reaction medium for conversion can be aqueous. In some embodiments, the reaction medium can be pure water, buffer or a combination thereof. In some embodiments, the reaction medium is a buffer. Suitable buffers include but are not limited to acetate buffer, citrate buffer, phosphate buffer and Bis-Tris buffer. In some embodiments, the reaction medium is an acetate buffer. In some embodiments, the reaction medium is a phosphate buffer. In some embodiments, the reaction medium is a Bis-Tris buffer. Alternatively, the reaction medium can also be an organic solvent. In some embodiments, glycerol, Tween-20, sucrose or sorbitol are added to the reaction medium as enzyme stabilizers. In some embodiments, the reaction medium is supplemented with 20% glycerol or 0.1% Tween or 2M sucrose or 2M sorbitol.

[0110] In some embodiments, measured by any of the above methods, KDG is completed at least about 2% to the conversion of dihydrofuran. In some embodiments, KDG is completed at least about 10% to the conversion of dihydrofuran, completed at least about 20%, completed at least about 30%, completed at least about 40%, completed at least about 50%, completed at least about 60%, completed at least about 70%, completed at least about 80%, completed at least about 90%, completed at least about 95%, completed at least about 100%. In some embodiments, KDG is completed greater than about 80% to the conversion of dihydrofuran. In some embodiments, at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% or about 90% of the KDG in the composition is converted into dihydrofuran.

[0111] In some embodiments, the reaction can be monitored by methods including, but not limited to, HPLC, LCMS, TLC, IR, UV, or NMR. In embodiments, the reaction is monitored using LCMS, UV, or LCMS and UV.

[0112] In some embodiments, the contact duration of the composition with the glycoside hydrolase and / or glycoside hydrolase polypeptide and KDG can be between 1 hour and 14 days, such as about 1 hour, about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, about 120 hours, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days or about 10 days. In some embodiments, the reaction is carried out for about 6 days. In some embodiments, the reaction is carried out for about 7 days.

[0113] Conversion of dihydrofuran to HMFA and FDCA

[0114] In some embodiments, dihydrofuran is further processed, e.g., purified and / or dehydrated, to produce HMFA and / or FDCA, e.g., Figure 1AAs shown, wherein HMFA is represented by 3. In some embodiments, dihydrofuran is chemically converted to HMFA under acidic conditions. In some embodiments, HMFA is purified. In some embodiments, HMFA is purified before subsequent reactions.

[0115] In some embodiments, provided herein is a method for producing HMFA, comprising the following steps: (a) providing a composition comprising greater than about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 99.6% of substrate ketose by weight on an anhydrous basis; (b) contacting the composition with a glycoside hydrolase polypeptide; (c) producing a composition comprising dihydrofuran; and (d) dehydrating dihydrofuran to HMFA under acidic conditions. In embodiments, a method is also provided, comprising contacting a composition comprising HMFA with a glycoside hydrolase polypeptide, thereby generating dihydrofuran. In an embodiment, dihydrofuran is dehydrated to HMFA under acidic conditions.

[0116] In some embodiments, dihydrofuran is converted to HMFA at high temperature. In some embodiments, the step of contacting the composition with a glycoside hydrolase polypeptide and KDG can be carried out at a temperature between about 0.5°C and about 110°C, such as about 10°C, about 20°C, about 30°C, about 40°C, about 50°C, about 60°C, about 70°C, about 80°C, about 90°C, about 100°C, or about 110°C. In a preferred embodiment, the reaction is carried out at about 74°C. In some embodiments, the reaction is carried out at about 69°C. In another embodiment, the reaction is carried out at about 63°C.

[0117] In some embodiments, dihydrofuran is contacted with an acid to achieve its conversion to HMFA. The acid may be selected from an inorganic acid, such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, and hydrobromic acid. The acid may be selected from an organic acid, such as a C1-6 carboxylic acid. In some embodiments, the contacting comprises dehydrating dihydrofuran using an acid selected from the group consisting of: formic acid, hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, hydrobromic acid, and a C1-6 carboxylic acid. In some embodiments, the acid comprises formic acid.

[0118] In some embodiments, dihydrofuran is converted to HMFA at low pH. In some embodiments, the pH may be acidic. In some embodiments, the pH is neutral or alkaline. In some embodiments, the pH is acidic and is 0-6. The pH may be 0, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, or up to about 10. In some embodiments, the pH is 4. In some embodiments, the pH is 4.5. In some embodiments, the pH is 5. In some embodiments, the pH is selected from the group consisting of 4, 4.5, and 5.

[0119] In some embodiments, the pH is about 4 and the temperature is about 63° C. In some embodiments, the pH is about 4.5 and the temperature is about 69° C. In some embodiments, the pH is about 5 and the temperature is about 72° C.

[0120] In some embodiments, the conversion of dihydrofuran (e.g., dehydration) to HMFA produces about 5%-100% HMFA. In some embodiments, about 5-10%, 10-30%, 25-40%, 30-50%, 35-60%, 40-70%, 45-85%, 50-80%, 50-90%, 55-90% or 60-100% is converted into HMFA. In some embodiments, the conversion of dihydrofuran to HMFA produces at least about or at most about 5%, 15%, 25%, 35%, 45%, 55%, 65%, 75%, 85%, 95% or 100% HMFA.

[0121] In some embodiments, HMFA can be further oxidized to FDCA by chemical and / or biological catalysis. In some embodiments, HMFA is purified prior to oxidation. In some embodiments, purification includes increasing the HMFA in the composition. In some embodiments, purification includes increasing the HMFA in the composition by at least about 1, 5, 10, 20, 30, 40, 50, 100, 150, 200, 300, or 500 times compared to an otherwise comparable composition that has not been purified.

[0122] In some embodiments, the resulting FDCA can be used as a polymer building block.FDCA has a lower carbon footprint than PET in the plastics industry.

[0123] Modified microorganisms

[0124] In some embodiments, any of the glycoside hydrolases or their motifs can be produced in a host (e.g., a microorganism). A modified microorganism may refer to a host cell that has been genetically modified by the cloning and transformation methods disclosed herein. Therefore, the term includes a host cell (e.g., bacteria, yeast cells, fungal cells, CHO, human cells, etc.) that has been genetically altered, modified, or transformed so that it exhibits a changed, modified, or different genotype and / or phenotype (e.g., when the genetic modification affects the encoding nucleic acid sequence of the microorganism) compared to the natural organism from which it is derived.

[0125] In some cases, microorganism is modified to produce glycoside hydrolase. In some embodiments, modified microorganism comprises and / or expresses any glycoside hydrolase provided herein. Modified microorganism can also comprise the polynucleotide of any glycoside hydrolase provided by encoding this paper. Therefore, this paper also provides modified microorganism and preparation method thereof comprising and / or expressing glycoside hydrolase.

[0126] In some embodiments, the DNA sequence encoding the glycoside hydrolase or its motif is cloned into an expression vector and inserted into a production host such as a microorganism (e.g., bacteria). The protein can be isolated from a cell extract based on the physical and chemical properties of the protein using techniques known in the art. In some embodiments, the sequence of the present disclosure can be introduced into a host cell using any of a variety of techniques, including transformation, transfection, transduction, viral infection, gene gun or Ti-mediated gene transfer (see Christie, PJ, and Gordon, JE, 2014 "The Agrobacterium Ti Plasmids" Microbiol SPectr. 2014; 2 (6); 10.1128). Specific methods include calcium phosphate transfection, DEAE-dextran-mediated transfection, lipofection or electroporation (Davis, L., Dibner, M., Battey, I., 1986 "Basic Methods in Molecular Biology"). Other transformation methods include, for example, lithium acetate transformation and electroporation, see, for example, Gietz et al., Nucleic Acids Res. 27: 69-74 (1992); Ito et al., J. Bacterol. 153: 163-168 (1983); and Becker and Guarente, Methods in Enzymology 194: 182-187 (1991). In addition, representative non-limiting techniques for isolating glycosyl hydrolases from modified microorganisms include centrifugation, electrophoresis, liquid chromatography, ion exchange chromatography, gel filtration chromatography and / or affinity chromatography.

[0127] In some embodiments, the microorganism is modified to contain and / or express an amino acid sequence having 80%-100% sequence identity to any one of SEQ ID NOs: 1-116. In some embodiments, the microorganism is modified to contain and / or express an amino acid sequence having at least 10% or at least 99.73% identity to an amino acid sequence selected from SEQ ID NOs: 1-116.

[0128] In some embodiments, the modified microorganism of the present disclosure comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the modified microorganism comprises a glycoside hydrolase polypeptide comprising an amino acid sequence selected from SEQ ID NO: 1-116. In some embodiments, the modified microorganism comprises a glycoside hydrolase polypeptide of SEQ ID NO: 19.

[0129] In some embodiments, the glycoside hydrolase polypeptide contained or expressed by the modified microorganism further comprises a tag amino acid sequence. In some embodiments, the tag amino acid sequence is His6.

[0130] In some embodiments, the modified microorganism is a bacterium. Bacteria include at least the following 11 different groups: (1) Gram-positive (gram+) bacteria, of which there are two major subgroups: (1) high G+C group (actinomycetes, mycobacteria, micrococci, others) (2) low G+C group (bacillus, clostridium, lactobacillus, staphylococcus, streptococcus, mycoplasma); (2) Proteobacteria, for example, purple photosynthetic + non-photosynthetic Gram-negative bacteria (including most "common" Gram-negative bacteria); (3) Cyanobacteria, for example, oxygen-producing phototrophic bacteria; (4) Spirochetes and related species; (5) Planctomyces; (6) Bacteroidetes, Flavobacterium; (7) Chlamydia; (8) Green sulfur bacteria; (9) Green non-sulfur bacteria (also anaerobic phototrophic bacteria); (10) Radioresistant Micrococcus and its related bacteria; (11) Thermotoga and Thermococcus. The bacterium can be any of the following: Escherichia coli, Saccharomyces, Aspergillus, Pichia, Pseudomonas, or Bacillus. In some embodiments, the bacterium is Escherichia coli.

[0131] Example

[0132] The following examples are given for the purpose of illustrating various embodiments of the present disclosure and are not intended to limit the present disclosure in any way. Those skilled in the art will recognize changes and other uses encompassed within the spirit of the present disclosure as defined by the scope of the claims.

[0133] Example 1: Production of glycoside hydrolases in vivo

[0134] Polynucleotides encoding the amino acid sequences SEQ ID NO: 1 to SEQ ID NO: 116 were synthesized (Twist Bioscience) and inserted into the pARZ4 expression vector. The recombinant vector was used to transform Escherichia coli NEBT7EL (New England Biolabs) in a heat shock method to prepare a recombinant microorganism.

[0135] The transformed modified microorganism was inoculated into 1 ml TB-kanamycin medium and cultured overnight at 37°C with shaking. 100uL of the culture was inoculated into 5 ml TB-kanamycin medium and cultured at 37°C for 2 hours, and then cultured at 25°C for 1 hour, 400RPM. The culture was induced with 1mM IPTG and expressed at 25°C, 400RPM for 20-24 hours. Finally, the culture was harvested by centrifugation at 2,200xg for 10 minutes, the supernatant was discarded, and the pellet was stored at -20°C.

[0136] Example 2: Purification of glycoside hydrolases

[0137] The microorganisms produced in Example 1 were thawed from storage at -20°C. After thawing, the precipitate was suspended in a lysis buffer (2 mg / mL lysozyme, 0.1 mg / mL DNAseI, 5% Protein extraction reagent, 20mMPO4 pH 7.5, 500mM NaCl and 20mM imidazole). The resuspended cells were incubated at 5 ° C and shaken at a speed of 200RPM for 30 minutes to break them. The broken lysate was centrifuged at 2200x g for 7 minutes. The supernatant obtained was loaded onto the Ni-NTA plate balanced by binding buffer. The plate was centrifuged at 100x g for 4 minutes, then washed twice with 500uL binding buffer (20mMPO4 pH 7.5, 500mM NaCl, 20mM imidazole) and centrifuged for two minutes (500x g). The protein was eluted with 150uL elution buffer (20mM PO4 pH 7.5, 500mM NaCl, 500mM imidazole), and then centrifuged at 500xg for 2 minutes. The recovered protein was desalted in a buffer solution for enzyme activity assessment (50mM acetate pH 5, 150mM NaCl).

[0138] Example 3: Measurement of glycoside hydrolase activity of ketose substrates by UV quantification

[0139] The purified enzyme in Example 2 was incubated in 50 mM acetate buffer (pH 5, 150 mM NaCl, 180 mM KDG (2-keto-3-deoxygluconic acid)) at 74°C for 3 hours. The reaction mixture was then filtered using a 10K MWCO filter plate to remove proteins from the product, substrate, and other reaction components. TM Product formation was detected by a Synergy HTX Multimode Microplate Reader. Products were measured at a wavelength of 250 nm in a UV-transparent plate. Quantification of products was achieved by calculation based on a KDG standard curve generated under the same reaction conditions.

[0140] Example 4: Measurement of glycoside hydrolase activity of ketose substrates by triple quadrupole (QQQ) quantitative method

[0141] The enzyme purified in Example 2 was incubated in 50 mM acetate buffer (pH 5, 150 mM NaCl, 180 mM KDG (2-keto-3-deoxygluconic acid)) at 74°C for 3 hours. The reaction mixture was then filtered with a 10K MWCO filter plate to remove proteins from the product, substrate, and other reaction components. Product formation was detected using an Agilent G6470A triple quadrupole mass spectrometer. The LC components consisted of an Agilent 1290 multisampler at 10°C, a 1290 infinity II pump, a 1290 Flexpump, and a 1290 MTC running a Waters 100x2.1 mm HSS T3 1.8 um C-18 column alternately at 40°C. In the received 384-well assay plate, 5ul of 2mM, 1mM, 0.5mM, 0.25mM stock solutions of HMFA in water were added to the selected 50ul wells for calibration of QQQ (yielding final concentrations of 200, 100, 50 and 25uM). Samples were run in an alternating manner, with one column being separated and analyzed while the other column was re-equilibrated with the starting mobile phase. This resulted in a run time of 2.5 minutes per sample. The QQQ was analyzed using a multiple reaction monitoring (MRM) method with the transitions for 13C labeled HMFA (m / z 147) and HMFA (m / z 141) set as follows, Figure 3 and Figure 4 .

[0142] Example 5: Obtaining or improving KDG dehydratase activity using rational design methods

[0143] A native enzyme with low levels of KDG dehydration activity was engineered to have higher activity levels.

[0144] To improve the activity of the parent scaffolds that showed some initial KDG dehydration activity, computational enzyme design techniques were used to improve substrate interactions in the active sites of SEQ ID NO: 1 and SEQ ID NO: 19, as summarized in Table 3. For these sequences, information on the native protein crystal structure provided an accurate picture of how the substrate or transition state fits into the active site. Loops suitable for flexible backbone design were identified in SEQ ID NO: 1 and 19. Overall, the efficiency of the enzymes was improved in five enzyme backbones by introducing 1 to 12 mutations into any position of the parental sequence through computational design.

[0145]

[0146] Site-saturation mutagenesis of glycoside hydrolases SEQ ID NO. 1 and 19.

[0147] To find amino acids at positions where point mutations on SEQ ID NOs: 1 and 19 could increase KDG dehydration activity, saturation mutagenesis was performed around the active site at positions 40, 41, 42, 88, 132, 133, 141, 147, 189, 211, 331, 332, 333, 334, 335, 336 and 352 of SEQ ID NO: 1 and positions 41, 42, 87, 88, 91, 134, 149, 152, 193, 211, 219, 221, 225, 337, 338, 339, 352 and 356 of SEQ ID NO: 19. A total of 323 and 342 protein variants (19 point mutants at each amino acid position) of SEQ ID NOs: 1 and 19 were tested for KDG dehydration activity.

[0148] Among the SEQ ID NO: 1 variants, 11 point mutations at 6 amino acid positions of SEQ ID NO: 1 resulted in up to 2-fold increase in KDG dehydration activity (SEQ ID NOs: 35, 37, 41, 42, 43, 48, 55, 62, 63, 64 and 68). The first 36 point mutations were: H42I, H42T, H42V, H42W, D88C, D88N, H132E, K133M, W141Y, H189A, H189V, V331K, G332E, G332M, G332V, S334A, S334C, S334D, S334E, S334G, S334I, S334K, S334M, S334N, S334Q, S334R, S334T, S334V, A335V, A335P, A335L, A335C, H352R, and H352V. Some flexible positions were found at which multiple neutral or beneficial amino acid changes were found. For example, 20 amino acids are found at positions 332, 334, and 335 of SEQ ID NO: 1. Positions in the loop region of amino acids 331-336 in SEQ ID NO: 1 are more susceptible to change.

[0149] In the SEQ ID NO: 19 variant, 8 point mutations at 3 amino acid positions resulted in an increase in KDG dehydration activity up to 1.4-fold (SEQ ID NO: 95, 96, 98, 99, 101, 102, 103 and 105). The point mutations that confer increased activity include: D41A, D41C, D41E, D41N, D41S, D41T, H87A, H87C, H87E, H87G, H87Q, H87R, H87S, L152M, L152N, W225Y, S337A, Y338V, H339A, H339N, W352F, Y356A, Y356C, Y356F, Y356H.

[0150] Homology modeling

[0151] Homology modeling was performed on SEQ ID NOs: 4-18 and 22-34 using the online SWISS-MODEL homology modeling server. For modeling, the target sequence was uploaded in fasta format. Template searches were performed using BLAST and HHblits. The searched templates were ranked according to global model quality evaluation (GMQE) and quaternary structure quality evaluation (QSQE). The top-ranked templates and alignments were compared to verify whether they covered different regions of the target protein and represented different conformational states. Multiple templates were automatically selected and different models were built accordingly. A template (e.g., the first template) was selected and used to build the model. The minimum percentage sequence identity for the template was 27%.

[0152] All references, articles, publications, patents, patent publications and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, reference to any reference, article, publication, patent, patent publication and patent application cited herein does not and should not be taken as an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

Claims

1. A biocatalytic method for producing dihydrofuran, the method comprising: Contacting 2-keto-3-deoxygluconic acid (KDG) with a glycoside hydrolase to produce dihydrofuran, wherein the contacting comprises: a. a pH of about 3 to about 7 as measured by a pH meter; or b. Temperature is 45°C to 74°C; or ca and b. both, thereby producing dihydrofuran.

2. The biocatalytic method of claim 1, comprising a., wherein the pH is about 4 to 5.

3. The biocatalytic method of claim 1, comprising b., wherein the temperature is 70°C to 74°C.

4. The biocatalytic process of any one of claims 1 to 3, comprising a. and b., wherein the pH is about 4 to 5, and the temperature is about 62°C to 72°C.

5. The biocatalytic method of any one of claims 1 to 4, comprising a. and b., wherein the pH and the temperature are selected from the group consisting of: a. pH of about 4 and temperature of about 63°C; b. pH of about 4.5 and temperature of about 69°C; and c. pH is about 5 and temperature is about 72°C.

6. The biocatalytic method of claim 5, comprising c.

7. The biocatalytic method of any one of claims 1 to 6, wherein the KDG is 100 mM to 2 M.

8. The biocatalytic method of claim 7, wherein the KDG is 100 mM to 750 mM.

9. The biocatalytic process of any one of claims 1-8, wherein the glycoside hydrolase comprises a protein having at least 80% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 1-116.

10. The biocatalytic method of claim 9, wherein the sequence identity is at least 85%, 90%, 95%, 98%, 99% or 100%.

11. The biocatalytic method of any one of claims 1 to 8, wherein the glycoside hydrolase comprises a first motif that binds to the KDG, and a second motif comprising a catalytic residue comprising aspartic acid, The first motif comprises at least two residues, the first residue comprises arginine and the second residue comprises tryptophan, phenylalanine or tyrosine, and The glycoside hydrolase was determined to be a homolog of SEQ ID NO: 1 or SEQ ID NO: 19 through SWISS-MODEL modeling.

12. The biocatalytic process of any one of claims 1-11, wherein the glycoside hydrolase comprises a protein having 100% sequence identity to SEQ ID NO:

27.

13. The biocatalytic process of any one of claims 1 to 12, wherein the contact time is from 72 hours to 14 days.

14. The biocatalytic method of claim 13, wherein the contacting time is from about 4 days to about 10 days.

15. The biocatalytic method of claim 14, wherein the contacting time is about 6 days.

16. The biocatalytic method according to any one of claims 1 to 15, further comprising: The dihydrofuran is dehydrated to form 5-hydroxymethyl-2-furoic acid (HMFA), wherein a HMFA yield of at least 40% is observed after said dehydration.

17. The biocatalytic method of claim 16, wherein the dehydration comprises: The dihydrofuran is contacted with an acid selected from the group consisting of formic acid, hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, hydrobromic acid, and a Ci-6 carboxylic acid.

18. The biocatalytic process of claim 17, wherein the acid comprises formic acid.

19. The method of any one of claims 16 to 18, further comprising: HMFA is oxidized to produce 2,5-furandicarboxylic acid (FDCA).

20. The biocatalytic method of claim 19, wherein the oxidation comprises a chemical oxidation reaction.

21. The biocatalytic method of claim 19, wherein the oxidation comprises an enzymatic oxidation reaction.

22. An isolated polypeptide having at least 85% identity to any one of SEQ ID NO:35 to SEQ ID NO:

116.

23. A biocatalytic method for producing dihydrofuran, the method comprising contacting 2-keto-3-deoxygluconic acid (KDG) with the isolated polypeptide of claim 22 to produce dihydrofuran.

24. The biocatalytic method of claim 23, wherein the isolated polypeptide comprises SEQ ID NO:

35.

25. The biocatalytic method of any one of claims 23-24, wherein the contacting comprises: a. a pH of about 3 to about 7 as measured by a pH meter; b. Temperature is 45°C to 74°C; or ca and b. both, thereby producing dihydrofuran.

26. The biocatalytic method of claim 25, comprising a., wherein the pH is about 4 to 5.

27. The biocatalytic process of claim 25, comprising b., wherein the temperature is 70°C to 74°C.

28. The biocatalytic process of any one of claims 25-27, comprising a. and b., wherein the pH is about 4 to 5, and the temperature is about 62°C to 72°C.

29. The biocatalytic process of any one of claims 25-28, comprising a. and b., wherein the pH and the temperature are selected from the group consisting of: a. pH of about 4 and temperature of about 63°C; b. pH of about 4.5 and temperature of about 69°C; and c. pH is about 5 and temperature is about 72°C.

30. The biocatalytic method of claim 29, comprising c.

31. The biocatalytic method of any one of claims 23-30, wherein the KDG is about 100 mM to 2 M.

32. The biocatalytic method of claim 31, wherein the KDG is between about 100 mM and 750 mM.

33. The biocatalytic process of any one of claims 23-32, wherein the contact time is from 72 hours to 14 days.

34. The biocatalytic method of claim 33, wherein the contacting time is from about 4 days to about 10 days.

35. The biocatalytic method of claim 33, wherein the contacting time is about 6 days.

36. The biocatalytic method of any one of claims 23-35, further comprising: The dihydrofuran is dehydrated to form 5-hydroxymethyl-2-furoic acid (HMFA), wherein a HMFA yield of at least 40% is observed after said dehydration.

37. The biocatalytic process of claim 36, wherein the dehydration comprises: The dihydrofuran is contacted with an acid selected from the group consisting of formic acid, hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, hydrobromic acid, and a Ci-6 carboxylic acid.

38. The biocatalytic process of claim 37, wherein the acid comprises formic acid.

39. The biocatalytic process of any one of claims 36-38, further comprising oxidizing HMFA to produce 2,5-furandicarboxylic acid (FDCA).

40. The biocatalytic method of claim 39, wherein the oxidation comprises a chemical oxidation reaction.

41. The biocatalytic method of claim 39, wherein the oxidation comprises an enzymatic oxidation reaction.

42. A modified microorganism comprising an exogenous glycoside hydrolase, wherein the exogenous glycoside hydrolase comprises a sequence having at least 85% sequence identity to a sequence selected from SEQ ID NOs: 1-116.

43. A modified microorganism as described in claim 42, wherein the exogenous glycoside hydrolase comprises a sequence having at least 90%, 95%, 97%, 98%, 99% or 100% sequence identity with a sequence selected from SEQ ID NOs: 1-116.

44. A modified microorganism as described in claim 43, wherein the exogenous glycoside hydrolase has at least 90%, 95%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO:

27.

45. A modified microorganism as described in any one of claims 42-44, wherein the modified microorganism is a bacterium.

46. ​​A modified microorganism as described in claim 45, wherein the bacteria is selected from the group consisting of Escherichia coli, Saccharomyces, Aspergillus, Pichia, Pseudomonas and Bacillus.

47. A modified microorganism as described in claim 46, wherein the bacterium is Escherichia coli.

48. A composition comprising the modified microorganism of any one of claims 42-47.

49. A composition comprising the isolated polypeptide of claim 22.

50. A method for producing polyethylene 2,5-furandicarboxylate (PEF), the method comprising the biocatalytic method of any one of claims 1-21 or 23-41.

51. An isolated non-natural glycoside hydrolase comprising: a first motif that binds to 2-keto-3-deoxygluconic acid, the first motif comprising at least two residues, the first residue being arginine and the second residue being tryptophan, phenylalanine or tyrosine; and a second motif comprising a catalytic residue, said catalytic residue being aspartic acid or glutamic acid, Wherein the isolated non-natural glycoside hydrolase has at least 20% identity to SEQ ID NO:1 or SEQ ID NO:

19.

52. The isolated non-natural glycoside hydrolase of claim 51, wherein the isolated non-natural glycoside hydrolase is at least 25%, 45%, 65%, 85%, 95% or 99% identical to SEQ ID NO: 1 or SEQ ID NO:

19.

53. The isolated non-natural glycoside hydrolase of claim 51, wherein the second of the at least two residues of the first motif is tryptophan and / or the catalytic residue of the second motif is aspartic acid.

54. The isolated non-natural glycoside hydrolase of claim 51, wherein the first motif has a sequence of RxQTW, x being serine or an aliphatic amino acid.

55. The isolated non-natural glycoside hydrolase of claim 51, wherein the first motif has a sequence of Rx1QTW(2x2)Yx2Y, x1 is serine, and x2 is an aliphatic amino acid.

56. The isolated non-natural glycoside hydrolase of claim 51, wherein the second motif has a sequence of xD, x being an aliphatic amino acid.

57. The isolated non-natural glycoside hydrolase of claim 51, wherein the second motif has a sequence of (2x)KSE(3x)DT(2M)xSxPFx, and x is an aliphatic amino acid.

58. The isolated non-natural glycoside hydrolase of claim 51, wherein the arginine of the first motif and the catalytic residue of the second motif are separated by about 70 residues.

59. An isolated glycoside hydrolase comprising: The sequence of Equation 1: P(19x)LPP(4x)HYHQGVxLxG(4x)(W / Y)(10x)Y(3x)(Y / W)x(D / E)(6x)G(9x)D(2x)Q(P / A)Gx(L / I)L(2x)L(7x)(R / K)Y(2x)(A / G)(3x)(L / I)(9 x)(T / N)xE(G / Q)G(F / Y)(W / F)H(K / N)(3x)Px(Q / E)(M / Q)WLDGLYMxG(5x)Y(A / G)(9x)D(4x)Q(6x)(H / K)(T / M)(R / K)(3x)TGL(2x)H(A / G)(W / F) (D / S)(2x)(R / K)(3x)W(A / S)(D / N)(2x)(T / S)Gx(S / A)PExW(G / A)R(S / A)xGW(9x)(D / E)x(I / L)P(2x)H(20x)Q(4x)GxWxQ(V / I)x(D / N)(K / R)( and has at least 50% homology to SEQ ID NO: 1, Where x is any amino acid.

60. The isolated glycoside hydrolase of claim 59, wherein the isolated glycoside hydrolase is at least 55%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or 100% homologous to SEQ ID NO:

1.

61. The isolated glycoside hydrolase of any one of claims 59-60, comprising Y at residue 41; D at residue 88; H at residue 132; W at residue 141; D at residue 143; M at residue 147; H at residue 189; W at residue 211; G at residue 212; R at residue 213; W at residue 217; S at residue 278; L or M at residue 282; C at residue 330; and H or K at residue 352, The residues are numbered according to SEQ ID NO:

1.

62. The isolated glycoside hydrolase of any one of claims 59-61, comprising a loop region having at least 21 residues.

63. The isolated glycoside hydrolase of any one of claims 59-61, comprising a loop region having at least one modification selected from the group consisting of 331 to 336 of SEQ ID NO:

1.

64. An isolated glycoside hydrolase as described in claim 63, wherein the at least one modification selected from the group consisting of 331 to 336 of SEQ ID NO: 1 comprises one or more of the following: V331K, G332E, G332M, G332V, S334A, S334C, S334D, S334E, S334G, S334I, S334K, S334M, S334N, S334Q, S334R, S334T, S334V, A335V, A335P, A335L and A335C.

65. The isolated glycoside hydrolase of claim 59, further comprising two amino acids appended to the n-terminus of the sequence and five amino acids appended to the c-terminus of the sequence.

66. A biocatalytic method for producing dihydrofuran, comprising: Contacting 2-keto-3-deoxygluconic acid (KDG) with a glycoside hydrolase to produce dihydrofuran comprises: a. a pH of about 3 to about 7 as measured by a pH meter; b. Temperature is 45°C to 74°C; or ca and b. both, thereby generating dihydrofuran, wherein the glycoside hydrolase has at least 50% homology to SEQ ID NO: 1, and wherein the glycoside hydrolase comprises a D at residue 143, an R at residue 213, and a W at residue 217 relative to SEQ ID NO:

1.

67. The biocatalytic process of claim 66, wherein residue 143 of the glycoside hydrolase is a catalytic residue and residue 213 and residue 217 are substrate binding residues.

68. An isolated glycoside hydrolase comprising: The sequence of equation 2: (F / Y)P(8x)(W / Y)(7x)W(T / M)(2x)F(2x)G(2x)(W / Y)(2x)Y(11x)(A / G)(10x)(L / I)(8x)(H / F)D(L / I)GF(4x)(S / T)(4x)(W / Y)(15x)(A / G)(13x)(L / I)(16x)IDx(L / M)(L / M)(N / S)(22x) H(3x)(T / S)(5x)RxDxS(S / T)(6x)(D / N)(10x)TxQG(4x)SxW(A / S / T)RG(Q / L)(A / T)W(2x)YG( 28x)PxD(4x)(Y / W)D(F / L)(12x)S(6x)(S / C)(33x)Y(30x)(W / F / Y)(G / A)DY(Y / F)(2x)ExL; and has at least 50% homology to SEQ ID NO: 19, Where x is any amino acid.

69. The isolated glycoside hydrolase of claim 68, wherein the isolated glycoside hydrolase is at least 55%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or 100% homologous to SEQ ID NO:

19.

70. The isolated glycoside hydrolase of any one of claims 68-69, comprising I or L at residue 26; H at residue 41; W at residue 42; M at residue 43; H at residue 87; D at residue 88; G at residue 134; D at residue 149; T at residue 150; M at residue 152; Q at residue 193; W at residue 219; R at residue 221; W at residue 225; S at residue 280; I at residue 284; and F at residue 352, wherein the residues are numbered according to SEQ ID NO:

19.

71. The isolated glycoside hydrolase of any one of claims 68 or 70, comprising a sequence having at least 90%, 95%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NOs: 24 to 27.

72. The isolated glycoside hydrolase of claim 68, further comprising thirteen amino acids appended to the n-terminus of the sequence and five amino acids appended to the c-terminus of the sequence.

73. An isolated glycoside hydrolase comprising: The sequence of formula 3: (W / Y)7x(A / G)(92x)WxD(35x)(L / M)(9x)(H / R)(22x)W(A / G / S)R(2x)(G / S)W(8x) (L / I)(27x)Q(3x)(G / K)xW(3x)(I / L)(9x)ExSx(S / T)(9x)(A / G)(52x)Gx(G / A); and It has at least 50% homology to SEQ ID NO:

2.

74. The isolated glycoside hydrolase of claim 73, wherein the isolated glycoside hydrolase is at least 55%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or 100% homologous to SEQ ID NO:

2.

75. The isolated glycoside hydrolase of claim 73, further comprising fifty-nine amino acids appended to the n-terminus of the sequence and thirteen amino acids appended to the c-terminus of the sequence.

76. A method for converting 2-keto-3-deoxygluconic acid (KDG) to dihydrofuran, comprising contacting 2-keto-3-deoxygluconic acid (KDG) with the isolated glycoside hydrolase of any one of claims 59-75.

77. A host cell heterologously expressing a nucleic acid encoding the glycoside hydrolase of any one of claims 59-75.

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