WO2022204246A1 - D-glucarate déshydratase thermostable qui est résistante à l'inhibition par soumission de séquence de tartrate - Google Patents
D-glucarate déshydratase thermostable qui est résistante à l'inhibition par soumission de séquence de tartrate Download PDFInfo
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- WO2022204246A1 WO2022204246A1 PCT/US2022/021490 US2022021490W WO2022204246A1 WO 2022204246 A1 WO2022204246 A1 WO 2022204246A1 US 2022021490 W US2022021490 W US 2022021490W WO 2022204246 A1 WO2022204246 A1 WO 2022204246A1
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- WIPO (PCT)
- Prior art keywords
- seq
- glucarate
- tartrate
- enzyme
- glucarate dehydratase
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- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical group OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 title claims abstract description 69
- 230000005764 inhibitory process Effects 0.000 title claims abstract description 41
- 101001009834 Escherichia coli (strain K12) Glucarate dehydratase Proteins 0.000 title description 5
- 108010000445 Glycerate dehydrogenase Proteins 0.000 claims abstract description 100
- 102000004190 Enzymes Human genes 0.000 claims abstract description 89
- 108090000790 Enzymes Proteins 0.000 claims abstract description 89
- 229940095064 tartrate Drugs 0.000 claims abstract description 61
- DSLZVSRJTYRBFB-LLEIAEIESA-N D-glucaric acid Chemical compound OC(=O)[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O DSLZVSRJTYRBFB-LLEIAEIESA-N 0.000 claims abstract description 48
- DNXDYHALMANNEJ-UHFFFAOYSA-N furan-2,3-dicarboxylic acid Chemical compound OC(=O)C=1C=COC=1C(O)=O DNXDYHALMANNEJ-UHFFFAOYSA-N 0.000 claims abstract description 28
- 238000004519 manufacturing process Methods 0.000 claims abstract description 21
- 230000000694 effects Effects 0.000 claims description 84
- 230000035772 mutation Effects 0.000 claims description 82
- DSLZVSRJTYRBFB-LLEIAEIESA-L D-glucarate(2-) Chemical compound [O-]C(=O)[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O DSLZVSRJTYRBFB-LLEIAEIESA-L 0.000 claims description 56
- 108090000623 proteins and genes Proteins 0.000 claims description 52
- 238000000034 method Methods 0.000 claims description 51
- AEMOLEFTQBMNLQ-BZINKQHNSA-N D-Guluronic Acid Chemical compound OC1O[C@H](C(O)=O)[C@H](O)[C@@H](O)[C@H]1O AEMOLEFTQBMNLQ-BZINKQHNSA-N 0.000 claims description 35
- 102000004169 proteins and genes Human genes 0.000 claims description 26
- AEMOLEFTQBMNLQ-UHFFFAOYSA-N beta-D-galactopyranuronic acid Natural products OC1OC(C(O)=O)C(O)C(O)C1O AEMOLEFTQBMNLQ-UHFFFAOYSA-N 0.000 claims description 22
- 230000002255 enzymatic effect Effects 0.000 claims description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 15
- 150000001413 amino acids Chemical class 0.000 claims description 15
- 238000002703 mutagenesis Methods 0.000 claims description 15
- 231100000350 mutagenesis Toxicity 0.000 claims description 15
- FEWJPZIEWOKRBE-LWMBPPNESA-L D-tartrate(2-) Chemical compound [O-]C(=O)[C@@H](O)[C@H](O)C([O-])=O FEWJPZIEWOKRBE-LWMBPPNESA-L 0.000 claims description 13
- 108700023413 Glucarate dehydratases Proteins 0.000 claims description 13
- FEWJPZIEWOKRBE-JCYAYHJZSA-L L-tartrate(2-) Chemical compound [O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O FEWJPZIEWOKRBE-JCYAYHJZSA-L 0.000 claims description 13
- 241000186660 Lactobacillus Species 0.000 claims description 13
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 13
- 229940039696 lactobacillus Drugs 0.000 claims description 11
- 238000006467 substitution reaction Methods 0.000 claims description 11
- FEWJPZIEWOKRBE-XIXRPRMCSA-N Mesotartaric acid Chemical compound OC(=O)[C@@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-XIXRPRMCSA-N 0.000 claims description 9
- 230000001747 exhibiting effect Effects 0.000 claims description 6
- SEUNICDWQTXURO-UHFFFAOYSA-N 2-methoxycarbonylfuran-3-carboxylic acid Chemical compound COC(=O)C=1OC=CC=1C(O)=O SEUNICDWQTXURO-UHFFFAOYSA-N 0.000 claims description 5
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 3
- 230000002194 synthesizing effect Effects 0.000 claims description 3
- 230000004075 alteration Effects 0.000 claims description 2
- 108090001042 Hydro-Lyases Proteins 0.000 abstract description 12
- 102000004867 Hydro-Lyases Human genes 0.000 abstract description 12
- 239000006166 lysate Substances 0.000 description 43
- 241000588724 Escherichia coli Species 0.000 description 36
- 239000000758 substrate Substances 0.000 description 33
- 239000003112 inhibitor Substances 0.000 description 32
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 31
- 239000008103 glucose Substances 0.000 description 29
- 238000007254 oxidation reaction Methods 0.000 description 25
- 238000006243 chemical reaction Methods 0.000 description 20
- 239000000203 mixture Substances 0.000 description 20
- 230000003647 oxidation Effects 0.000 description 20
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 13
- 230000035484 reaction time Effects 0.000 description 13
- 239000000463 material Substances 0.000 description 11
- 239000011541 reaction mixture Substances 0.000 description 10
- 235000002906 tartaric acid Nutrition 0.000 description 10
- 239000011975 tartaric acid Substances 0.000 description 10
- 238000003556 assay Methods 0.000 description 9
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- 238000012239 gene modification Methods 0.000 description 8
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- 235000013617 genetically modified food Nutrition 0.000 description 8
- 241000204396 Acetonema Species 0.000 description 7
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- 238000006297 dehydration reaction Methods 0.000 description 7
- -1 for example Chemical class 0.000 description 7
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- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- FEWJPZIEWOKRBE-LWMBPPNESA-N levotartaric acid Chemical compound OC(=O)[C@@H](O)[C@H](O)C(O)=O FEWJPZIEWOKRBE-LWMBPPNESA-N 0.000 description 4
- 108090000765 processed proteins & peptides Proteins 0.000 description 4
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
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- 108020004705 Codon Proteins 0.000 description 2
- NPTTZSYLTYJCPR-HRFVKAFMSA-N D-arabinaric acid Chemical compound OC(=O)[C@@H](O)C(O)[C@H](O)C(O)=O NPTTZSYLTYJCPR-HRFVKAFMSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 238000010923 batch production Methods 0.000 description 2
- IJOOHPMOJXWVHK-UHFFFAOYSA-N chlorotrimethylsilane Chemical compound C[Si](C)(C)Cl IJOOHPMOJXWVHK-UHFFFAOYSA-N 0.000 description 2
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- 238000006911 enzymatic reaction Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
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- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- VBUYCZFBVCCYFD-JJYYJPOSSA-N 2-dehydro-D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C(=O)C(O)=O VBUYCZFBVCCYFD-JJYYJPOSSA-N 0.000 description 1
- IZSRJDGCGRAUAR-MROZADKFSA-M 5-dehydro-D-gluconate Chemical compound OCC(=O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O IZSRJDGCGRAUAR-MROZADKFSA-M 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- RGHNJXZEOKUKBD-SQOUGZDYSA-M D-gluconate Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O RGHNJXZEOKUKBD-SQOUGZDYSA-M 0.000 description 1
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 1
- RBNPOMFGQQGHHO-UWTATZPHSA-N D-glyceric acid Chemical compound OC[C@@H](O)C(O)=O RBNPOMFGQQGHHO-UWTATZPHSA-N 0.000 description 1
- DSLZVSRJTYRBFB-MMPJQOAZSA-N D-idaric acid Chemical compound OC(=O)[C@@H](O)[C@H](O)[C@@H](O)[C@H](O)C(O)=O DSLZVSRJTYRBFB-MMPJQOAZSA-N 0.000 description 1
- 241001495410 Enterococcus sp. Species 0.000 description 1
- 241001134569 Flavonifractor plautii Species 0.000 description 1
- BDAGIHXWWSANSR-UHFFFAOYSA-M Formate Chemical compound [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- DSLZVSRJTYRBFB-UHFFFAOYSA-N Galactaric acid Natural products OC(=O)C(O)C(O)C(O)C(O)C(O)=O DSLZVSRJTYRBFB-UHFFFAOYSA-N 0.000 description 1
- RGHNJXZEOKUKBD-SQOUGZDYSA-N Gluconic acid Natural products OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 108010093096 Immobilized Enzymes Proteins 0.000 description 1
- IAJILQKETJEXLJ-SQOUGZDYSA-N L-guluronic acid Chemical compound O=C[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O IAJILQKETJEXLJ-SQOUGZDYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- XCOBLONWWXQEBS-KPKJPENVSA-N N,O-bis(trimethylsilyl)trifluoroacetamide Chemical compound C[Si](C)(C)O\C(C(F)(F)F)=N\[Si](C)(C)C XCOBLONWWXQEBS-KPKJPENVSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 241001169825 Oribacterium sp. Species 0.000 description 1
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 1
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- 241000589516 Pseudomonas Species 0.000 description 1
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- 150000007513 acids Chemical class 0.000 description 1
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- HSJKGGMUJITCBW-UHFFFAOYSA-N beta-hydroxybutyraldehyde Natural products CC(O)CC=O HSJKGGMUJITCBW-UHFFFAOYSA-N 0.000 description 1
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- DSLZVSRJTYRBFB-DUHBMQHGSA-N galactaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)C(O)=O DSLZVSRJTYRBFB-DUHBMQHGSA-N 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 229940050410 gluconate Drugs 0.000 description 1
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- ROBFUDYVXSDBQM-UHFFFAOYSA-L hydroxymalonate(2-) Chemical compound [O-]C(=O)C(O)C([O-])=O ROBFUDYVXSDBQM-UHFFFAOYSA-L 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
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- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 150000004715 keto acids Chemical class 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
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- 108700041430 link Proteins 0.000 description 1
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- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 229940049920 malate Drugs 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N malic acid Chemical compound OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
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- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical group [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
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- DUIOPKIIICUYRZ-UHFFFAOYSA-N semicarbazide Chemical compound NNC(N)=O DUIOPKIIICUYRZ-UHFFFAOYSA-N 0.000 description 1
- 150000007659 semicarbazones Chemical class 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 159000000000 sodium salts Chemical group 0.000 description 1
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- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- ILJSQTXMGCGYMG-UHFFFAOYSA-N triacetic acid Chemical compound CC(=O)CC(=O)CC(O)=O ILJSQTXMGCGYMG-UHFFFAOYSA-N 0.000 description 1
- 239000005051 trimethylchlorosilane Substances 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/58—Aldonic, ketoaldonic or saccharic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/01—Hydro-lyases (4.2.1)
- C12Y402/0104—Glucarate dehydratase (4.2.1.40)
Definitions
- FDCA furan dicarboxylic acid
- FDME furan dicarboxylic methyl ester
- Glucose as with other aldol hexoses, may be oxidized by chemical catalysis to form glucaric acid, which can be subsequently dehydrated in two steps by known methods, such as acid catalysis to form FDCA or dehydrated and esterified with methanol to form FDME.
- Enzymatic dehydration of glucaric acid to 5-keto-4-deoxy glucaric acid (KDG) by the enzyme glucarate dehydratase provides an enzymatic route for producing KDG that in turn can be subject to further acid catalyzed dehydration to FDCA.
- the dehydration of glucaric acid to KDG by glucarate dehydratase is therefore a key enabling step for a combined chemo-enzymatic process for making FDCA/FDME from dextrose.
- Studies of the enzymatic conversion of glucaric acid to KDG have been primarily directed towards understanding the mechanism of the glucarate dehydratase enzyme and its role in degradation of glucarate by microorganisms.
- thermostable enzymes that can operate at temperatures of greater than 50°C, more preferably at greater than 60 °C, still more preferably greater than 65 °C and most preferably greater than 70°C. Operating at higher temperature will increase the catalytic activity of the enzyme and can use the heat derived from exothermic process of oxidizing glucose to form glucaric acid.
- thermostable glucarate dehydratase enzymes that are resistant to inhibition by tartrate and other byproducts present in a crude glucose oxidation mixture containing glucaric acid thereby eliminating the need to purify the glucaric acid prior to dehydration.
- glucarate dehydratases having increased thermostability and or resistance to inhibition by tartrate, which may be used in methods of making 5-keto-4-deoxy glucaric acid (KDG), which in turn provides improved methods of making FDCA or FDME.
- KDG 5-keto-4-deoxy glucaric acid
- KDG 5-keto-4-deoxy glucaric acid
- a glucarate dehydratase enzyme at a temperature of at least 50°C where the enzyme has at least one characteristic selected from the group consisting of: (i) exhibiting thermostability at a temperature of at least 50 °C and (i) exhibiting at least 50% of its enzymatic activity converting glucarate to KDG in the presence of 10 mM glucarate and 10 mM tartrate as it exhibits in the absence of tartrate.
- the glucarate dehydratase enzyme is selected from the group consisting of (i) a naturally occurring enzyme having a protein sequence according to any one of SEQ ID NOS 141 -148; (b) a variant sequence of any one of SEQ ID NOS 141 -148 made by substituting at least 3 amino acids of SEQ ID NOS: 141 -148 and that retains or has improved thermostability or resistance to tartrate.; (c) a protein sequence according to any one of SEQ ID NO 2-140; and (d) a variant of SEQ ID NO 2-140 made by substituting at least one amino acid present in any of SEQ ID NO 2-140 and hat retains or has improved thermostability or resistance to tartrate.
- the glucarate dehydratase enzyme is from Th. carboxydivorans having a protein sequence according to SEQ ID NO: 147 or from Lactobacillus pentous having a protein sequence according to SEQ ID NO: 146 or a variant derived by alteration of at least one amino acid in SEQ ID NO 146 or 147 that retains or has improved thermostability or resistance to tartrate.
- the tartrate is at least one of D-tartrate, L-tartrate, and meso-tartrate.
- the glucarate dehydratase enzyme exhibits enzymatic activity at a pH range of 5.5-7.0. In certain embodiments the glucarate dehydratase exhibits enzymatic activity at greater than 60°C, greater than 65°C and in some embodiments greater than 70°C. In some particular embodiments, the glucarate dehydratases exhibits at least 72% of enzymatic activity at temperatures above 50°C as exhibited at 37-45°C and that is resistant to inhibition by D-tartrate and guluronate relative to the parent and other naturally occurring glucarate dehydratases.
- the forging can be used of making furan dicarboxylic acid (FDCA) comprising obtaining KDG made by the forging methods and dehydrating the KDG to form FDCA.
- the FDCA is esterified with methanol to furan dicarboxylic methyl ester (FDME).
- FDME furan dicarboxylic methyl ester
- the glucarate dehydratase enzyme is immobilized on a column.
- glucarate dehydratase enzymes with improved thermostability and resistance to tartrate and guluronate.
- a glucarate dehydratase enzyme comprising the amino acid sequence according to SEQ ID NO 146 except having at least one mutation selected from the group of individual mutations present in SEQ ID NOS: 2-140 and that exhibit improved thermostability and/or resistance to in comparison to the glucarate dehydratase enzyme according to SEQ ID NO 146.
- the glucarate dehydratases have at least two mutations selected from the group of individual mutations present in SEQ ID NOS: 2-24. In certain embodiments the glucarate dehydratase has at least three mutations selected group of individual mutations present is SEQ ID NOS: 25-87. In further embodiments, the glucarate dehydratase enzyme has at least four mutations selected from the group of individual mutations present in SEQ ID NOS: 25-87. In still further embodiments, the glucarate dehydratase enzyme has at least five mutations selected from the group of individual mutations present in SEQ ID NOS: 88-140.
- the glucarate dehydratase has an amino acid sequence according any one of SEQ ID. NO 2-140.
- a method of obtaining a mutant glucarate dehydratase that exhibits at least one of thermostability and resistance to inhibition by tartrate and/or guluronic acid that includes: a) subjecting a parent glucarate dehydratase having a protein sequence according to any of SEQ ID NOs: 141-148 to a first round of mutagenesis to generate a first set of variants of the parent glucarate dehydratase that include at least three amino substitutions; b) determining the glucarate dehydratase activity of the first set of variants at a desired temperature of at least 50°C and/or determining activity of the variants in the presence of tartrate and/or guluronic acid; c) selecting a variant that has higher activity at the desired temperature and/or that has higher activity in the presence of tartrate and/or guluronic acid than the parent glucarate dehydratase, and optionally d) subjecting at least one member from the first set
- the mutagenesis is conducted by synthesizing genes that encode a protein sequence with amino acid mutations of the parent or selected variant glucarate dehydratases, and the method of selecting includes expressing the synthesized genes from a host organism and assaying the expressed genes for glucarate dehydratase activity.
- the variants synthesized in subsequent rounds of mutagenesis retain at least one amino acid substitution present in the variants selected after the first round of mutagenesis.
- the forgoing enzymes are used in another aspect of the present invention, which is a method of making 5-keto-4-deoxy glucaric acid (KDG) comprising contacting glucaric acid with any of the forgoing new glucarate dehydratase enzymes.
- the glucarate dehydratase enzyme is immobilized on a column.
- the forging method can be used in a further method for making furan dicarboxylic acid (FDCA) comprising obtaining KDG made using the new glucarate dehydratase enzymes and dehydrating the KDG to form FDCA.
- the method may further include is esterifying the FDCA with methanol to furan dicarboxylic methyl ester (FDME).
- FIG. 1A and FIG. IB are graphs showing the effect of increasing L- tartrate concentration on glucarate activity between dehydratase variant 350378 (SEQ ID NO:2) (FIG. 1A) and wild-type dehydratase (336438, SEQ ID NO: 146) (FIG. IB).
- the Y axis is KDG production rate (mM KDG/min)
- the X axis is glucarate concentration (mM).
- the L-tartrate concentration (mM) of each curve is identified by the legend, and correspond to L-tartrate concentrations of 0, 0.5, 1, 2, 5, 10, 20 and 40 mM.
- FIG. 2A and FIG. 2B are graphs showing the effect of increasing D- tartrate concentration on glucarate activity between dehydratase variant 350378 (SEQ ID NO:2) (FIG. 2A) and wild-type dehydratase (336438, SEQ ID NO: 146) (FIG. 2B).
- the Y axis is KDG production rate (mM KDG/min)
- the X axis is glucarate concentration (mM).
- the D-tartrate concentration (mM) of each curve is identified by the legend, and correspond to D-tartrate concentrations of 0, 0.5, 1, 2, 5, 10, 20 and 40 mM.
- FIG. 3 A and FIG. 3B are graphs showing the effect of increasing meso-tartrate concentration on glucarate activity between dehydratase variant 350378 (SEQ ID NO:2) (FIG. 3A) and wild-type dehydratase (336438, SEQ ID NO: 146) (FIG. 3B).
- the Y axis is KDG production rate (mM KDG/min)
- the X axis is glucarate concentration (mM).
- the meso-tartrate concentration (mM) of each curve is identified by the legend, and correspond to meso-tartrate concentrations of 0, 0.5, 1, 2, 5, 10, 20 and 40 mM.
- FIG. 4 is a graph showing tartrate inhibition of variants 368238 (SEQ ID NO:90), 368265 (SEQ ID NO:94), 368298 (SEQ ID NO:98), and 357047 (SEQ ID NO:82). Left bar of each pair 40 mM D-tartrate, right bar of each pair 20 mM L-tartrate.
- FIG. 5 A, 5B, and 5C are graphs showing guluronate inhibition at 0 mM guluronate (FIG. 5A), 40 mM guluronate (FIG. 5B), and 80 mM guluronate (FIG. 5C) for variants 368238 (SEQ ID NO:90), 368265 (SEQ ID NO:94), 368298 (SEQ ID NO:98), and 357047 (SEQ ID NO:82).
- the R number in the prefix preceding the variant number indicates the round of mutation from which the variant was generated.
- Each of the R3 variants retained the mutations from the R2 variant 357047.
- FIG. 6A, 6B, 6C, and 6D are graphs showing glucarate dehydratase activity assays at 66° C (FIG. 6A), 68° C (FIG. 6B), 70° C (FIG. 6C), and 72° C (FIG. 6D) for variants 374373 (SEQ ID NO: 104), 374377 (SEQ ID NO: 107), 374388 (SEQ ID NO: 111), 374398 (SEQ ID NO: 116), 374413 (SEQ ID NO: 123), 374414 (SEQ ID NO: 124), 374423 (SEQ ID NO: 128), 368238 (SEQ ID NO:90), and 357047 (SEQ ID NO:82).
- the R number in the prefix preceding the variant number indicates the round of mutation from which the variant was generated.
- FIG. 7 is a graph showing the results of forming KDG from glucarate using a glucarate dehydratase enzyme immobilized on a column for continuous production of the KDG reaction product from a glucaric acid feedstock passed over the column at a rate of
- FIG. 8 is a bar graph illustrating results of screening naturally occurring glucarate dehydratases present in crude extracts made from E. coli cultures engineered to heterolgously express candidate enzymes.
- the X axis shows assigned gene numbers and Y axis shows measured glucarate dehydratase activity. Notable candidate sources are identified.
- the present disclosure provides the discovery a handful of naturally occurring glucarate dehydratase enzymes that may be employed in a method for making KDG from glucaric at higher temperatures, i.e, at a temperatures of least 50°C, at least 60°C, at least 65°C or even at least 70°C. Some of these naturally occurring enzymes also show better resistance to tartrate inhibition than previously described enzymes.
- Glucarate dehydratase has been identified from E. coli and has been well characterized. The properties of the E. coli D-glucarate dehydratase were first described by H. J. Blumenthal in Method of enzymology, 1966, 9, 660. The authors conclude that glucarate dehydratase is inhibited by galactaric acid, D-idaric acid, and tartaric acid. Roger Jeffcoat Eur. J. Biochem. 25 (1972) 515-523 has further characterized the enzyme from Pseudomonas and has shown that (+)-tartrate is a competitive inhibitor of that enzyme at a concentration of 2.5 mM. The E.
- coli D-glucarate dehydratase was used as the base enzyme to compare to other enzymes discovered or developed in accordance with the present disclosure.
- the E.coli enzyme was purified by fast protein liquid chromatography (FPLC) and then assayed for enzyme activity in the presence of various inhibitors known to exist in a crude glucose oxidation mixture containing glucaric acid.
- FPLC fast protein liquid chromatography
- Example 1 shows the inhibitory properties of byproducts present in a typical glucose oxidation mixture to the glucarate conversion activity of the E. coli dehydratase enzyme.
- E. coli glucarate dehydratase Genes encoding E. coli glucarate dehydratase and a library of 94 homologues (all previously uncharacterized) were synthesized with optimized codons and expressed in E. coli. Some members in the library of 94 homologues to the E. coli glucarate dehydratase have as little as 30% protein sequence homology to the E. coli sequence yet still exhibited glucarate dehydratase activity. Crude lysates were made from E. coli cultures expressing the library of gene and used to screen for specific activity, temperature tolerance, pH tolerance, and resistance to the competitive inhibitor tartrate.
- thermostability means the enzyme exhibits a specific activity (e.g ., KDG made from glucarate per minute / mg protein) at a temperature of 50°C or greater that is at least 75% of the specific activity exhibited by the E. coli enzyme in converting glucarate to KDG at a 30°C.
- the naturally occurring enzymes exhibiting higher thermostability are from Enterococcus sp., Oribacterium sp., Elalomonas sp., Elalomonas sp., F.
- Acetonema enzyme Another feature of the Acetonema enzyme disclosed herein is its resistance to the presence of tartaric acid. While most enzymes in the library that was screened were indeed very sensitive to D-tartrate, the Acetonema enzyme was discovered to be much less sensitive to D-tartrate than the E. coli enzyme. This is a substantial and surprising result. There are literature reports ( e.g http://onjinelibrary. wiley .com/doi/ 723.x/epdf) that document the sensitivity of this class of enzymes to the competitive inhibitor D-tartrate
- the present disclosure provides for a method to generate a broad family of variant glucarate dehydratase enzymes derived from any of the forgoing parent natural enzymes that exhibit enzyme activity at least 50°C, at least 60°C, at least 65°C or in some embodiments at least 70°C.
- the variant enzymes are resistant to inhibition by the competitive inhibitors tartaric acid and in some embodiments also resistant to inhibition by guluronic acid.
- the enzymes disclosed herein are catalytically active in a crude glucose oxidation mixture containing glucarate, tartrate and guluronic acid.
- the crude glucose oxidation mixture used herein for illustrative purposes is a mixture containing glucaric acid, tartaric acid and other diacids resulting from oxidation of glucose made according to the method described in US patent number 9,156,766.
- the enzymes disclosed herein would, however, be active with any crude glucose oxidation mixtures containing glucaric acid and inhibitors of the dehydratase such as tartaric and guluronic acid.
- Figures 5A through 5C illustrate exemplary variants that show increased resistance to guluronic acid, where the mutations present in a second-round variant were preserved in third round variants, all of which showed increased resistance to inhibition by guluronic acid.
- the method of improving at least one of thermostability and resistance to inhibition by tartrate by glucarate dehydratase includes subjecting a parent glucarate dehydratase having a protein sequence according to any of SEQ ID NOs: 141-148 to a first round of mutagenesis to generate variant glucarate dehydratases including at least three amino substitutions, determining the glucarate dehydratase activity of the variants at a temperature of at least 50°C, or at least 60°C , or at last 65°C or at last 70°C and selecting a variant that has higher activity at the desired temperature than the parent enzyme.
- the mutagenesis is exemplified herein by synthesizing a gene that encodes selected random mutations that span the entire peptide sequence of the parent enzyme, expressing the mutated genes from a suitable host, and making crude lysates of cultures expressing the genes, but the method may also be practiced by making purely random mutations in the coding sequence and expressing the mutant genes from a suitable host.
- the method is illustrated herein using E. coli harboring the mutant genes in an expression vector, but other hosts with suitable expression sequences may be used.
- the activity of the variants expressed may be determined in the presence of tartrate or guluronic acid at temperatures of at 50°C, or at least 60°C or at last
- a leas 65 °C and variants are selected that have also greater activity than the parent enzyme in the presence of these inhibitors.
- concentration of inhibitors used to test for resistance to inhibition should be 1/4* to four times the amount of glucaric acid used in the assay.
- assays are performed with about 10 mM glucaric acid in the presence of 5 mM to 40 mM of the inhibitors. Reactions containing 10 mM glucarate and 10 mM of tartrate or other inhibitors are suitable for use in screening for resistance to the inhibitors.
- a crude glucose oxidation mixture is used as the substrate for enzymatic conversion of glucarate, and the amount of inhibitors present is as inherently exist in a crude glucose oxidation mixture, which is shown in Example 1 of this disclosure.
- At least one mutation introduced to make variants in the first round of mutations is preserved and at least a second round of mutation is made in the parent sequence wherein the second round includes at least one preserved mutation from the first round of mutations that showed increased activity at the desired temperatures and optionally in the presence of tartrate or guluronic acid.
- at least two, or at least three mutations from the first round of mutations that show improved thermos tolerance and or inhibitor resistance are preserved.
- the second round introduces new mutations, and the thermostability and/or resistance to inhibitors are determined for the new variants at the desired temperature optionally in the presence of the inhibitors and those with increased thermostability and/or resistance to the inhibitors are again selected.
- at least four rounds of mutation are conducted, each creating variants that preserve at least one beneficial mutation selected from earlier rounds.
- at least two, or most preferably at least three mutations from prior rounds of mutation are preserved in subsequent rounds.
- the tables from Examples 5, and 7-9 illustrate examples of variants made in each of 4 rounds of mutation.
- the variants made in the first round of mutation contained 3 mutations altering up to three amino acids and those mutations exhibiting the same or higher thermostability and/or resistance to tartrate (SEQ ID NOS: 2-24) were retained for use in a second round.
- Each of the variants made in the second round retained at least two and often all three of the mutations made in the first round and those mutations that exhibited thermostability or resistance inhibition (SEQ ID NOS 25-87) were retained for use in a third round of mutation which new mutations therefore also included at least three mutations from the first and second round.
- beneficial mutations identified in the third round were preserved for combination with new mutations made in the fourth round, and those that exhibited thermostability or resistance to inhibition in the fourth round (SEQ ID NOS: 88-140) therefore contained at least four of the mutations made in earlier rounds. Mutations could further be made in subsequent rounds that would preserve at least 5 of the individual mutations present in SEQ ID NOS: 88-140.
- the forgoing method provides a third aspect of the present disclosure, which is a variant glucarate dehydratase derived from a parent glucarate dehydratase according to
- SEQ ID NOS 141-148 that has improved thermostability at 50°C , 60°C, 65°C or 70°C and/or improved resistance to inhibition by tartaric acid or guluronic acid.
- the Examples that follow illustrate this aspect of the disclosure by providing variant glucarate dehydratases having peptides sequences according to SEQ ID NOS: 2-140, which were generated by the forgoing method after one, two, three or four rounds of mutation of the glucarate dehydratase from Lactobacillus pentous where the parent protein had the peptide sequence according to SEQ ID NO: 146.
- Variants made in each subsequent round of mutation preserved at least one, two, three or more mutations discovered to be beneficial for thermostability or resistance to inhibitors from at least one previous round of mutation. Often, at least two and most often at least 3 mutations from prior rounds were preserved in subsequent rounds of invention. Production of these variants is illustrated in Examples 5 through 9 of the present disclosure. All of the variants should retain at least 94%, more preferably at least 96% and most preferably at least 98% sequence identity to the parent enzyme. The variants exemplified herein retain at least 97.5% sequence identity to the parent sequence.
- the glucarate dehydratase from L. pentous having the peptide sequence according to SEQ ID NO: 146 was selected to illustrate this aspect of the invention because although the naturally occurring enzyme from Acetoma having the protein sequence according to SEQ ID NO: 147 was judged to have the best thermostability and good resistance to inhibition by tartrate using purified substrate and inhibitors, the Acetoma enzyme was also judged to be less resistance to inhibition using as substrate a crude glucose oxidation mixture than the enzyme from L. pentous , as shown in Table 2 in Example 2. Nonetheless, the present invention can be practiced using any of the naturally occurring sequences according to SEQ ID NOS: 141-148 as the starting parent enzyme for making subsequent rounds of mutation.
- Figures 6A through 6D shows the activity of exemplary variants that are both resistant to guluronic acid and continue to exhibit thermostability at 66°C, at 68°C, at 70°C and at 72°C.
- These illustrative enzymes exhibit enzymatic activity at 72°C that is at least 72% of the enzymatic activity at reference temperatures of 37-45 °C. Not all variants from SEQ ID NOS 2-140 were assayed at these higher temperatures, but most if not, all are expected to also show both guluronic acid and increased thermostability.
- the variants made in round 3 and round 4 of mutations should show similar thermostability and resistance to guluronic acid because the variants preserved the beneficial mutations from round 2 variants that exhibited these features.
- glucarate dehydratase may be used in a fourth aspect of the present invention, which is, a method of making furan dicarboxylic acid (FDCA) that comprises contacting glucaric acid with a glucarate dehydratase enzyme having protein sequences according to SEQ ID NOS 141-148 or a variant thereof made according to the methods described herein at a temperature of above 50°C to form 5-keto-4-deoxy glucaric acid (KDG) and further dehydrating the KDG to form FDCA,
- FDCA furan dicarboxylic acid
- KDG 5-keto-4-deoxy glucaric acid
- the variants retain at least 94%, at least 96% and more preferably at least 98% sequence identity with the parent.
- the dehydration to KDG may be performed at a pH of 5.5-7.5 wherein the glucarate dehydratase enzyme is thermostable at a temperature of at least 50°C and /or is resistant to inhibition by tartrate.
- Particular embodiments of variant glucarate dehydratase are illustrated by SEQ ID NOS 2- 140.
- the glucaric acid is in a salt form selected from the group consisting of a potassium salt form, a sodium salt form, and an ammonium salt form. All of the embodiments of the method may be practiced using as the glucarate substrate, a crude glucose oxidation mixture, exemplified by a mixture made according to the method described in US Pat. No. 9,156,766.
- gluconate glycerate, gycolate, formate, 2-keto-gluconate, glucoronate, chloride, 2-furonate, 5-keto-gluconate, malate, maleate, tartrate, tartronate, oxalate, glucose, fructose, and arabinaric acid.
- gluconate glycerate, gycolate, formate, 2-keto-gluconate, glucoronate, chloride, 2-furonate, 5-keto-gluconate, malate, maleate, tartrate, tartronate, oxalate, glucose, fructose, and arabinaric acid.
- Glucarate dehydratase activity was quantified using a continuous enzyme-coupled spectroscopic assay.
- 100 pL of reaction mixture contained 100 mM phosphate buffer at pH 7.5, 10 mM MgCk, 0.25 mM to 50 mM D-glucarate, 0.1 mg glucarate dehydratase and byproducts/inhibitors (1-20 mM) incubated at 30°C for 10 minutes.
- Semicarbazide (0.1 M) in triacetate was added to the reaction mass and incubated at room temperature for 10 minutes. The amount of ketoacid was quantitated by detection of its semicarbazone at 250 nm. Values of kcat and kcat/Km were determined by varying the concentration of the sugar acid substrate.
- Table 1 shows that for purified E. coli enzyme, DL-tartronic, tartaric and L- guluronic acid inhibit the D-glucarate dehydratase compared to other compounds typically present in a glucose oxidation reaction mixture to convert glucose to glucarate.
- Drum A material which is the product of a glucose oxidation according to US patent number 9,156,766.
- the composition of Drum A material is shown below.
- the reaction was carried at a concentration that formed 10% w/w dissolved solids from Drum A material as the substrate.
- the reactions did not go to completion likely due to inhibition by tartrate and the high amount of guluronic acid shown above to be competitive inhibitor of glucarate with the E. coli enzyme. Therefore, a need existed to discover glucarate dehydratase enzymes that were resistant to inhibition to by the byproducts present in the glucose oxidation reaction mixture.
- Drum A contains amongst other byproducts, tartaric acid. , the previously known inhibitor of the E. coli glucarate dehydratase enzyme and also contains a relatively high concentration of guluronic acid shown in Example 1 to also be an inhibitor of the E. coli enzyme. Therefore, using Drum A material as a substrate for screening glucarate dehydratases for industrial use inherently screens for enzymes more likely be resistant to inhibition by these compounds.
- lysates containing the candidate enzymes were screened for activity at 50°C for a period of 2 hrs. and 19 hrs. Reactions were conducted at pH 6.5 with a 10% volume of crude lysate containing the expressed protein product with 20 % w/w dissolved solids from Drum A as the substrate.
- Fig. 8 One result of such screening is shown in Fig. 8.
- Table 2 shows a summary comparison of activity ranking for 8 particularly useful naturally occurring glucarate dehydratases. The indicated performance values for each feature are expressed on a relative scale of 1 through 9, with the highest ranking being 1 and poorest being 9. For reference, on this relative scale the E. coli enzyme would be at least 10 for crude tolerance and thermo tolerance.
- thermostability was Gene No. 333729, source Th. Carboxydivorans (a.k.a. Aectonema). Expressed activity and specific activity, however, are dependent at least in part on the E. coli host expression system and is not necessarily indicative of the best inherent activity of the enzyme itself.
- the sequence with the best combination of thermostability and crude resistance was gene No. 336438, source Lactobacillus pentous, which had the second-best crude tolerance with a medial level of thermostability.
- the dehydratase form Acetonema ⁇ i.e., Th. Carboxydivorans) was expressed in E. coli and cultured in a shake flask conditions LB medium with a IPTG inducer. Crude extracts were prepared by lysing the cell pellet with Bugbuster® and the activity in the crude extract was used to evaluate the conversion of potassium glucarate in a 100 uL reaction mixture (10% glucarate, 50 mM Tris pH 7.5, 2 mM Magnesium sulfate) at lysate to substrate dosages of 1:1. The reaction mixture was incubated at 50 C for 4 hr and the product mixture was analyzed by GC-MS.
- Each sample was diluted in dimethylformamide and trimethylsilylated with N,0-bis(trimethylsilyl)trifluoroacetamide containing 1 % trimethylchloro silane.
- Derivatized samples were analyzed by GC-MS with components separated through a 5 % phenyl-modified polydimethyl arylene siloxane capillary column.
- the concentration of glucaric acid obtained from a crude glucose oxidation reaction mixture was enriched by precipitating as mono potassium salt and re-dissolving in water.
- the precipitated glucaric acid sample included ⁇ 400-500 ppm of tartaric acid, -500 ppm of arabinaric acid, and - 250 ppm of gluconic acid.
- the dehydratase form Acetonema ⁇ i.e., Th. Carboxydivorans) was expressed in E. coli and cultured under fed batch fermentation conditions with a lactose inducer. Crude extracts were prepared by homogenizing the culture and the activity in the crude extract was used to evaluate the conversion of glucarate in the semi-crude reaction mixture (10% dissolved solids content in assay) at varying enzyme dosages at pH 7.0 at 55°C, and the results are shown in Table 3.
- Crude lysates from heterlogously expressed glucarate dehydratase enzymes 333729 (SEQ ID NO: 147), 336431 (SEQ ID NO: 148), and 368238 (SEQ ID NO: 146) were immobilized onto a silica resin with by infusing the column with the crude lysate and incubation with polyethyleneimine and glutaraldehyde for a time sufficient to cross link protein to the silica resin. The resin was then washed with buffer and evaluated for use as a reactor for making KDG from glucarate using potassium glucarate as the substrate feed stock providing the results shown in Table 4 below.
- the immobilized enzyme on the column was used for continuous conversion of glucaric acid into the KDG reaction product.
- the potassium glucarate feedstock was passed over the column at 0.5 BV/hr at 55°C and the material eluted from the column was measures over _ 80 _ column volumes with the results shown in FIG. 7. It was found that immobilization of the enzyme retained the activity as shown in Table 4.
- Lactobacillus pentous Glucarate dehydratase The Lactobacillus pentous glucarate dehydratase (gene No. 336438, SEQ ID NO
- Table 5 shows a summary of properties of variants obtained after round 1 of genetic modification. Amino acid changes are shown as the one letter abbreviation for the naturally occurring amino acid followed by its position in the wild type gene ID 33643 (SEQ ID NO: 146) followed by the one letter abbreviation for the amino acid in the created in variant. The reaction conditions for the various assays are shown in the footnotes of Table 5. The performance of the variants from this first round of mutations is indicated on a relative scale sorted in descending order from highest activity with pure glucarate which was exhibited by variant gene ID number 350378 (SEQ ID NO:2).
- variant 350378 SEQ ID NO:2
- SEQ ID NO: 1436 The kinetics and tartrate inhibition of variant 350378 (SEQ ID NO:2) as compared to its wild-type parent hydratase (336438, SEQ ID NO: 146) was investigated. Reaction conditions were as follows: 5 minutes, with pure glucarate at 60°C, 1% lysate, 4mM MgS0 4 , 100 mM MES pH 6.5. The results are shown in FIG. 1A, FIG. IB, FIG. 2A, FIG. 2B, FIG. 3A, and Fig. 3B. For each graph depicted in these figures, the Y axis is KDG production rate (mM KDG/min), and the X axis is glucarate concentration (mM). The tartrate concentration (mM) of each curve is identified by the legend, and correspond to tartrate concentrations of 0, 0.5, 1, 2, 5, 10, 20 and 40 mM, respectively.
- FIG. 1A and IB show the effect of increasing F-tartrate concentration on glucarate activity between hydratase variant 350378 (SEQ ID NO:2) (FIG. 1A) and wild-type hydratase (336438, SEQ ID NO: 146) (FIG. IB).
- the F-tartrate concentration (mM) of each curve is identified by the legend, and correspond to F-tartrate concentrations of 0, 0.5, 1, 2, 5, 10, 20 and 40 mM, respectively.
- a comparison of FIG. 1A to FIG. IB shows that Variant 350378 (SEQ ID NO:2) has much greater resistance to F-tartrate than wild-type hydratase (336438, SEQ ID NO: 146). At 10 mM F-tartrate the variant retains greater than 50% the activity exhibited in the absence of F-tartrate.
- FIG. 2A and FIG. 2B are graphs showing the effect of increasing D- tartrate concentration on glucarate activity between hydratase variant 350378 (SEQ ID NO:2) (FIG. 2A) and wild-type hydratase (336438, SEQ ID NO: 146) (FIG. 2B).
- the D- tartrate concentration (mM) of each curve is identified by the legend, and correspond to D- tartrate concentrations of 0, 0.5, 1, 2, 5, 10, 20 and 40 mM, respectively.
- a comparison of FIG. 2A to FIG. 2B shows that Variant 350378 (SEQ ID NO:2) has much greater resistance to D-tartrate than wild-type hydratase (336438, SEQ ID NO: 146). At 10 mM D-tartrate the variant retains greater than 70% the activity exhibited in the absence of D-tartrate.
- FIG. 3 A and FIG. 3B are graphs showing the effect of increasing meso-tartrate concentration on glucarate activity between hydratase variant 350378 (SEQ ID NO:2) (FIG. 3A) and wild-type hydratase (336438, SEQ ID NO: 146) (FIG. 3B).
- the meso-tartrate concentration (mM) of each curve is identified by the legend, and correspond to meso-tartrate concentrations of 0, 0.5, 1, 2, 5, 10, 20 and 40 mM, respectively.
- variant 350378 (SEQ ID NO:2) has much greater resistance to meso-tartrate than wild-type hydratase (336438, SEQ ID NO: 146). At 20 mM L-tartrate the variant retains greater than 50% the activity exhibited in the absence of L-tartrate.
- Variant 350378 produced in in first round of mutation contained mutations A183S, T282V, and D412E in comparison wild-type hydratase 336438.
- a second set 96 variants were synthesized constituting a second round of spanning mutations, each or which preserved at least two and most often all three of these first-round mutations and further introduced three other mutations spanning the protein sequence. These second-round mutations expressed and assayed as with the first round of mutations at 60°C or 65°C under different conditions of pH i.e, at pH 5.5 or 6.0
- Table 6 shows a summary of properties of variants obtained after this second genetic modification sorted in descending order by tartrate resistance. The conditions of temperature and pH for the various assays are shown in the footnotes to the table. All amino acid changes including those preserved from the first round are again shown relative to the wild-type amino acid sequence (gene ID 336438, SEQ ID NO: 146).
- a third-round genetic variants produced were produced including at least one mutation selected from A183S, T282V and D412E shown to be beneficial for glucarate conversion activity in the first round of mutations and including at least one mutation that showed increased tartrate resistance from the second round of mutations.
- Table 7 A is a summary of properties of variants obtained after round 3 of genetic modification.
- the assays for this round of mutation were conducted at 66°C or 69°C at pH
- Table 7 A provides properties for variant 335047 made during the second round of mutations for sake of comparison.
- Table 7B shows amino acid changes of these variants again relative to the wild-type amino acid sequence (gene ID 336438, SEQ ID NO: 146).
- Tables 8 A is a summary of properties of variants obtained after round 4 of genetic modification. All assays were at 66 C at pH 6.0. Conditions are shown in the footnote to table 7A. Table 8B shows all amino acid changes of these variants relative to the wild- type amino acid sequence (gene ID 336438, SEQ ID NO: 146).
- FIG. 4 shows tartrate inhibition of variants 368238 (SEQ ID NO:90), 368265 (SEQ ID NO:94), 368298 (SEQ ID NO:98), and 357047 (SEQ ID NO:82). It was found that variant 368238 (SEQ ID NO:90) had overall similar qualities as variant 368265 (SEQ ID NO:94), even though it is a simpler variant, having fewer substitutions than variant 368265 (SEQ ID NO:94).
- Test conditions were 10 mM glucarate, pH 6.0, and 66° C, with 1% lysate, and the results are shown in FIG. 5A, 5B, and 5C.
- FIG. 6A, 6B, 6C, and 6D are graphs showing KDG production rates over time, at pH 6.0, with 10% lysate at 66° C (FIG. 6A), 68° C (FIG. 6B), 70° C (FIG. 6C), and 72° C (FIG. 6D) for variants 374373 (SEQ ID NO: 104), 374377 (SEQ ID NO: 107), 374388 (SEQ ID NO: 111), 374398 (SEQ ID NO: 104), 374377 (SEQ ID NO: 107), 374388 (SEQ ID NO: 111), 374398 (SEQ ID NO: 104), 374377 (SEQ ID NO: 107), 374388 (SEQ ID NO: 111), 374398 (SEQ ID NO: 104), 374377 (SEQ ID NO: 107), 374388 (SEQ ID NO: 111), 374398 (SEQ ID NO: 104), 374377 (SEQ ID NO: 107), 374388
- Table 9 summarizes the amino acid substitutions of 139 variants made through various rounds of mutations of parent SEQ ID NO: 146 from Lactobacillis pentous, all of which exhibit thermostability in retaining at 60°C, at least 50% of the activity exhibited by the parent enzyme at 30-40°C in converting glucarate to KDG and which exhibit resistance to inhibition by tartrate by retaining at least 50% of the activity in converting glucarate to KDG in the presence of 10 mM tartrate in comparison to the activity exhibited in the absence of tartrate.
- compositions disclosed herein can be used in an integrated process with a particular crude feedstock.
- the integrated process may comprise a two-step process.
- the first step may comprise oxidizing glucose with a metal catalyst to glucaric acid.
- the second step may comprise converting glucaric acid to KDG using a thermostable glucarate dehydratase.
- inhibitors e.g., tartaric acid
- the glucarate dehydratase enzymes disclosed in the present disclosure can be immobilized on a substrate in a column and the dehydration occurs by flowing the crude glucose oxidation mixture over the column and collecting KDG from the eluate.
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- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
Priority Applications (5)
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KR1020237036981A KR20240017781A (ko) | 2021-03-26 | 2022-03-23 | 타르트레이트 서열 제출에 의한 저해에 내성인 열안정성 d-글루카레이트 데하이드라타제 |
EP22776545.0A EP4314311A1 (fr) | 2021-03-26 | 2022-03-23 | D-glucarate déshydratase thermostable qui est résistante à l'inhibition par soumission de séquence de tartrate |
JP2023558863A JP2024513175A (ja) | 2021-03-26 | 2022-03-23 | 酒石酸塩による阻害に耐性のある熱安定性d-グルカル酸デヒドラターゼ |
CN202280025261.1A CN118843696A (zh) | 2021-03-26 | 2022-03-23 | 对酒石酸盐的抑制有抗性的热稳定的d-葡糖二酸脱水酶序列提交 |
BR112023019608A BR112023019608A2 (pt) | 2021-03-26 | 2022-03-23 | Método para produzir ácido 5-ceto-4-deoxi glucárico (kdg), método de produção de ácido furano dicarboxílico (fdca), enzima glucarato desidratase, método para obter uma glucarato desidratase mutante, e variante de glucarato desidratase |
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US202163166361P | 2021-03-26 | 2021-03-26 | |
US63/166,361 | 2021-03-26 |
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WO2022204246A1 true WO2022204246A1 (fr) | 2022-09-29 |
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PCT/US2022/021490 WO2022204246A1 (fr) | 2021-03-26 | 2022-03-23 | D-glucarate déshydratase thermostable qui est résistante à l'inhibition par soumission de séquence de tartrate |
Country Status (6)
Country | Link |
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EP (1) | EP4314311A1 (fr) |
JP (1) | JP2024513175A (fr) |
KR (1) | KR20240017781A (fr) |
CN (1) | CN118843696A (fr) |
BR (1) | BR112023019608A2 (fr) |
WO (1) | WO2022204246A1 (fr) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110124065A1 (en) * | 2008-04-04 | 2011-05-26 | Massachusetts Institute Of Technology | Cellular production of glucaric acid |
US20170015643A1 (en) * | 2012-12-20 | 2017-01-19 | Archer Daniels Midland Company | Use of carboxylic acids and furanic molecules for esterification |
-
2022
- 2022-03-23 BR BR112023019608A patent/BR112023019608A2/pt unknown
- 2022-03-23 WO PCT/US2022/021490 patent/WO2022204246A1/fr active Application Filing
- 2022-03-23 EP EP22776545.0A patent/EP4314311A1/fr active Pending
- 2022-03-23 JP JP2023558863A patent/JP2024513175A/ja active Pending
- 2022-03-23 KR KR1020237036981A patent/KR20240017781A/ko unknown
- 2022-03-23 CN CN202280025261.1A patent/CN118843696A/zh active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110124065A1 (en) * | 2008-04-04 | 2011-05-26 | Massachusetts Institute Of Technology | Cellular production of glucaric acid |
US20170015643A1 (en) * | 2012-12-20 | 2017-01-19 | Archer Daniels Midland Company | Use of carboxylic acids and furanic molecules for esterification |
Non-Patent Citations (2)
Title |
---|
DATABASE UniprotKB [https://www.uniprot.org/uniprot/A0A200IHY5] 25 October 2017 (2017-10-25), "Enterococcus sp. 6D12_DiV0197 Glucarate dehydratase", XP055974889, retrieved from Uniprot * |
DONALD C.FISH; HAROLD J.BLUMENTHAL: "[13a] d-Glucaric and some related acids", METHODS IN ENZYMOLOGY, vol. 9, 30 November 1965 (1965-11-30), US, pages 53 - 56, XP009540240, ISSN: 0076-6879, DOI: 10.1016/0076-6879(66)09016-5 * |
Also Published As
Publication number | Publication date |
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CN118843696A (zh) | 2024-10-25 |
JP2024513175A (ja) | 2024-03-22 |
EP4314311A1 (fr) | 2024-02-07 |
KR20240017781A (ko) | 2024-02-08 |
BR112023019608A2 (pt) | 2023-12-19 |
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