Novel glucaric acid biosynthesis path and method for fermentation production of glucaric acid

Abstract

Relates to the technical field of genetic engineering and fermentation engineering, in particular to a novel glucaric acid biosynthesis path and a method for producing glucaric acid through microbial fermentation. The glucaric acid is converted into the glucaric acid under the catalysis of glucose isomerase, mannose isomerase, hexaldehyde dehydrogenase, D-mannose oxidoreductase, uronic acid isomerase and uronic acid dehydrogenase. The method is easy to operate and relatively environment-friendly, and has a large-scale production prospect.

Description

A novel biosynthetic pathway for glucaric acid and a method for producing glucaric acid by fermentation

[0001] This application claims priority to the prior application with application number: 2023104355149, filed with the Patent Office of China on April 21, 2023, and entitled “A novel biosynthetic pathway of glucaric acid and a method for producing glucaric acid through fermentation”. The full text of the application is incorporated into this application by reference. Technical Field

[0002] The present invention relates to the technical field of genetic engineering and fermentation engineering, and in particular to a novel biosynthetic pathway of glucaric acid and a method for producing glucaric acid by fermentation. Background Art

[0003] Glucaric acid is a naturally occurring uronic acid, typically found in small quantities in fruits, vegetables, and mammals. Its highest concentrations are found in grapefruit, apples, citrus fruits, and cruciferous vegetables. Glucaric acid has a wide range of applications in the chemical industry. It serves as a building block for a variety of polymers, including novel nylons and hyperbranched polyesters. It is also used in electroplating as a chelating agent for metal corrosion protection. It can also be used in household detergents to replace environmentally harmful phosphates. It is also available as a dietary supplement in the form of calcium D-gluconate and has been studied for therapeutic purposes, including cholesterol reduction and cancer chemotherapy. The U.S. Department of Energy lists glucaric acid as a high-value-added chemical derived from biomass.

[0004] Glucaric acid is a highly functionalized compound with four chiral carbon atoms. Currently, it is produced through chemical oxidation of glucose using nitric acid and 2,2,6,6-tetramethylpiperidinyloxy (TEMPO) as oxidants. This process suffers from low yields, numerous byproducts, high costs, and the need for post-processing. Compared to chemical synthesis, microbial fermentation offers advantages such as readily available raw materials, environmental friendliness, and mild production conditions. Therefore, microbial fermentation has the potential to produce higher yields of glucaric acid.

[0005] Although glucaric acid can be synthesized in mammalian and plant cells, the synthesis pathway requires more than 10 transformation steps. Currently, research has been conducted on the production of glucaric acid via fermentation, but issues such as low yield and conversion efficiency persist. Therefore, improving the fermentation strains and technical processes for glucaric acid production is of great significance for increasing glucaric acid production and product market competitiveness.

[0006] Summary of the Invention

[0007] The specific technical solutions of the present invention include:

[0008] The first aspect of the present invention provides a method for synthesizing glucaric acid, comprising the steps of:

[0009] 5) Fructuronate is converted into D-glucuronic acid by biological methods, preferably under the action of isomerase;

[0010] 6) D-glucuronic acid is converted into glucaric acid by biological methods, preferably under the action of dehydrogenase;

[0011] Preferably, the method for synthesizing glucaric acid further comprises: 4) converting D-mannonic acid into fructose ketoate by a biological method, preferably under the action of an oxidoreductase;

[0012] Preferably, the method for synthesizing glucaric acid further comprises: 3) converting D-mannose into D-mannonic acid by a biological method, preferably under the action of a dehydrogenase;

[0013] Preferably, the method for synthesizing glucaric acid further comprises: 2) converting D-fructose into D-mannose by a biological method, preferably under the action of an isomerase;

[0014] Preferably, the method for synthesizing glucaric acid further comprises: 1) converting D-glucose into D-fructose by a biological method, preferably under the action of an isomerase;

[0015] Further preferably, steps 1) and 2) are direct conversion of D-glucose into D-mannose under the action of isomerase.

[0016] In a specific embodiment of the present invention,

[0017] The biological method described in step 5) is carried out under the action of the following enzyme: uronic acid isomerase (EC 5.3.1.12);

[0018] and / or

[0019] The biological method described in step 6) is carried out under the action of the following enzyme: uronate dehydrogenase (EC 1.1.1.19 or EC 1.1.1.203).

[0020] In a specific embodiment of the present invention, the biological method described in step 4) is carried out under the action of the following enzyme: D-mannonate oxidoreductase (EC 1.1.1.57).

[0021] In a specific embodiment of the present invention, the biological method described in step 3) is carried out under the action of the following enzyme: aldose dehydrogenase (EC 1.1.1.121 or EC 1.1.1.122).

[0022] In a specific embodiment of the present invention, the biological method described in step 2) is carried out under the action of the following enzymes: mannose isomerase (D-mannose isomerase, EC 5.3.1.7);

[0023] and / or

[0024] The biological method described in step 1) is carried out under the action of the following enzyme: D-glucose isomerase (EC 5.3.1.5);

[0025] and / or

[0026] In the biological method described in steps 1)-2), glucose is directly converted into mannose under the action of cellobiose 2-epimerase (EC 5.1.3.11) or D-mannose 2-epimerase (EC 5.1.3).

[0027] The second aspect of the present invention provides a method for synthesizing a glucaric acid intermediate from D-glucose:

[0028] The second aspect of the present invention first provides a method for synthesizing D-mannonic acid, comprising: 1) converting D-glucose into D-fructose; 2) converting D-fructose into D-mannose; and 3) converting D-mannose into D-mannonic acid by a biological method, preferably under the action of a dehydrogenase, preferably under the action of an aldose dehydrogenase.

[0029] The second aspect of the present invention further provides a method for synthesizing fructoic acid, comprising first obtaining D-mannonic acid using the above method, and then 4) converting the D-mannonic acid into fructose by a biological method, preferably under the action of an oxidoreductase, preferably under the action of D-mannonate oxidoreductase.

[0030] The second aspect of the present invention again provides a method for synthesizing D-glucuronic acid, first using the above method to obtain fructuronate, and then 5) fructuronate is converted into D-glucuronic acid by a biological method, preferably under the action of an isomerase, preferably under the action of uronic acid isomerase.

[0031] The third aspect of the present invention provides a method for synthesizing a glucaric acid intermediate:

[0032] The third aspect of the present invention first provides a method for synthesizing D-glucuronic acid, comprising the steps of:

[0033] 5) Fructuronate is converted into D-glucuronic acid by biological methods, preferably under the action of an isomerase, preferably under the action of uronic acid isomerase.

[0034] In a specific embodiment of the present invention, the method for synthesizing D-glucuronic acid further comprises: 4) converting D-mannonic acid into fructose ketoate by a biological method, preferably under the action of an oxidoreductase, preferably under the action of D-mannonate oxidoreductase;

[0035] In a specific embodiment of the present invention, the method for synthesizing D-glucuronic acid further comprises: 3) obtaining D-mannose by a biological method, preferably converting D-mannose under the action of aldose dehydrogenase.

[0036] In a specific embodiment of the present invention, the method for synthesizing D-glucuronic acid further comprises: 2) converting D-fructose into D-mannose by a biological method, preferably under the action of an isomerase, preferably under the action of mannose isomerase.

[0037] The third aspect of the present invention also provides a method for synthesizing fructoic acid, comprising the following steps:

[0038] 4) D-mannonic acid Fructose ketoate is obtained by biological methods, preferably by conversion under the action of D-mannonate oxidoreductase.

[0039] In a specific embodiment of the present invention, the method for synthesizing fructoic acid further comprises: 3) converting D-mannose into D-mannonic acid by a biological method, preferably under the action of a dehydrogenase, preferably under the action of aldose dehydrogenase.

[0040] In a specific embodiment of the present invention, the method for synthesizing fructoic acid further comprises: 2) converting D-fructose into D-mannose by a biological method, preferably under the action of an isomerase, preferably under the action of mannose isomerase.

[0041] In a specific embodiment of the present invention, any step of the method is optionally performed inside or outside a microbial cell;

[0042] Preferably, the microorganism is a genetically engineered strain;

[0043] The genetically engineered strain expresses:

[0044] 1) at least one DNA molecule encoding glucose isomerase;

[0045] 2) at least one DNA molecule encoding mannose isomerase;

[0046] 3) at least one DNA molecule encoding aldohexose dehydrogenase;

[0047] 4) at least one DNA molecule encoding D-mannose oxidoreductase;

[0048] 5) at least one DNA molecule encoding uronate isomerase;

[0049] 6) at least one DNA molecule encoding uronate dehydrogenase;

[0050] Optional combination of;

[0051] Preferably, it is a combination of 5)-6), a combination of 4)-6), a combination of 3)-6), a combination of 2)-6) or a combination of 1)-6);

[0052] Preferably, it is: a combination of 1)-3), a combination of 1)-4) or a combination of 1)-5);

[0053] Preferably, it is a combination of: 4)-5), a combination of 3)-5), or a combination of 2)-5);

[0054] Preferably: a combination of 3)-4) or a combination of 2)-4);

[0055] Preferably, at least one of the DNA molecules is endogenously overexpressed or exogenously introduced;

[0056] Preferably, the genetically engineered strain is selected from genetically engineered Escherichia coli, Bacillus, Corynebacterium, yeast or Streptomyces; alternatively, the genetically engineered strain is selected from genetically engineered Escherichia coli, Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Corynebacterium glutamicum, Saccharomyces cerevisiae, Candida utilis or Pichia pastoris; preferably, it is selected from genetically engineered Escherichia coli.

[0057] In a specific embodiment of the present invention, the competitive pathway of glucaric acid biosynthesis in the genetically engineered strain is blocked to increase the glucaric acid yield.

[0058] Preferably, one or a combination of the following genes is knocked out:

[0059] 1) at least one enzyme of the glucose phosphotransferase system, such as the gene encoding the glucose-specific PTS enzyme IIBC component, ptsG;

[0060] 2) at least one enzyme of the mannose phosphotransferase system, such as the gene encoding the mannose-specific PTS enzyme IIAB component, manX, the gene encoding the mannose-specific PTS enzyme IIC component, manY, or the gene encoding the mannose-specific PTS enzyme IID component, manZ;

[0061] 3) fructose-specific PTS multiphosphoryl transfer protein encoding gene fruA, fruB or fruK;

[0062] 4) uxuA, the gene encoding D-mannonate dehydratase;

[0063] 5) L-gulonate 5-dehydrogenase encoding gene rspB;

[0064] 6) D-glucarate dehydratase encoding gene gudD;

[0065] 7) Glucokinase encoding gene glk;

[0066] 8) Fructokinase encoding gene mak.

[0067] In a specific embodiment of the present invention, any step of the method is performed in a cell; optionally, each step of the method is performed in the same microbial cell or in different microbial cells.

[0068] In a specific embodiment of the present invention, any step of the method is performed outside the cell; optionally, the reaction is carried out under whole-cell catalysis or cell-free enzyme catalysis conditions.

[0069] A fourth aspect of the present invention provides a genetically engineered strain for synthesizing glucaric acid or an intermediate thereof, the strain comprising:

[0070] 1) at least one DNA molecule encoding glucose isomerase;

[0071] 2) at least one DNA molecule encoding mannose isomerase;

[0072] 3) at least one DNA molecule encoding aldohexose dehydrogenase;

[0073] 4) at least one DNA molecule encoding D-mannose oxidoreductase;

[0074] 5) at least one DNA molecule encoding uronate isomerase;

[0075] 6) at least one DNA molecule encoding uronate dehydrogenase;

[0076] Optional combination of;

[0077] Preferably, at least one of the DNA molecules is endogenously overexpressed or exogenously introduced;

[0078] Preferably, it is a combination of 5)-6), a combination of 4)-6), a combination of 3)-6), a combination of 2)-6) or a combination of 1)-6);

[0079] Preferably, it is: a combination of 1)-3), a combination of 1)-4) or a combination of 1)-5);

[0080] Preferably, it is a combination of: 4)-5), a combination of 3)-5), or a combination of 2)-5);

[0081] Preferred are: a combination of 3)-4) or a combination of 2)-4).

[0082] In a specific embodiment of the present invention, the competitive pathway of glucaric acid biosynthesis in the genetically engineered strain is blocked to increase the glucaric acid yield.

[0083] Preferably, one or a combination of the following genes is knocked out:

[0084] 1) at least one enzyme of the glucose phosphotransferase system, such as the gene encoding the glucose-specific PTS enzyme IIBC component, ptsG;

[0085] 2) at least one enzyme of the mannose phosphotransferase system, such as the gene encoding the mannose-specific PTS enzyme IIAB component, manX, the gene encoding the mannose-specific PTS enzyme IIC component, manY, or the gene encoding the mannose-specific PTS enzyme IID component, manZ;

[0086] 3) fructose-specific PTS multiphosphoryl transfer protein encoding gene fruA, fruB or fruK;

[0087] 4) uxuA, the gene encoding D-mannonate dehydratase;

[0088] 5) L-gulonate 5-dehydrogenase encoding gene rspB;

[0089] 6) D-glucarate dehydratase encoding gene gudD;

[0090] 7) Glucokinase encoding gene glk;

[0091] 8) Fructokinase encoding gene mak.

[0092] A fifth aspect of the present invention provides a biomaterial or a combination thereof for synthesizing glucaric acid or an intermediate thereof, wherein the biomaterial or the combination thereof comprises:

[0093] 1) at least one DNA molecule encoding glucose isomerase;

[0094] 2) at least one DNA molecule encoding mannose isomerase;

[0095] 3) at least one DNA molecule encoding aldohexose dehydrogenase;

[0096] 4) at least one DNA molecule encoding D-mannose oxidoreductase;

[0097] 5) at least one DNA molecule encoding uronate isomerase;

[0098] 6) at least one DNA molecule encoding uronate dehydrogenase;

[0099] Optional combination of;

[0100] Preferably, it is a combination of 5) and 6), a combination of 4)-6), a combination of 3)-6), a combination of 2)-6), or a combination of 1)-6);

[0101] Preferably, it is: a combination of 1)-3), a combination of 1)-4) or a combination of 1)-5);

[0102] Preferably, it is a combination of: 4)-5), a combination of 3)-5), or a combination of 2)-5);

[0103] Preferably: a combination of 3)-4) or a combination of 2)-4);

[0104] The DNA molecules are optionally in the same or different biological materials;

[0105] In a specific embodiment of the present invention, the biological material is an expression cassette, a transposon, a plasmid vector, a phage vector or a viral vector.

[0106] The sixth aspect of the present invention provides an enzyme combination for synthesizing glucaric acid or an intermediate thereof, the enzyme combination comprising:

[0107] 1) Glucose isomerase;

[0108] 2) mannose isomerase;

[0109] 3) Aldohexose dehydrogenase;

[0110] 4) D-mannose oxidoreductase;

[0111] 5) uronate isomerase;

[0112] 6) uronate dehydrogenase;

[0113] Optional combination of

[0114] Preferably, it is a combination of 5) and 6), a combination of 4)-6), a combination of 3)-6), a combination of 2)-6), or a combination of 1)-6);

[0115] Preferably, it is: a combination of 1)-3), a combination of 1)-4) or a combination of 1)-5);

[0116] Preferably, it is a combination of: 4)-5), a combination of 3)-5), or a combination of 2)-5);

[0117] Preferred are: a combination of 3)-4) or a combination of 2)-4).

[0118] A seventh aspect of the present invention provides a method for synthesizing glucaric acid or a chemical containing glucaric acid, comprising:

[0119] 1) culturing the genetically engineered strain according to the fourth aspect of the present invention in a culture medium containing D-glucose, D-fructose or D-mannose to obtain glucaric acid;

[0120] Preferably, the genetically engineered strain expresses the following DNA combinations: a combination of 5)-6), a combination of 4)-6), a combination of 3)-6), a combination of 2)-6), or a combination of 1)-6);

[0121] or alternatively,

[0122] 1-1) using D-glucose, D-fructose or D-mannose as a substrate, and reacting to obtain glucaric acid under the catalysis of the enzyme combination described in the sixth aspect of the present invention;

[0123] 2) Recovering glucaric acid or preparing chemicals containing glucaric acid.

[0124] An eighth aspect of the present invention provides use of the method, genetically engineered strain, biological material, or a combination thereof, or an enzyme combination, in the preparation of glucaric acid or chemicals containing glucaric acid.

[0125] In a specific embodiment of the present invention, in the method, genetically engineered strain, biological material or combination thereof or enzyme combination:

[0126] 1) Glucose isomerase selected from the group consisting of: xylA, xylA2, sle_59150, xsi, HaOG217244, or enzymes having at least 90%, 95%, or 99% sequence identity with the above enzymes and retaining activity;

[0127] 2) mannose isomerase selected from: Toce_1877, Z5688, lyxi, manI, Marme_2490, or an enzyme having at least 90%, 95%, or 99% sequence identity with the above enzymes and retaining activity;

[0128] 3) an aldohexose dehydrogenase selected from the group consisting of Ta0754, gdh, ycdF, gcd, dhg, TVG0075046, and FAD_1020, or an enzyme having at least 90%, 95%, or 99% sequence identity thereto and retaining activity;

[0129] 4) D-mannose oxidoreductase selected from uxuB, yeiQ, SH2646, PPA2328, GDI3276, por, or an enzyme having at least 90%, 95%, or 99% sequence identity with the above enzymes and retaining activity;

[0130] 5) uronate isomerase selected from uxaC, ygjX, ygjY, ygjZ, yjmA, or an enzyme having at least 90%, 95%, or 99% sequence identity with the above enzymes and retaining activity;

[0131] 6) The uronate dehydrogenase is selected from udh, HNR61_001967, FHX33004122, GGQ72003410, or an enzyme having at least 90%, 95%, or 99% sequence identity with the above enzymes and retaining activity.

[0132] A ninth aspect of the present invention also provides a method for synthesizing D-fructose, comprising the steps of:

[0133] 1) Glucose-6-phosphate is converted into fructose-6-phosphate by the action of glucose-6-phosphate isomerase; 2) Fructose-6-phosphate is converted into D-fructose by the action of phosphatase.

[0134] In a specific embodiment of the present invention, any step or combination of the methods is performed in a genetically engineered strain cell, or under whole-cell catalysis;

[0135] Preferably, the genetically engineered strain is selected from genetically engineered Escherichia coli, Bacillus, Corynebacterium, yeast or Streptomyces; optionally, the genetically engineered strain is selected from genetically engineered Escherichia coli, Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Corynebacterium glutamicum, Saccharomyces cerevisiae, Candida utilis or Pichia pastoris; preferably, it is selected from genetically engineered Escherichia coli.

[0136] A tenth aspect of the present invention also provides a method for synthesizing D-mannose, comprising the following steps:

[0137] 1) Glucose-6-phosphate is converted into fructose-6-phosphate by the action of glucose-6-phosphate isomerase; 2) Fructose-6-phosphate is converted into mannose-6-phosphate by the action of mannose-6-phosphate isomerase; 3) Mannose-6-phosphate is converted into D-mannose by the action of phosphatase.

[0138] In a specific embodiment of the present invention, any step or combination of the methods is performed in a genetically engineered strain cell, or under whole-cell catalysis;

[0139] Preferably, the genetically engineered strain is selected from genetically engineered Escherichia coli, Bacillus, Corynebacterium, yeast or Streptomyces; optionally, the genetically engineered strain is selected from genetically engineered Escherichia coli, Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Corynebacterium glutamicum, Saccharomyces cerevisiae, Candida utilis or Pichia pastoris; preferably, it is selected from genetically engineered Escherichia coli.

[0140] Beneficial technical effects:

[0141] The present invention designs a new biosynthetic pathway for glucaric acid, with high enzyme activity in each step, which can achieve higher yield and conversion rate; and is easy to operate, relatively environmentally friendly, and has the prospect of large-scale production. Description of the drawings:

[0142] Figure 1: Reaction mechanism diagram of the present invention;

[0143] Figure 2: HPLC analysis of glucaric acid in the fermentation broth of the genetically engineered strain. DETAILED DESCRIPTION

[0144] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is further described below with reference to specific embodiments.

[0145] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the corresponding fields and are common procedures. To facilitate a better understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0146] As used herein, the term "amplification" means that the intracellular activity of one or more enzymes encoded by appropriate DNA in a microorganism is increased, for example, by increasing the gene copy number, using a strong promoter, or using a gene encoding an appropriate enzyme with high activity, and optionally combining these methods.

[0147] Unless specifically stated, the terms "first" and "second" do not denote any order or importance, but rather the terms first, second, etc. are used to distinguish one object from another.

[0148] As used herein, the term "gene synthesis" refers to the production of DNA or amino acid sequences using recombinant DNA technology or obtained using techniques for synthesizing DNA or amino acid sequences available and known in the art. "Encoding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is equivalent to the mRNA sequence and is typically provided in a sequence listing, and the non-coding strand, which serves as a template for transcription of a gene or cDNA, may be said to encode a protein or other product of that gene or cDNA.

[0149] As used herein, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter. The terms "overexpression" or "enhanced expression" refer to an increase in the level of mRNA encoding a protein and / or an increase in the level of a protein in a cell compared to a similar, unmodified cell expressing a basal level of mRNA or protein. For example, endogenous or exogenous genes can be introduced into a microorganism to increase the expression level of the corresponding enzyme. Alternatively, endogenous or exogenous gene expression can be enhanced by replacing a strong promoter with a strong promoter or introducing an enhancer.

[0150] As used herein, the term "vector" or "recombinant vector" is a genetic material composition that includes an isolated nucleic acid and that can be used to deliver the isolated nucleic acid to the interior of a cell. The transferred nucleic acid is typically linked to, for example, inserted into, a vector nucleic acid molecule. The vector may contain sequences that direct autonomous replication in the cell or may contain sequences sufficient to allow integration into the host cell DNA. Many vectors are known in the art, including but not limited to plasmids, phagemids, artificial chromosomes, bacterial phages, and animal viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The host may be not only a wild strain, but also a mutant strain thereof or an artificial genetic recombinant; in some specific embodiments of the present invention, the Escherichia coli mutant strain BW25113 is used as the host.

[0151] The term "recovery" refers to separation and purification of glucaric acid from the production culture medium by a known method, such as crystallization, chromatography, ion exchange resin, activated carbon adsorption elution, and solvent extraction.

[0152] As used herein and unless otherwise indicated, the term "about" when referring to a measurable value such as an amount, a time period, etc., is meant to include variations of ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the given value, provided that such variations are appropriate to practice the disclosed methods.

[0153] As described above, the purpose of the present invention is to provide a novel biosynthetic pathway for glucaric acid and a genetically engineered strain. Some embodiments disclose a method for preparing glucaric acid, wherein D-glucose, D-fructose, or D-mannose is added to a culture medium as a substrate, and bacteria or fungi ferment or biotransform the fermentation substrate in vitro to obtain glucaric acid.

[0154] Since the supply of D-glucose, D-fructose or D-mannose directly affects the biosynthesis of glucaric acid, the technical problem to be solved by the present invention is to provide a novel glucaric acid biosynthesis pathway and a genetically engineered strain, and to cultivate the genetically engineered strain by adding a culture medium containing D-glucose, D-fructose or D-mannose to achieve a method for producing glucaric acid.

[0155] One of the purposes of the present invention is to provide a method for preparing glucaric acid, wherein D-glucose is fermented by bacteria or yeast into D-fructose or D-mannose or D-mannonic acid or fructosolic acid or D-glucuronic acid or glucaric acid.

[0156] A second object of the present invention is to provide a method for preparing D-fructose, comprising: converting D-glucose into D-fructose through bacterial or yeast fermentation.

[0157] A third object of the present invention is to provide a method for preparing D-mannose, comprising: 1) converting D-glucose into D-fructose by bacterial or yeast fermentation; and 2) converting D-fructose into D-mannose by bacterial or yeast fermentation.

[0158] A fourth object of the present invention is to provide a method for preparing D-mannonic acid, comprising: 1) converting D-glucose into D-fructose by bacterial or yeast fermentation; 2) converting D-fructose into D-mannose by bacterial or yeast fermentation; and 3) converting D-mannose into D-mannonic acid by bacterial or yeast fermentation.

[0159] A fifth object of the present invention is to provide a method for preparing fructosolic acid, comprising: 1) converting D-glucose into D-fructose by bacterial or yeast fermentation; 2) converting D-fructose into D-mannose by bacterial or yeast fermentation; 3) converting D-mannose into D-mannonic acid by bacterial or yeast fermentation; and 4) converting the D-mannonic acid into fructosolic acid by bacterial or yeast fermentation.

[0160] A sixth object of the present invention is to provide a method for preparing D-glucuronic acid, comprising: 1) converting D-glucose into D-fructose by bacterial or yeast fermentation; 2) converting D-fructose into D-mannose by bacterial or yeast fermentation; 3) converting D-mannose into D-mannonic acid by bacterial or yeast fermentation; 4) converting the D-mannonic acid into fructose ketoate by bacterial or yeast fermentation; and 5) converting the fructose ketoate into D-glucuronic acid by bacterial or yeast fermentation.

[0161] In the present invention, serial numbers are used to describe combinations of reaction steps, DNA molecules, biomaterials, or enzymes. For example, 4)-6) includes a combination of the three aspects numbered 4), 5), and 6). In the present invention, the combination of reaction steps indicates that the final synthesis is completed by utilizing the reaction steps therein, but does not limit the manner in which the reactions of each step are carried out. Each reaction step can be carried out step by step or simultaneously, can be carried out inside a cell or outside a cell, or can be carried out under the catalysis of a cell-free enzyme. Unless otherwise stated, the combination of DNA molecules, biomaterials, or enzymes does not mean that the combination of DNA molecules, biomaterials, or enzymes is in the same organism or biomaterial, but the present invention preferably expresses the combination of DNA molecules and enzymes in the same organism.

[0162] In the present invention, "at least one" refers to at least one of the same or similar references, and is not necessarily present. For example, a combination comprising "at least one DNA molecule encoding glucose isomerase" refers to comprising one or more DNA molecules encoding glucose isomerase. However, if there is an expression of "optionally comprising" or "optionally in combination", the "DNA molecule encoding glucose isomerase" may not exist.

[0163] In the present invention, the term "amino acid mutation" or "nucleotide mutation" includes "substitution, duplication, deletion or addition of one or more amino acids or nucleotides". In the present invention, the term "mutation" refers to a change in the nucleotide sequence or amino acid sequence. In a specific embodiment, the term "mutation" refers to "substitution".

[0164] In the present invention, the term "native state" used refers to the activity of the polypeptide in the microorganism in an unmodified state, that is, the activity in the natural state.

[0165] In the present invention, the term "maintaining activity" used has the same or similar meaning as commonly understood by those skilled in the art, and both refer to that the amino acid sequence of a fragment is part of the amino acid sequence of a complete protein or polypeptide and has the same or similar function or activity as the complete protein or polypeptide.

[0166] Therefore, it is readily apparent that further mutations of the enzymes of the present invention can be made to produce further mutants that still possess the corresponding functions and activities. For example, it is well known to those skilled in the art that adding or subtracting a number of amino acid residues, such as preferably 1-20, more preferably 1-15, more preferably 1-10, more preferably 1-3, and most preferably 1, at either end of a polypeptide will not affect the function of the resulting mutant. For example, to facilitate purification, technicians often attach a 6×His tag to either end of the resulting protein, and such a protein has the same function as a protein without the 6×His tag. Therefore, the present invention encompasses conservative mutations obtained based on the present invention.

[0167] In the present invention, the term "conservative mutation" refers to a mutation that can maintain the normal function of a protein. A representative example of a conservative mutation is a conservative substitution.

[0168] As used in the present invention, the term "conservative substitution" refers to replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, and include residues having basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), β-branched chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).

[0169] As used herein, a "conservative substitution" generally replaces an amino acid at one or more sites in a protein. Such substitutions may be conservative. Examples of substitutions considered conservative include substitutions of Ala for Ser or Thr, Arg for Gln, His or Lys, Asn for Glu, Gln, Lys, His or Asp, Asp for Asn, Glu or Gln, Cys for Ser or Ala, Gln for Asn, Glu, Lys, His, Asp or Arg substitution, Glu to Gly, Asn, Gln, Lys or Asp substitution, Gly to Pro substitution, His to Asn, Lys, Gln, Arg or Tyr substitution, Ile to Leu, Met, Val or Phe substitution, Leu to Ile, Met, Val or Phe substitution, Lys to Asn, Glu, Gln, His or Arg substitution, Met to Ile, Leu, Val or Phe substitution, Phe to Trp, Tyr, Met, Ile or Leu substitution, Ser to Thr or Ala substitution, Thr to Ser or Ala substitution, Trp to Phe or Tyr substitution, Tyr to His, Phe or Trp substitution, and Val to Met, Ile or Leu substitution. In addition, conservative mutations also include naturally occurring mutations due to individual differences, strain differences, species differences, etc. from which the gene is derived.

[0170] The term "corresponding to" as used herein has the meaning commonly understood by those skilled in the art. Specifically, "corresponding to" refers to a position in one sequence that corresponds to a specified position in the other sequence after alignment for homology or sequence identity. Thus, for example, with respect to "the amino acid residue corresponding to position 40 of the amino acid sequence set forth in SEQ ID NO: 1," if a 6×His tag is added to one end of the amino acid sequence set forth in SEQ ID NO: 1, then the position corresponding to position 40 in the resulting mutant may be position 46.

[0171] In a specific embodiment, the homology or sequence identity may be greater than 90%, preferably greater than 95%, more preferably 96%, 97%, 98%, or 99%.

[0172] Methods for determining sequence homology or identity that are well known to those of ordinary skill in the art include, but are not limited to, Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987, and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991, and Carillo, H. and Lipman, D., SIAM J. Applied Biology. Math., 48:1073 (1988). Preferred methods for determining identity are to obtain the largest match between the sequences tested. Methods for determining identity are codified in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S, F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith-Waterman algorithm can also be used to determine identity.

[0173] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0174] The present invention provides a method for producing glucaric acid by fermentation, which is characterized in that D-glucose is added to a culture medium, the above-mentioned Escherichia bacteria are cultured, glucaric acid is generated and accumulated in the culture medium, and then glucaric acid is extracted from the culture medium.

[0175] At the same time, the present invention also provides a method for optimizing the production of glucaric acid by genetically engineered strains using different sugars, which is characterized in that some sugar competition pathways, such as the glucokinase encoding gene glk and the fructokinase encoding gene mak, are knocked out in the genetically engineered strain to improve the production and accumulation of glucaric acid, and then the glucaric acid is extracted from the culture medium.

[0176] The reaction mechanism of preparing glucaric acid according to the present invention is shown in FIG1 .

[0177] Example

[0178] Materials and Methods

[0179] Phanta Max Super-Fidelity DNA Polymerase, ligase-independent single-fragment rapid cloning kit used in the present invention One Step was purchased from Nanjing Novozymes Biotechnology Co., Ltd.

[0180] LB medium composition: tryptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L, solid medium supplemented with 1.5% agar powder.

[0181] The antibiotic concentrations were: ampicillin 100 μg / mL and kanamycin 50 μg / mL.

[0182] Fermentation medium components:

[0183] Detection method of glucaric acid: HPLC-DAD equipped with Agilent chromatographic columns (Agilent InfinityLab Poroshell 120 Columns) was used to quantitatively detect glucaric acid. The glucaric acid yield in the example table is shown in g / L.

[0184] Example 1 Construction of recombinant plasmids pZE-xylA-Toce_1877-Ta0754 and pZA-uxuB-uxaC-udh

[0185] The primers used in the present invention are shown in the following table:

[0186] The uxuB and uxaC gene fragments were amplified by PCR using the genome of Escherichia coli MG1655 as a template, and the udh gene fragment was amplified by PCR using the genome of Pseudomonas syringae as a template. They were ligated into the vector pZAlac containing an IPTG-inducible promoter using a ligase-independent single-fragment rapid cloning kit. The resulting fragments were then transformed into DH5α competent cells, plated on kanamycin sulfate-resistant plates, and cultured overnight. Positive clones were selected for sequencing verification, and the correct recombinant vector was named pZA-uxuB-uxaC-udh.

[0187] The xylA gene fragment was synthesized based on the sequence of Acidothermus cellulolyticus 11B, the Toce_1877 gene fragment was synthesized based on the sequence of Thermosediminibacter oceani DSM 16646, and the Ta0754 gene fragment was synthesized based on the sequence of Thermoplasma acidophilum. The corresponding gene fragments were obtained by PCR and digested with restriction endonucleases. Then, they were ligated to the pZElac vector containing an IPTG-inducible promoter using ligase. The recombinant vectors were then transformed into DH5α competent cells and plated on ampicillin-resistant plates for overnight culture. Positive clones were selected for sequencing verification, and the correct recombinant vector was named pZE-xylA-Toce_1877-Ta0754.

[0188] The recipient bacteria C177, C187, and C195 used in the present invention were constructed by the following method: using Escherichia coli BW25113 as the background bacteria, the target genes (ptsG, fruAKB, manXYZ, uxuA, rspB, and gudD genes) were sequentially knocked out by homologous recombination, and the strains were spread on kanamycin sulfate-resistant plates and cultured overnight. Positive clones were selected for verification, and the resulting strain was named C177.

[0189] Using the C177 strain as the background bacteria, the target gene (mak gene) was knocked out by homologous recombination. The strain was plated on kanamycin sulfate resistance plates and cultured overnight. Positive clones were selected for verification. The final strain obtained was named C187.

[0190] Using the C187 strain as the background bacteria, the target gene (glk gene) was knocked out by homologous recombination. The strain was plated on kanamycin sulfate resistance plates and cultured overnight. Positive clones were selected for verification, and the final strain obtained was named C195.

[0191] The biological materials or plasmids constructed by the present invention are as follows:

[0192] The gene information of the enzyme involved in the present invention is as follows:

[0193] In the context of the present disclosure, the enzyme referred to includes mutants that retain enzyme activity, wherein the mutant has an amino acid sequence that is at least 98%, or at least 99%, identical to the amino acid sequence represented by the protein accession number in the table or the amino acid sequence encoded by the gene accession number (or SEQ ID NO: 1-6 in the present invention).

[0194] Example 2: Genetically engineered bacterial fermentation method for producing glucaric acid using D-glucuronic acid

[0195] Recombinant plasmids pZA-uxuB-uxaC-udh and pZE-xylA-Toce_1877-Ta0754 were electroporated into recipient strain C177 (ΔptsG ΔfruAKB ΔmanXYZ ΔuxuA ΔrspB ΔgudD) to produce genetically engineered bacteria. Each of the genetically engineered bacteria was inoculated into 2 mL of LB liquid medium containing 100 μg / mL ampicillin and 50 μg / mL kanamycin sulfate and cultured overnight at 37°C, 240 rpm (approximately 16 hours). A 5% inoculum was then inoculated into fermentation medium (using D-glucuronic acid as the carbon source) containing an appropriate amount of antibiotics and cultured at 30°C, 240 rpm. After 4 hours of incubation, IPTG was added to a final concentration of 1 mM, and fermentation was continued for 66 hours before cessation. HPLC analysis revealed the presence of glucaric acid in the fermentation broth of the genetically engineered strain (see Figure 2). The results of glucaric acid production by fermentation are shown in Table 1. D-glucuronic acid is converted into glucaric acid under the action of the udh gene, and the D-glucaric acid yield is 80.19±1.45%. The yield calculation formula is: (actual yield / theoretical yield)×100%.

[0196] Table 1

[0197] Example 3: Production of Glucaric Acid Using D-Mannoic Acid by Genetically Engineered Bacteria Fermentation

[0198] Recombinant plasmids pZA-uxuB-uxaC-udh and pZE-xylA-Toce_1877-Ta0754 were electroporated into recipient strain C177 (ΔptsG ΔfruAKB ΔmanXYZ ΔuxuA ΔrspB ΔgudD) to generate genetically engineered bacteria. Each strain was inoculated into 2 mL of LB liquid medium containing 100 μg / mL ampicillin and 50 μg / mL kanamycin sulfate and cultured overnight at 37°C, 240 rpm (approximately 16 hours). A 5% inoculum of the culture was then inoculated into fermentation medium (using D-mannose as the carbon source) containing an appropriate amount of antibiotics and cultured at 30°C, 240 rpm. After an initial 4-hour incubation, IPTG was added to a final concentration of 1 mM and fermentation was continued for 66 hours before cessation. The results of the fermentation for glucaric acid production are shown in Table 2. D-mannonic acid is converted into fructoic acid under the action of the uxuB gene, fructoic acid is converted into D-glucuronic acid under the action of the uxaC gene, and D-glucuronic acid is converted into glucaric acid under the action of the udh gene.

[0199] Table 2

[0200] Example 4: Production of Glucaric Acid Using D-Mannose by Genetically Engineered Bacteria Fermentation

[0201] Recombinant plasmids pZA-uxuB-uxaC-udh and pZE-xylA-Toce_1877-Ta0754 were electroporated into recipient strain C177 (ΔptsG ΔfruAKB ΔmanXYZ ΔuxuA ΔrspB ΔgudD) to generate genetically engineered bacteria. Each strain was inoculated into 2 mL of LB liquid medium containing 100 μg / mL ampicillin and 50 μg / mL kanamycin sulfate and cultured overnight at 37°C, 240 rpm (approximately 16 hours). A 5% inoculum of the culture was then inoculated into fermentation medium (using D-mannose as the carbon source) containing an appropriate amount of antibiotics and cultured at 30°C, 240 rpm. After an initial 4-hour incubation, IPTG was added to a final concentration of 1 mM and fermentation was continued for 66 hours before cessation. The results of the fermentation for glucaric acid production are shown in Table 3. D-mannose is converted into D-mannonic acid under the action of the Ta0754 gene, D-mannonic acid is converted into fructosolic acid under the action of the uxuB gene, fructosolic acid is converted into D-glucuronic acid under the action of the uxaC gene, and D-glucuronic acid is converted into glucaric acid under the action of the udh gene.

[0202] Table 3

[0203] Example 5: Production of Glucaric Acid Using D-Mannose by Genetically Engineered Bacteria Fermentation

[0204] Recombinant plasmids pZA-uxuB-uxaC-udh and pZE-xylA-Toce_1877-Ta0754 were electroporated into recipient strain C187 (ΔptsG ΔfruAKB ΔmanXYZ ΔuxuA ΔrspB ΔgudD Δmak) to generate genetically engineered bacteria. Each strain was inoculated into 2 mL of LB liquid medium containing 100 μg / mL ampicillin and 50 μg / mL kanamycin sulfate and cultured overnight at 37°C, 240 rpm (approximately 16 hours). A 5% inoculum of the culture was then inoculated into fermentation medium (using D-mannose as the carbon source) containing an appropriate amount of antibiotics and cultured at 30°C, 240 rpm. After an initial 4-hour culture, IPTG was added to a final concentration of 1 mM and fermentation continued. The results of fermentation for glucaric acid production are shown in Table 4. D-mannose is converted into D-mannonic acid under the action of the Ta0754 gene, D-mannonic acid is converted into fructosolic acid under the action of the uxuB gene, fructosolic acid is converted into D-glucuronic acid under the action of the uxaC gene, and D-glucuronic acid is converted into glucaric acid under the action of the udh gene.

[0205] Table 4

[0206] Example 6: Production of Glucaric Acid Using D-Fructose by Genetically Engineered Bacteria Fermentation

[0207] Recombinant plasmids pZA-uxuB-uxaC-udh and pZE-xylA-Toce_1877-Ta0754 were electroporated into recipient strain C187 (ΔptsG ΔfruAKB ΔmanXYZ ΔuxuA ΔrspB ΔgudD Δmak) to generate genetically engineered bacteria. Each strain was inoculated into 2 mL of LB liquid medium containing 100 μg / mL ampicillin and 50 μg / mL kanamycin sulfate and cultured overnight at 37°C, 240 rpm (approximately 16 hours). A 5% inoculum of the culture was then inoculated into fermentation medium (using D-fructose as the carbon source) containing an appropriate amount of antibiotics and cultured at 30°C, 240 rpm. After an initial 4-hour culture, IPTG was added to a final concentration of 1 mM and fermentation continued. The results of fermentation production of glucaric acid are shown in Table 5. D-fructose is converted into D-mannose by the action of mannose isomerase, D-mannose is converted into D-mannonic acid by the action of Ta0754 gene, D-mannonic acid is converted into fructosolic acid by the action of uxuB gene, fructosolic acid is converted into D-glucuronic acid by the action of uxaC gene, and D-glucuronic acid is converted into glucaric acid by the action of udh gene.

[0208] Table 5

[0209] Example 7: Production of Glucaric Acid Using D-Glucose by Genetically Engineered Bacteria Fermentation

[0210] Recombinant plasmids pZA-uxuB-uxaC-udh and pZE-xylA-Toce_1877-Ta0754 were electroporated into recipient strain C195 (ΔptsG ΔfruAKB ΔmanXYZ ΔuxuA ΔrspB ΔgudD ΔmakΔglk) to generate genetically engineered bacteria. Each engineered strain was inoculated into 2 mL of LB liquid medium containing 100 μg / mL ampicillin and 50 μg / mL kanamycin sulfate and cultured overnight at 37°C, 240 rpm (approximately 16 hours). A 5% inoculum of the culture was then inoculated into fermentation medium (using D-glucose as the carbon source) containing an appropriate amount of antibiotics and cultured at 30°C, 240 rpm. After an initial 4-hour culture, IPTG was added to a final concentration of 1 mM and fermentation continued. D-glucose is converted into D-fructose by the action of glucose isomerase xylA, D-fructose is converted into D-mannose by the action of mannose isomerase Toce_1877, D-mannose is converted into D-mannonic acid by the action of Ta0754 gene, D-mannonic acid is converted into fructosolic acid by the action of uxuB gene, fructosolic acid is converted into D-glucuronic acid by the action of uxaC gene, and D-glucuronic acid is converted into glucaric acid by the action of udh gene.

[0211] Table 6

[0212] The present invention provides a new synthesis pathway for glucaric acid and a method for producing glucaric acid. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters. The present invention is not limited to the above embodiments. The above embodiments and descriptions are only illustrative of the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the invention claimed. The scope of protection claimed in the present invention is defined by the attached claims and their equivalents.

Claims

1. [Corrected 12.07.2024 in accordance with Rule 91] A method for synthesizing glucaric acid comprising the following steps: 5) Fructuronate is converted into D-glucuronic acid by biological methods, preferably under the action of isomerase; 6) D-glucuronic acid is preferably converted into glucaric acid by a biological method under the action of dehydrogenase; Preferably, the method for synthesizing glucaric acid further comprises: 4) D-mannose acid is converted into fructose ketoate by a biological method preferably under the action of an oxidoreductase; Preferably, the method for synthesizing glucaric acid further comprises: 3) converting D-mannose into D-mannonic acid by a biological method, preferably under the action of a dehydrogenase; Preferably, the method for synthesizing glucaric acid further comprises: 2) converting D-fructose into D-mannose by a biological method, preferably under the action of isomerase; Preferably, the method for synthesizing glucaric acid further comprises: 1) converting D-glucose into D-fructose by a biological method, preferably under the action of isomerase; Further preferably, steps 1) and 2) are steps of directly converting D-glucose into D-mannose under the action of isomerase.

2. [Corrected 12.07.2024 in accordance with Rule 91] A method for synthesizing glucaric acid as claimed in claim 1, wherein The biological method described in step 5) is carried out under the action of the following enzymes: uronic acid isomerase; and / or The biological method described in step 6) is carried out under the action of the following enzyme: uronate dehydrogenase.

3. [Corrected 12.07.2024 in accordance with Rule 91] A method for synthesizing glucaric acid as claimed in claim 1, wherein The biological method described in step 4) is carried out under the action of the following enzyme: D-mannonate oxidoreductase.

4. [Corrected 12.07.2024 in accordance with Rule 91] A method for synthesizing glucaric acid as claimed in claim 1, wherein The biological method described in step 3) is carried out under the action of the following enzyme: aldose dehydrogenase.

5. [Corrected 12.07.2024 in accordance with Rule 91] A method for synthesizing glucaric acid as claimed in claim 1, wherein The biological method described in step 2) is carried out under the action of the following enzymes: mannose isomerase (D-mannose isomerase); and / or The biological method described in step 1) is carried out under the action of the following enzymes: D-glucose isomerase; and / or In the biological method described in steps 1)-2), glucose is directly converted into mannose under the action of cellobiose 2-epimerase (EC 5.1.3.11) or D-mannose 2-epimerase (EC 5.1.3).

6. [Corrected 12.07.2024 in accordance with Rule 91] A process for the synthesis of D-mannose acid, comprising: 1) Convert D-glucose into D-fructose; 2) Convert D-fructose into D-mannose; 3) D-mannose is converted into D-mannonic acid by a biological method preferably under the action of a dehydrogenase, preferably under the action of aldose dehydrogenase.

7. [Corrected 12.07.2024 according to Rule 91] A method for synthesizing fructosolic acid, firstly obtaining D-mannonic acid using the synthesis method described in claim 6, and then 4) converting D-mannonic acid into fructose by a biological method, preferably under the action of an oxidoreductase, preferably under the action of D-mannonic acid oxidoreductase.

8. [Corrected 12.07.2024 according to Rule 91] A method for synthesizing D-glucuronic acid, firstly obtaining fructuronate using the synthesis method described in claim 7, and then 5) converting fructuronate into D-glucuronic acid by a biological method, preferably under the action of an isomerase, preferably under the action of uronic acid isomerase.

9. [Corrected 12.07.2024 in accordance with Rule 91] A method for synthesizing D-glucuronic acid, comprising the following steps: 5) Fructuronate is converted into D-glucuronic acid by a biological method, preferably under the action of an isomerase, preferably under the action of uronic acid isomerase.

10. [Corrected 12.07.2024 in accordance with Rule 91] The method for synthesizing D-glucuronic acid as claimed in claim 9, further comprising: 4) D-mannonic acid is preferably converted into fructose ketoate by a biological method under the action of an oxidoreductase, preferably under the action of D-mannonic acid oxidoreductase.

11. [Corrected 12.07.2024 in accordance with Rule 91] The method for synthesizing D-glucuronic acid as claimed in claim 10, further comprising: 3) D-mannose is converted into D-mannonic acid by a biological method preferably under the action of a dehydrogenase, preferably under the action of aldose dehydrogenase.

12. [Corrected on 12.07.2024 in accordance with Rule 91] The method for synthesizing D-glucuronic acid as claimed in claim 11, further comprising: 2) D-fructose is converted into D-mannose by a biological method, preferably under the action of an isomerase, preferably under the action of mannose isomerase.

13. [Corrected 12.07.2024 in accordance with Rule 91] A method for synthesizing fructosolic acid, comprising the following steps: 4) D-mannonic acid Fructose ketoate is obtained by biological methods, preferably by conversion under the action of D-mannonic acid oxidoreductase.

14. [Corrected 12.07.2024 in accordance with Rule 91] The method for synthesizing fructosolic acid as claimed in claim 13, further comprising: 3) D-mannose is converted into D-mannonic acid by a biological method preferably under the action of a dehydrogenase, preferably under the action of aldose dehydrogenase.

15. [Corrected 12.07.2024 in accordance with Rule 91] The method for synthesizing fructosolic acid as claimed in claim 14, further comprising: 2) D-fructose is converted into D-mannose by a biological method, preferably under the action of an isomerase, preferably under the action of mannose isomerase.

16. [Corrected 12.07.2024 according to Rule 91] The method according to any one of claims 1 to 15, wherein any step of the method is optionally carried out inside or outside a microbial cell; Preferably, the microorganism is a genetically engineered strain that expresses: 1) at least one DNA molecule encoding glucose isomerase; 2) at least one DNA molecule encoding mannose isomerase; 3) at least one DNA molecule encoding aldohexose dehydrogenase; 4) at least one DNA molecule encoding D-mannose acid oxidoreductase; 5) at least one DNA molecule encoding uronate isomerase; 6) at least one DNA molecule encoding uronate dehydrogenase; An optional combination of; Preferably, it is: a combination of 5)-6), a combination of 4)-6), a combination of 3)-6), a combination of 2)-6) or a combination of 1)-6); Preferably, it is: a combination of 1)-3), a combination of 1)-4) or a combination of 1)-5); Preferably: a combination of 4)-5), a combination of 3)-5) or a combination of 2)-5); Preferably: a combination of 3)-4) or a combination of 2)-4); Preferably, at least one of the DNA molecules is endogenously overexpressed or exogenously introduced; Preferably, the genetically engineered strain is selected from genetically engineered Escherichia coli, Bacillus, Corynebacterium, yeast or Streptomyces; optionally, the genetically engineered strain is selected from genetically engineered Escherichia coli, Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Corynebacterium glutamicum, Saccharomyces cerevisiae, Candida utilis or Pichia pastoris; preferably, it is selected from genetically engineered Escherichia coli.

17. [Corrected 12.07.2024 according to Rule 91] The method according to claim 16, blocking the competitive pathway of glucaric acid biosynthesis in the genetically engineered strain to increase the yield of glucaric acid, preferably, knocking out one or a combination of any of the following genes: 1) at least one enzyme in the glucose phosphotransferase system, such as the gene encoding the glucose-specific PTS enzyme IIBC component ptsG; 2) at least one enzyme in the mannose phosphotransferase system, such as the gene encoding the mannose-specific PTS enzyme IIAB component manX, the gene encoding the mannose-specific PTS enzyme IIC component manY, or the gene encoding the mannose-specific PTS enzyme IID component manZ; 3) fructose-specific PTS multiphosphoryl transfer protein encoding gene fruA, fruB or fruK; 4) uxuA, the gene encoding D-mannonate dehydratase; 5) L-gulonic acid 5-dehydrogenase encoding gene rspB; 6) D-glucarate dehydratase encoding gene gudD; 7) Glucokinase encoding gene glk; 8) Fructokinase encoding gene mak.

18. [Corrected on 12.07.2024 according to Rule 91] The method according to claim 16, wherein any step of the method is carried out in a cell; optionally, each step of the method is carried out in the same microbial cell or in different microbial cells.

19. [Corrected on 12.07.2024 according to Rule 91] The method according to claim 16, wherein any step of the method is carried out outside the cell; optionally under whole-cell catalysis or cell-free enzyme catalysis conditions.

20. [Corrected 12.07.2024 in accordance with Rule 91] A genetically engineered strain for the synthesis of glucaric acid or an intermediate thereof, the strain expressing: 1) at least one DNA molecule encoding glucose isomerase; 2) at least one DNA molecule encoding mannose isomerase; 3) at least one DNA molecule encoding aldohexose dehydrogenase; 4) at least one DNA molecule encoding D-mannose acid oxidoreductase; 5) at least one DNA molecule encoding uronate isomerase; 6) at least one DNA molecule encoding uronate dehydrogenase; An optional combination of; Preferably, it is: a combination of 5)-6), a combination of 4)-6), a combination of 3)-6), a combination of 2)-6) or a combination of 1)-6); Preferably, it is: a combination of 1)-3), a combination of 1)-4) or a combination of 1)-5); Preferably: a combination of 4)-5), a combination of 3)-5) or a combination of 2)-5); Preferably: a combination of 3)-4) or a combination of 2)-4); Preferably, at least one of the DNA molecules is endogenously overexpressed or exogenously introduced; Blocking the competitive pathway of the microbial glucaric acid biosynthesis to increase the glucaric acid yield, preferably, knocking out one or a combination of any of the following genes: 1) at least one enzyme in the glucose phosphotransferase system, such as the gene encoding the glucose-specific PTS enzyme IIBC component ptsG; 2) at least one enzyme in the mannose phosphotransferase system, such as the gene encoding the mannose-specific PTS enzyme IIAB component manX, the gene encoding the mannose-specific PTS enzyme IIC component manY, or the gene encoding the mannose-specific PTS enzyme IID component manZ; 3) fructose-specific PTS multiphosphoryl transfer protein encoding gene fruA, fruB or fruK; 4) uxuA, the gene encoding D-mannonate dehydratase; 5) L-gulonic acid 5-dehydrogenase encoding gene rspB; 6) D-glucarate dehydratase encoding gene gudD; 7) Glucokinase encoding gene glk; 8) Fructokinase encoding gene mak.

21. [Corrected 12.07.2024 in accordance with Rule 91] A biological material or a combination thereof for synthesizing glucaric acid or an intermediate thereof, the biological material or the combination thereof comprising: 1) at least one DNA molecule encoding glucose isomerase; 2) at least one DNA molecule encoding mannose isomerase; 3) at least one DNA molecule encoding aldohexose dehydrogenase; 4) at least one DNA molecule encoding D-mannose acid oxidoreductase; 5) at least one DNA molecule encoding uronate isomerase; 6) at least one DNA molecule encoding uronate dehydrogenase; An optional combination of; Preferably, it is a combination of 5) and 6), a combination of 4)-6), a combination of 3)-6), a combination of 2)-6) or a combination of 1)-6); Preferably, it is: a combination of 1)-3), a combination of 1)-4) or a combination of 1)-5); Preferably: a combination of 4)-5), a combination of 3)-5) or a combination of 2)-5); Preferably: a combination of 3)-4) or a combination of 2)-4); The DNA molecules are optionally in the same or different biological material; Preferably, the biological material is an expression cassette, a transposon, a plasmid vector, a phage vector or a viral vector.

22. [Corrected 12.07.2024 in accordance with Rule 91] An enzyme combination for synthesizing glucaric acid or an intermediate thereof, the enzyme combination comprising: 1) Glucose isomerase; 2) Mannose isomerase; 3) Aldohexose dehydrogenase; 4) D-mannose acid oxidoreductase; 5) uronate isomerase; 6) uronate dehydrogenase; An optional combination of; Preferably, it is a combination of 5) and 6), a combination of 4)-6), a combination of 3)-6), a combination of 2)-6) or a combination of 1)-6); Preferably, it is: a combination of 1)-3), a combination of 1)-4) or a combination of 1)-5); Preferably: a combination of 4)-5), a combination of 3)-5) or a combination of 2)-5); Preferred are: a combination of 3)-4) or a combination of 2)-4).

23. [Corrected 12.07.2024 in accordance with Rule 91] A process for the synthesis of glucaric acid or a chemical containing glucaric acid, comprising: 1) culturing the genetically engineered strain according to claim 21 in a culture medium containing D-glucose, D-fructose or D-mannose to obtain glucaric acid; Preferably, the genetically engineered strain has a DNA combination: a combination of 5)-6), a combination of 4)-6), a combination of 3)-6), a combination of 2)-6) or a combination of 1)-6); or alternatively, 1-1) using D-glucose, D-fructose or D-mannose as substrate, reacting glucaric acid under the catalysis of the enzyme combination of claim 23; Preferably, the enzyme combination is: a combination of 5)-6), a combination of 4)-6), a combination of 3)-6), a combination of 2)-6) or a combination of 1)-6); 2) Recovering glucaric acid or preparing chemicals containing glucaric acid.

24. [Corrected 12.07.2024 according to Rule 91] Use of the method, genetically engineered strain, biological material or a combination thereof or an enzyme combination as claimed in the above claims in the preparation of glucaric acid or chemicals containing glucaric acid.

25. [Corrected 12.07.2024 in accordance with Rule 91] In the method, genetically engineered strain, biological material or combination thereof, enzyme combination or use as claimed in any of the above claims: 1) Glucose isomerase is selected from: xylA, xylA2, sle_59150, xsi, HaOG217244, or an enzyme having at least 90%, 95%, or 99% sequence identity with the above enzymes and maintaining activity; 2) mannose isomerase is selected from: Toce_1877, Z5688, lyxi, manI, Marme_2490, or an enzyme having at least 90%, 95% or 99% sequence identity with the above enzymes and maintaining activity; 3) an aldohexose dehydrogenase selected from the group consisting of Ta0754, gdh, ycdF, gcd, dhg, TVG0075046, FAD_1020, or an enzyme having at least 90%, 95%, or 99% sequence identity with the above enzymes and maintaining activity; 4) D-mannose oxidoreductase is selected from uxuB, yeiQ, SH2646, PPA2328, GDI3276, por, or an enzyme having at least 90%, 95%, or 99% sequence identity with the above enzymes and retaining activity; 5) uronate isomerase is selected from uxaC, ygjX, ygjY, ygjZ, yjmA, or an enzyme having at least 90%, 95%, or 99% sequence identity with the above enzymes and retaining activity; 6) The uronate dehydrogenase is selected from udh, HNR61_001967, FHX33_004122, GGQ72_003410, or an enzyme having at least 90%, 95%, or 99% sequence identity with the above enzymes and retaining activity.

26. [Corrected 12.07.2024 in accordance with Rule 91] A method for synthesizing D-fructose, comprising: 1) Glucose-6-phosphate is converted into fructose-6-phosphate by glucose-6-phosphate isomerase; 2) Fructose-6-phosphate is converted into D-fructose by phosphatase; Preferably, any step or combination of the method is carried out in a genetically engineered strain cell, or under whole-cell catalysis; Preferably, the genetically engineered strain is selected from genetically engineered Escherichia coli, Bacillus, Corynebacterium, yeast or Streptomyces; optionally, the micro-engineered strain is selected from genetically engineered Escherichia coli, Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Corynebacterium glutamicum, Saccharomyces cerevisiae, Candida utilis or Pichia pastoris; preferably, it is selected from genetically engineered Escherichia coli.

27. [Corrected 12.07.2024 in accordance with Rule 91] A method for synthesizing D-mannose, comprising: 1) Glucose-6-phosphate is converted into fructose-6-phosphate by glucose-6-phosphate isomerase; 2) Fructose-6-phosphate is converted into mannose-6-phosphate by mannose-6-phosphate isomerase; 3) Mannose-6-phosphate is converted into D-mannose by phosphatase. Preferably, any step or combination of the method is carried out in a genetically engineered strain cell, or under whole-cell catalysis; Preferably, the genetically engineered strain is selected from genetically engineered Escherichia coli, Bacillus, Corynebacterium, yeast or Streptomyces; optionally, the micro-engineered strain is selected from genetically engineered Escherichia coli, Bacillus subtilis, Bacillus megaterium, Bacillus amyloliquefaciens, Corynebacterium glutamicum, Saccharomyces cerevisiae, Candida utilis or Pichia pastoris; preferably, it is selected from genetically engineered Escherichia coli.

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