Process for the multi-enzymatic coupling conversion of aromatic compounds

Abstract

The application discloses a method for coupling conversion of aromatic compounds by multiple enzymes. The method takes carboxylation-dioxygenation coupling reaction as a core, overcomes the thermodynamic reversibility of enzyme catalysis carboxylation reaction, greatly improves the efficiency of CO2 fixation, and builds a new naphthalene degradation reaction path taking the CO2 fixation as a core. Naphthalene is converted into 1-naphthol by an oxygenase, and then into 2-carboxybenzopropanoic acid by the carboxylation-dioxygenation coupling reaction, and then into o-carboxybenzaldehyde by an aldehyde condensation reaction or into phthalic acid by an aldehyde condensation-dehydrogenation coupling reaction, so that the cleavage ring opening and CO2 fixation of naphthalene, and the preparation of o-carboxybenzaldehyde and phthalic acid are realized.

Description

Technical Field

[0001] This invention relates to the field of biochemistry, and more specifically to a method for the multi-enzyme coupling conversion of aromatic compounds. Background Technology

[0002] Polycyclic aromatic hydrocarbons (PAHs) are a class of compounds composed of multiple benzene rings in their structure. They possess carcinogenic and mutagenic activities and are subject to strict quarantine worldwide. Most PAHs are byproducts of fossil fuels. Since fossil fuels are the energy foundation of human socio-economic activities, the hazards of PAHs will continue to receive widespread attention in the foreseeable future. Due to π-electron conjugation, aromatic hydrocarbons have low molecular potential energy and are exceptionally chemically stable. Furthermore, aromatic compounds are nonpolar substances, most of which are slightly soluble or insoluble in water. The stability and polarity of aromatic hydrocarbons increase with the number of benzene rings in their structure. Therefore, the degradation of aromatic substances is a slow and complex process.

[0003] Naphthalene is the simplest PAH (polycyclic aromatic hydrocarbon) compound, consisting of a molecule composed of two benzene rings. Naphthalene and its derivatives are important chemical raw materials, widely used in pesticide synthesis, dyeing, and rubber processing. The petroleum lightening and coal coking processes generate large quantities of light recycled hydrocarbons (LCO) containing naphthalene and its derivatives; the annual LCO production from the crude oil industry is 50 million tons. LCO is a valuable resource, but its cracking produces light aromatic hydrocarbons (benzene, toluene, and xylene). However, LCO also poses a significant threat to ecology and health. Based on the principles of sustainable development in the petrochemical industry and the implementation of environmental protection, the generation and treatment of naphthalene and other aromatic hydrocarbons are subject to strict regulation. However, the aromatic hydrocarbons such as naphthalene generated in the petrochemical industry are mainly produced through metal catalytic cracking in high-temperature, high-pressure environments. This method is complex and suffers from high pollution and high energy consumption. In comparison, biological cracking of PAHs has low energy consumption and is environmentally friendly.

[0004] Another serious environmental threat posed by the use of fossil fuels is CO2 emissions and the greenhouse effect. Due to the greenhouse effect, global climate anomalies have led to a significant increase in the frequency of extreme weather events. Therefore, reducing CO2 emissions or improving CO2 fixation efficiency has become an urgent need. The Kolbe–Schmitt reaction, catalyzed by non-oxidative carboxylases, involves the insertion of a carboxyl group at the ortho position of the hydroxyl group in phenolic compounds (Kolbe–Schmitt reaction), is a typical carbon fixation reaction. The Kolbe–Schmitt reaction has a significant reverse reaction, resulting in a very low conversion rate. High temperatures and pressures (~90 bar, 120–300 °C) are typically used to improve the conversion rate. This process is energy-intensive, highly polluting, and produces significant carbon emissions. Enzymatic catalysis of this process can be carried out under milder conditions and does not require a large energy input, but the significant reverse reaction and low conversion rate are unavoidable. To improve the efficiency of enzyme-catalyzed CO2 fixation, the limitation of the reverse reaction must be addressed.

[0005] o-Carboxybenzaldehyde is an important intermediate in the synthesis of antipyretic and analgesic drugs. It is typically obtained from phenol via bromination and hydrolysis. The process involves heating phenol, introducing bromine, reacting the bromine, adding water, hydrolyzing, and then cooling to precipitate o-carboxybenzaldehyde. This process is cumbersome, lacks controllability, and results in low yields and poor purity of o-carboxybenzaldehyde. Phthalic acid is an intermediate in the production of fuels, polyester resins, polyester fibers, pharmaceuticals, and plasticizers. Traditionally, the preparation of phthalic acid is complex, using linoleic acid, o-xylene, or naphthalene as raw materials, and involving metal catalysis and strong oxidants. Naphthalene can be used to prepare o-carboxybenzaldehyde and phthalic acid; however, the safety and economic efficiency of this process need further improvement. Summary of the Invention

[0006] The purpose of this invention is to provide a method for the multi-enzyme coupling conversion of aromatic compounds.

[0007] In a first aspect, the present invention claims protection for a method for ring-opening an aromatic compound.

[0008] The ring-opening method for the aromatic compound claimed in this invention may include the following steps:

[0009] (A1) Aromatic compounds react with monooxygenases to form corresponding phenolic compounds;

[0010] (A2) The phenolic compounds are catalyzed by carboxylase to react and generate carboxyl-containing aromatic compounds;

[0011] (A3) The carboxyl-containing aromatic compound is catalyzed by a dioxygenase to react and generate a carboxylic acid compound;

[0012] (A4) The carboxylic acid compounds are catalyzed by aldolase to react and generate pyruvate and aromatic aldehyde compounds;

[0013] (A5) The pyruvate and aromatic aldehyde compounds react with dehydrogenase to generate dicarboxylic acid compounds.

[0014] In the method, if the aromatic compound contains a phenolic hydroxyl group, step (A1) is skipped and step (A2) is performed directly.

[0015] In the method, the catalytic reactions of various enzymes are carried out in any of the following ways: 1) the corresponding enzyme is added directly to the reaction system; 2) cells capable of expressing the corresponding enzyme are added to the reaction system.

[0016] In a specific embodiment of the present invention, the cells capable of expressing the monooxygenase are *E. coli* strains capable of expressing the monooxygenase, such as BL21 Gold(DE3); the cells capable of expressing the carboxylase are *E. coli* strains capable of expressing the carboxylase, such as BL21 Gold(DE3); the cells capable of expressing the dioxygenase are *E. coli* strains capable of expressing the dioxygenase, such as BL21 Gold(DE3); the cells capable of expressing the aldolase are *E. coli* strains capable of expressing the aldolase, such as BL21 Gold(DE3); and the cells capable of expressing the dehydrogenase are *E. coli* strains capable of expressing the dehydrogenase, such as BL21 Gold(DE3).

[0017] In step (A1), reducing power is required from NAD(P)H.

[0018] Furthermore, the provision of reducing power by NAD(P)H can be achieved in either of the following ways: 1) directly adding NAD(P)H to the reaction system; 2) forming the coenzyme NAD(P)H / NAD(P) in the reaction system. + cycle.

[0019] Furthermore, the coenzyme NAD(P)H / NAD(P) is formed in the reaction system. + The cycle can be achieved by either of the following methods: 1) adding alcohol dehydrogenase ADH and NAD(P) to the reaction system. + ;2) Add cells that express alcohol dehydrogenase ADH and NAD(P) to the reaction system. + 3) Add alcohol dehydrogenase ADH and NAD(P)H to the reaction system; 4) Add cells capable of expressing alcohol dehydrogenase ADH and NAD(P)H to the reaction system; 5) When steps (A1) and (A5) are completed in the same reaction system, add NAD(P)H or NAD(P)H to the reaction system. + Regarding 5), monooxygenase catalysis can convert NAD(P)H to NAD(P). + Dehydrogenases catalyze the reaction of NAD(P) +The conversion to NAD(P)H involves a monooxygenase in step (A1) and a dehydrogenase in step (A5). Therefore, when steps (A1) and (A5) are in the same reaction system, a coenzyme cycle can be formed, requiring only the addition of either NAD(P)H or NAD(P). + That's fine. The same applies below.

[0020] In a specific embodiment of the present invention, the cells capable of expressing alcohol dehydrogenase ADH are *Escherichia coli* species capable of expressing alcohol dehydrogenase ADH, such as BL21 Gold(DE3). The amount of cells capable of expressing alcohol dehydrogenase ADH added to the reaction system is 0.5 g cell wet weight / mL, NAD(P) + Alternatively, the amount of NAD(P)H added may be 30 mM.

[0021] In step (A2), HCO3 - Alternatively, CO2 can be used as another substrate.

[0022] In step (A5), the reaction requires NAD(P). + .

[0023] Furthermore, NAD(P) in the reaction + NAD(P) can be introduced in either of the following ways: 1) Directly add NAD(P) to the reaction system. + ;2) Coenzyme NAD(P)H / NAD(P) is formed in the reaction system. + cycle.

[0024] Furthermore, the coenzyme NAD(P)H / NAD(P) is formed in the reaction system. + The cycle can be achieved by: 1) adding NAD(P)H oxidase and NAD(P) to the reaction system. + 2) Add NAD(P)H oxidase and NAD(P)H to the reaction system; 3) When steps (A1) and (A5) are completed in the same reaction system, add NAD(P)H or NAD(P) to the reaction system. + .

[0025] In a specific embodiment of the present invention, the amount of NAD(P)H oxidase (Nox) added is 4.5 U / mL, NAD(P) + The amount added was 0.5 mM.

[0026] The aromatic compound may be an aromatic hydrocarbon compound.

[0027] Furthermore, the aromatic hydrocarbon compound may be a polycyclic aromatic hydrocarbon;

[0028] Furthermore, the polycyclic aromatic hydrocarbon may be naphthalene.

[0029] In a specific embodiment of the present invention, the aromatic compound is naphthalene.

[0030] Secondly, the present invention claims protection for a method for degrading naphthalene and / or fixing CO2.

[0031] The method for degrading naphthalene and / or fixing CO2 claimed in this invention may include the following steps:

[0032] (a1) Naphthalene reacts with monooxygenase to produce 1-naphthol;

[0033] (a2) 1-Naphthol reacts with carboxylase to produce 1-hydroxy-2-benzoic acid;

[0034] (a3) 1-Hydroxy-2-benzoic acid reacts with dioxygenase to produce 2-carboxybenzopyruvic acid;

[0035] (a4)2-Carboxybenzopyruvate reacts with aldolase to produce o-carboxybenzaldehyde;

[0036] (a5) o-Carboxybenzaldehyde reacts with dehydrogenase to produce phthalic acid.

[0037]

[0038] In step (a1), reducing power is required from NAD(P)H.

[0039] Furthermore, the provision of reducing power by NAD(P)H can be achieved in either of the following ways: 1) directly adding NAD(P)H to the reaction system; 2) forming the coenzyme NAD(P)H / NAD(P) in the reaction system. + cycle.

[0040] Furthermore, the coenzyme NAD(P)H / NAD(P) is formed in the reaction system. + The cycle can be achieved by either of the following methods: 1) adding alcohol dehydrogenase ADH and NAD(P) to the reaction system. + ;2) Add cells that express alcohol dehydrogenase ADH and NAD(P) to the reaction system. + 3) Add alcohol dehydrogenase ADH and NAD(P)H to the reaction system; 4) Add cells capable of expressing alcohol dehydrogenase ADH and NAD(P)H to the reaction system; 5) When steps (a1) and (a5) are completed in the same reaction system, add NAD(P)H or NAD(P)H to the reaction system. + Regarding 5), monooxygenase catalysis can convert NAD(P)H to NAD(P). + Dehydrogenases catalyze the reaction of NAD(P)+ The conversion to NAD(P)H involves a monooxygenase in step (a1) and a dehydrogenase in step (a5). Therefore, when steps (a1) and (a5) are in the same reaction system, a coenzyme cycle can be formed, requiring only the addition of either NAD(P)H or NAD(P). + That's all.

[0041] In a specific embodiment of the present invention, the cells capable of expressing alcohol dehydrogenase ADH are *Escherichia coli* species capable of expressing alcohol dehydrogenase ADH, such as BL21 Gold(DE3). The amount of cells capable of expressing alcohol dehydrogenase ADH added to the reaction system is 0.5 g cell wet weight / mL, NAD(P) + Alternatively, the amount of NAD(P)H added may be 30 mM.

[0042] In step (a2), HCO3 - Alternatively, CO2 can be used as another substrate.

[0043] In the method described, the catalytic reactions of various enzymes can be carried out in any of the following ways: 1) directly adding the corresponding enzyme to the reaction system; 2) adding cells capable of expressing the corresponding enzyme to the reaction system.

[0044] In step (a5), the reaction requires NAD(P). + .

[0045] Furthermore, NAD(P) in the reaction + NAD(P) can be introduced in either of the following ways: 1) Directly add NAD(P) to the reaction system. + ;2) Coenzyme NAD(P)H / NAD(P) is formed in the reaction system. + cycle.

[0046] Furthermore, the coenzyme NAD(P)H / NAD(P) is formed in the reaction system. + The cycle can be achieved by: 1) adding NAD(P)H oxidase and NAD(P) to the reaction system. + ;2) Add NAD(P)H oxidase and NAD(P)H to the reaction system;3) When steps (a1) and (a5) are completed in the same reaction system, add NAD(P)H or NAD(P) to the reaction system. + .

[0047] In a specific embodiment of the present invention, the amount of NAD(P)H oxidase (Nox) added is 4.5 U / mL, NAD(P) + The amount added was 0.5 mM.

[0048] In the method, steps (a1)-(a3) can be completed in one step in the same reaction system, which is referred to as reaction system I.

[0049] Furthermore, the reaction system I contains 1) naphthalene, 2) a monooxygenase or a cell capable of expressing the monooxygenase, 3) a carboxylase or a cell capable of expressing the carboxylase, 4) a dioxygenase or a cell capable of expressing the dioxygenase, 5) NAD(P)H, and 6) HCO3-. - Or CO2, 7) reaction buffer I.

[0050] Furthermore, in the reaction system I, the final concentration of naphthalene is 15 mM, the final concentration of NAD(P)H is 60 mM, and the final concentration of HCO3- is... - The final concentration is 50 mM or CO2 is continuously introduced into reaction system I during the reaction. The monooxygenase is 0.5 mg / mL or 0.5 g wet cell weight / mL of cells capable of expressing the monooxygenase; the carboxylase is 0.03-1 U / mL (e.g., 1 U / mL, 0.03 U / mL, 0.14 U / mL) or 0.5 g wet cell weight / mL of cells capable of expressing the carboxylase; the dioxygenase is 9 U / mL or 0.5 g wet cell weight / mL of cells capable of expressing the dioxygenase; the remainder is reaction buffer I.

[0051] Furthermore, the pH of the reaction buffer I can be 6.5-8.0 (e.g., pH 7.0-7.5). Specifically, it can be a potassium phosphate buffer (pH 7.0 or pH 7.5, 100mM).

[0052] Furthermore, the reaction temperature can be 25-35℃ (e.g., 30℃), and the reaction time can be 3-12h (e.g., 12h).

[0053] In the method, steps (a4)-(a5) can be completed in one step in the same reaction system, which is referred to as reaction system II.

[0054] Furthermore, the reaction system II contains 1) 2-carboxybenzopyruvate, 2) aldolase or cells capable of expressing the aldolase, 3) dehydrogenase or cells capable of expressing the dehydrogenase, 4) NAD(P)H, 5) KHCO3, and 6) reaction buffer II.

[0055] In a specific embodiment of the present invention, the reaction system II also contains NAD(P)H oxidase, but NAD(P)H oxidase is not essential; its function is to produce NAD(P). + Specifically, during the catalysis of dehydrogenases, NAD(P) is released. + Transformed into NAD(P)H, if NAD(P)+ It is not enough; it requires NAD(P)H oxidase to catalyze the production of NAD(P)H.

[0056] Furthermore, in reaction system II, the final concentration of 2-carboxybenzopyruvate is 10 mM, the final concentration of NAD(P)H is 15 mM, and the final concentration of KHCO3 is 50 mM. The concentration of the aldolase is 0.75 mg / ml, the concentration of the dehydrogenase is 18 U / ml, and the remainder is reaction buffer II.

[0057] In a specific embodiment of the present invention, the concentration of NAD(P)H oxidase in reaction system II is 4.5 U / ml.

[0058] Furthermore, the pH of the reaction buffer II is 6.5-8.0 (e.g., pH 7.0-7.5). Specifically, it can be a potassium phosphate buffer (pH 7.0 or pH 7.5, 100 mM).

[0059] Furthermore, the reaction temperature can be 25-35℃ (e.g., 30℃), and the reaction time can be 3-12h (e.g., 5.5h).

[0060] In the method, steps (a2)-(a5) can be completed in one step in the same reaction system, which is referred to as reaction system III.

[0061] Furthermore, the reaction system III contains 1) 1-naphthol, 2) a carboxylase or a cell capable of expressing the carboxylase, 3) a dioxygenase or a cell capable of expressing the dioxygenase, 4) an aldolase or a cell capable of expressing the aldolase, 5) a dehydrogenase or a cell capable of expressing the dehydrogenase, 6) NAD(P)H oxidase or a cell capable of expressing the NAD(P)H oxidase, and 7) NAD(P) + 8) KHCO3, 9) Reaction buffer III.

[0062] Furthermore, in reaction system III, the final concentration of 1-naphthol is 7.5 mM, and the concentration of NAD(P) is... + The final concentration of the reagent is 0.5 mM, and the final concentration of the KHCO3 is 90 mM. The concentration of the carboxylase is 0.03-1 U / mL (e.g., 1 U / mL, 0.03 U / mL, 0.14 U / mL) or 0.5 g wet weight / mL of cells capable of expressing the carboxylase; the concentration of the dioxygenase is 9 U / mL or 0.5 g wet weight / mL of cells capable of expressing the dioxygenase; the concentration of the aldolase is 0.75 mg / mL; the concentration of the dehydrogenase is 18 U / mL; the concentration of the NAD(P)H oxidase is 4.5 U / mL; the remainder is the reaction buffer III.

[0063] Furthermore, the pH of the reaction buffer III is 6.5-8.0 (e.g., pH 7.0-7.5). Specifically, it can be a potassium phosphate buffer (pH 7.0 or pH 7.5, 100 mM).

[0064] Furthermore, the reaction temperature is 25-35℃ (e.g., 30℃), and the reaction time is 3-12h (e.g., 12h).

[0065] Thirdly, the present invention claims protection for any of the following methods:

[0066] Method I: A method for generating o-carboxybenzaldehyde from naphthalene as a substrate, comprising steps (a1)-(a4) of the method described in the second aspect above;

[0067] Method II: A method for generating phthalic acid from naphthalene as a substrate, comprising steps (a1)-(a5) of the method described in the second aspect above;

[0068] Method III, a method for generating o-carboxybenzaldehyde using 1-naphthol as a substrate, comprising steps (a2)-(a4) of the method described in the second aspect above;

[0069] Method IV: A method for generating phthalic acid from 1-naphthol as a substrate, comprising steps (a2)-(a5) of the method described in the second aspect above.

[0070] Fourthly, this invention claims protection for complete sets of enzymes.

[0071] The enzyme kits claimed in this invention are as follows: (B1) or (B2) or (B3) or (B4):

[0072] (B1) is composed of carboxylase, dioxygenase and aldolase;

[0073] (B2) consists of monooxygenase, alcohol dehydrogenase (ADH), carboxylase, dioxygenase, and aldolase;

[0074] (B3) is composed of carboxylase, dioxygenase, aldolase, dehydrogenase and NAD(P)H oxidase;

[0075] (B4) consists of monooxygenase, alcohol dehydrogenase (ADH), carboxylase, dioxygenase, aldolase, dehydrogenase, and NAD(P)H oxidase.

[0076] Fifthly, the present invention claims protection of complete cell sets.

[0077] The complete cell assembly claimed in this invention is as follows (C1) or (C2) or (C3) or (C4):

[0078] (C1) consists of cells that can express carboxylase, cells that can express dioxygenase, and cells that can express aldolase.

[0079] (C2) consists of cells that can express monooxygenase, cells that can express alcohol dehydrogenase (ADH), cells that can express carboxylase, cells that can express dioxygenase, and cells that can express aldolase.

[0080] (C3) consists of cells that can express carboxylase, cells that can express dioxygenase, cells that can express aldolase, cells that can express dehydrogenase, and cells that can express NAD(P)H oxidase.

[0081] (C4) consists of cells that can express monooxygenase, cells that can express alcohol dehydrogenase (ADH), cells that can express carboxylase, cells that can express dioxygenase, cells that can express aldolase, cells that can express dehydrogenase, and cells that can express NAD(P)H oxidase.

[0082] Sixthly, the present invention claims protection for any of the following applications P1-P2:

[0083] P1. The application of the method described in the first aspect above, or the set of enzymes described in the fourth aspect above, or the set of cells described in the fifth aspect above in the degradation of aromatic compounds and / or fixation of CO2 and / or preparation of o-carboxybenzaldehyde and / or phthalic acid.

[0084] P2. The application of the method described in the first aspect above, or the set of enzymes described in the fourth aspect above, or the set of cells described in the fifth aspect above, in the degradation of naphthalene and / or fixation of CO2 and / or preparation of o-carboxybenzaldehyde and / or phthalic acid.

[0085] In all of the above aspects, the monooxygenase may be a monooxygenase mutant derived from Bacillus megaterium [P450 BM-3(A74G / F87V / L188Q)]. Further, the amino acid sequence of the monooxygenase mutant derived from Bacillus megaterium [P450 BM-3(A74G / F87V / L188Q)] is shown in SEQ ID No. 1.

[0086] In the foregoing aspects, the carboxylase may be a carboxylase derived from *Aspergillus oryzae*, a carboxylase derived from *Rhizobium sp.*, or a carboxylase derived from *Trichosporon moniliiforme*. Further, the amino acid sequence of the carboxylase derived from *Aspergillus oryzae* is shown in SEQ ID No. 2; the amino acid sequence of the carboxylase derived from *Rhizobium sp.* is shown in SEQ ID No. 3; and the amino acid sequence of the carboxylase derived from *Trichosporon moniliiforme* is shown in SEQ ID No. 4.

[0087] In all of the above aspects, the dioxygenase may be a dioxygenase derived from Mycobacterium vanbaalenii PYR-1. Further, the amino acid sequence of the dioxygenase derived from Mycobacterium vanbaalenii PYR-1 is as shown in SEQ ID No. 5.

[0088] In all of the above aspects, the aldolase may be an aldolase derived from *Pseudomonas putida*. Further, the amino acid sequence of the aldolase derived from *Pseudomonas putida* is shown in SEQ ID No. 6.

[0089] In all of the above aspects, the dehydrogenase may be a dehydrogenase derived from Nocardioides sp. KP7. Further, the amino acid sequence of the dehydrogenase derived from Nocardioides sp. KP7 is shown in SEQ ID No. 7.

[0090] In all the above aspects, the NAD(P)H oxidase may be an NAD(P)H oxidase derived from Lactiplantibacillus pentosus. Further, the amino acid sequence of the NAD(P)H oxidase derived from Lactiplantibacillus pentosus is shown in SEQ ID No. 8.

[0091] In all the above aspects, the amino acid sequence of the alcohol dehydrogenase ADH is shown in SEQ ID No. 17.

[0092] In this invention, the monooxygenase, carboxylase, dioxygenase, aldolase, dehydrogenase, and NAD(P)H oxidase are all obtained by prokaryotic expression using *E. coli* as the host bacterium, followed by Ni column purification. Specifically, they can be prepared according to the following steps: *E. coli* (e.g., BL21 Gold(DE3)) expressing the monooxygenase, carboxylase, dioxygenase, aldolase, dehydrogenase, or NAD(P)H oxidase is induced to express the enzyme using IPTG at a final concentration of 50 μM under the condition of 20–30 °C for 24 h; cells are collected by centrifugation, resuspended, and lysed; the supernatant is collected by centrifugation, filtered, purified by Ni column, desalted, and freeze-dried to obtain the final product.

[0093] This invention discloses a method for CO2 fixation and naphthalene cleavage coupling reaction. This method takes carboxylation-dioxygenation coupling reaction as the core, overcomes the thermodynamic reversibility of enzyme-catalyzed carboxylation reaction, greatly improves the efficiency of CO2 fixation, and builds a new naphthalene degradation reaction pathway based on this. Naphthalene is converted into 1-naphthol by oxygenase, then into 2-carboxybenzopyruvate through carboxylation-dioxygenation coupling reaction, and then into o-carboxybenzaldehyde through aldol condensation reaction or into phthalic acid through aldol condensation-dehydrogenation coupling reaction. This realizes the ring-opening cleavage of naphthalene and CO2 fixation, as well as the preparation of o-carboxybenzaldehyde and phthalic acid.

[0094] Compared with existing non-oxidative carboxylation reactions, the present invention has the following advantages:

[0095] 1. By coupling carboxylase and oxygenase, product inhibition is eliminated, and the carboxylase-catalyzed reaction can proceed under normal environmental conditions, which greatly reduces the cost of C fixation by carboxylase and improves efficiency.

[0096] 2. Using naphthalene as a substrate, a new reaction pathway was constructed to couple the degradation of naphthalene with the fixation of CO2.

[0097] 3. Using naphthalene as a substrate, a green and mild method for synthesizing o-carboxybenzaldehyde and phthalic acid was designed.

[0098] 4. The method of the present invention does not rely on substrate and temperature for driving, and the reaction conditions are mild and the operation is convenient. Attached Figure Description

[0099] Figure 1The enzyme purification effect was detected by SDS-PAGE electrophoresis. A shows the purification effect of the five enzymes involved in Example 2; B shows the purification effect of the three enzymes involved in Example 10. In the figure, M: standard protein marker; L1: P450BM-3 (A74G / F87V / L188Q); L2: 2,3-DHBD; L3: 1HNDO; L4: SAD; L5: 2,6-DHBD; L6: NsaE; L7: Nox; L8: PhdK. The arrows indicate the target protein.

[0100] Figure 2 This is for the detection of pure 2-carboxybenzopyruvate using liquid chromatography-mass spectrometry (LC-MS) and the detection of 1-naphthol using liquid chromatography (LC). A is the pure 2-carboxybenzopyruvate for LC-MS detection; B is the 1-naphthol standard for LC-chromatographic detection.

[0101] Figure 3 A direct enzymatic reaction method was developed for the liquid-phase detection of carboxylase 2,3-DHBD and dioxygenase 1HNDO coupled to catalyze the cleavage of 1-naphthol.

[0102] Figure 4 A direct enzymatic reaction method was developed for the liquid-phase detection of the cleavage of 1-naphthol catalyzed by the coupling reaction of carboxylase 2,6-DHBD and dioxygenase 1HNDO.

[0103] Figure 5 A direct enzymatic reaction method was developed for the liquid-phase detection of the cleavage of 1-naphthol catalyzed by the coupling reaction of carboxylase SAD and dioxygenase 1HNDO.

[0104] Figure 6 To detect the cleavage of 1-naphthol by coupling carboxylase 2,3-DHBD and dioxygenase 1HNDO in liquid chromatography (resting cell reaction method).

[0105] Figure 7 A direct enzymatic reaction method was developed for the liquid-phase detection of carboxylase 2,3-DHBD and dioxygenase 1HNDO coupled to catalyze the cleavage of 1-naphthol and fixation of CO2.

[0106] Figure 8 To detect the degradation of naphthalene by coupling monooxygenase P450 BM-3 (A74G / F87V / L188Q), carboxylase 2,3-DHBD, and dioxygenase 1HNDO in liquid chromatography (direct enzymatic reaction method).

[0107] Figure 9 To detect the degradation of naphthalene by coupling monooxygenase P450 BM-3 (A74G / F87V / L188Q), carboxylase 2,3-DHBD, and dioxygenase 1HNDO in liquid chromatography (resting cell coenzyme cycle reaction method).

[0108] Figure 10This study describes the liquid chromatography (LC) detection of o-carboxybenzaldehyde as a standard and the NsaE aldolase-catalyzed cleavage of 2-carboxybenzopyruvate to o-carboxybenzaldehyde. A represents the LC detection of o-carboxybenzaldehyde as a standard; B represents the LC detection of the NsaE aldolase-catalyzed cleavage of 2-carboxybenzopyruvate to o-carboxybenzaldehyde.

[0109] Figure 11 Pyruvic acid and phthalic acid are used as standards for ion chromatography detection.

[0110] Figure 12 This method is used for the ion chromatography detection of phthalic acid and pyruvate produced by the cleavage of 2-carboxybenzopyruvate catalyzed by aldolase NsaE, dehydrogenase PhdK, and NAD(P)H oxidase.

[0111] Figure 13 This method is used for the ion chromatography detection of carboxylase 2,3-DHBD, dioxygenase 1-HNDO, aldolase NsaE, dehydrogenase PhdK, and NAD(P)H oxidase catalyzing the cleavage of 1-naphthol to phthalic acid and pyruvate. Detailed Implementation

[0112] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0113] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0114] Example 1: Expression Host Construction

[0115] Following gene optimization, Suzhou Genewise Biotechnology Co., Ltd. was commissioned to artificially synthesize a monooxygenase mutant [P450 BM-3] derived from Bacillus megaterium. (A74G / F87V / L188Q) The gene encoding the protein shown in SEQ ID No. 9 (SEQ ID No. 9 encodes the protein shown in SEQ ID No. 1) was inserted into the NdeⅠ restriction site of pET28a. The resulting recombinant vector was named pET28a::bm3 after being verified by sequencing.

[0116] The gene encoding the carboxylase (2,3-DHBD) from Aspergillus oryzae was synthesized as shown in SEQ ID No. 10 (SEQ ID No. 10 encodes the protein shown in SEQ ID No. 2). This gene was inserted into the NcoⅠ restriction site of the pRSFDuet1 vector (New England Biolabs). The resulting recombinant vector was named pRSFDuet1::2,3-dhnd after being verified by sequencing.

[0117] The gene encoding the carboxylase (2,6-DHBD) derived from Rhizobium sp. was synthesized as shown in SEQ ID No. 11 (SEQ ID No. 11 encodes the protein shown in SEQ ID No. 3). This gene was inserted into the NcoⅠ restriction site of the pRSFDuet1 vector (New England Biolabs). The resulting recombinant vector was correctly sequenced and named pRSFDuet1::2,6-dhnd.

[0118] The gene encoding the carboxylase (SAD) derived from *Trichosporon moniliiforme* was synthesized as shown in SEQ ID No. 12 (SEQ ID No. 12 encodes the protein shown in SEQ ID No. 4). This gene was inserted into the NcoⅠ restriction site of the pRSFDuet1 vector (New England Biolabs). The resulting recombinant vector was correctly sequenced and named pRSFDuet1::sad.

[0119] The gene encoding the dioxygenase (1HNDO) derived from Mycobacterium vanbaalenii PYR-1 was synthesized as shown in SEQ ID No. 13 (SEQ ID No. 13 encodes the protein shown in SEQ ID No. 5). This gene was inserted into the NcoI restriction site of the pRSFDuet1 vector (New England Biolabs). The resulting recombinant vector was sequenced and verified to be correct, and named pRSFDuet1::1hndo.

[0120] The above expression vectors were transformed into Escherichia coli BL21 Gold(DE3), and positive clones were screened and cultured on LB plates containing kanamycin. Plasmids were extracted and sequenced to confirm successful vector construction.

[0121] Example 2: Expression and purification of enzymes

[0122] Each of the positive engineered bacteria constructed in Example 1 was inoculated into 5 mL of LB medium and cultured at 37°C and 200 rpm for 12 h. Then, 1 mL of seed culture was transferred to 100 mL of fresh LB medium and cultured at 37°C and 220 rpm for approximately 2 h until OD (digestive activity) was reached. 600 When the expression level reached 0.4–0.6, IPTG at a final concentration of 50 μM was added to induce expression. The expression conditions were: low temperature induction at 20–30 °C, 220 r / min, and 24 h.

[0123] After expression, cells were collected by centrifugation, resuspended in binding buffer, and concentrated 20-fold. Cells were then homogenized under high pressure, centrifuged at 12,000 rpm / min, and the supernatant was collected. The supernatant was filtered through a 0.45 μm filter and then loaded onto a Ni column. Washing buffer was used to elute contaminating proteins, and Eution buffer was used to elute and recover the enzyme. The enzyme was then desalted, freeze-dried, and stored at -80°C. The protein buffer used for Ni column purification is shown in Table 1.

[0124] Table 1. Protein buffer for Ni column purification

[0125] Binding buffer 20mM phosphate, 0.5M NaCl, 20mM imidazole, pH 8.0 Washing buffer 20mM phosphate, 0.5M NaCl, 40mM imidazole, pH 8.0 Elution buffer 20mM phosphate, 0.5M NaCl, 500mM imidazole, pH 8.0

[0126] The purification effects of the five enzymes involved in this embodiment are shown in [reference needed]. Figure 1 A. As can be seen from the figure, except for P450 BM-3 (A74G / F87V / L188Q) In addition, the purity of the other four enzymes all reached over 90%, meeting the experimental requirements. Monooxygenase [P450BM-3] (A74G / F87V / L188Q) The purity is 70%.

[0127] In addition, the purified product was subjected to enzyme activity assay using a fluorescence method, continuously detecting changes in the fluorescence signal of the reaction sample. Excitation light was 350 nm, and emission light was 420 nm. Carboxylase: The final concentration of KHCO3 in the reaction sample was 200 mM, and the final concentration of 1-naphthol was 10 mM. An appropriate amount of carboxylase was added, and the volume was brought up to 120 μL with potassium phosphate buffer (pH 7.0 or pH 7.5, 100 mM). The mixture was transferred to a 96-well fluorescent microplate, and the increase in fluorescence was detected using a microplate reader (the product 1-hydroxy-2-naphthoic acid exhibits fluorescence). The enzyme activity required to generate 1 μM of 1-hydroxy-2-naphthoic acid per minute was defined as 1 U. Dioxygenase: The final concentration of 1-hydroxy-2-naphthoic acid in the reaction sample was 0.2 mM. An appropriate amount of carboxylase was added, and the volume was brought up to 120 μL with potassium phosphate buffer (pH 7.0 or pH 7.5, 100 mM). The mixture was then transferred to a 96-well fluorescent microplate, and the decrease in fluorescence was detected using a microplate reader. Definition: The enzyme activity required to convert 1 μM of 1-hydroxy-2-naphthoic acid per minute is 1 U. Enzyme activity results: 2,3-DHBD: 0.65 U / mg 蛋白 SAD: 0.09 U / mg 蛋白 2,6-DHBD: 0.002 U / mg 蛋白 1HNDO: 12U / mg 蛋白 Monooxygenase activity was found to be very low, so subsequent reactions generally use whole cells.

[0128] Example 3: Carbon fixation via coupling reaction of 2,3-DHBD and 1HNDO

[0129] 1. Weigh 1-naphthol and dissolve it in N,N-dimethylformamide (DMF) to prepare a 500 mM solution. Prepare potassium phosphate buffer (pH 7.0, 100 mM). Prepare 3 M KHCO3 solution.

[0130] 2. Prepare enzyme solutions by dissolving the carboxylase 2,3-DHBD and dioxygenase 1HNDO prepared in Example 2 in potassium phosphate buffer (pH 7.0, 100mM).

[0131] 3. Premix the reaction components to prepare a reaction solution, as shown in Table 2.

[0132] Table 2. Coupling reaction solution (1 mL)

[0133]

[0134]

[0135] Note: The concentrations of each substance in the table are the final concentrations in the reaction solution.

[0136] Reaction conditions: 200 rpm, 30℃ for 12 h.

[0137] The reaction results were detected by high performance liquid chromatography.

[0138] Spectral column: PAH;

[0139] Eluents: A: 5mM ammonium acetate, B: acetonitrile;

[0140] Elution procedure: as shown in Table 3.

[0141] Table 3. High Performance Liquid Chromatography Elution Program 1

[0142] 0.0 0.6 5.0 7.0 0.6 5.0 12.0 0.8 45.0 24.0 0.8 50.0 30.0 0.6 5.0

[0143] Detection: Ultraviolet absorption method, 300nm.

[0144] The liquid chromatography results for 2-carboxybenzopyruvic acid and 1-naphthol standards are shown in the figure. Figure 2 (A: The LC-MS analysis results of the prepared 2-carboxybenzopyruvic acid after separation and purification show that the structure and molecular weight of the substance are correct; B: Liquid chromatography analysis results of 1-naphthol standard).

[0145] The results are as follows Figure 3 As shown, comparison Figure 2 It can be seen that the substrate 1-naphthol is almost entirely converted into the product 2-carboxybenzopyruvic acid.

[0146] Example 4: Carbon fixation via coupling reaction of 2,6-DHBD and 1HNDO

[0147] 1. Weigh 1-naphthol and dissolve it in N,N-dimethylformamide (DMF) to prepare a 500 mM solution. Prepare potassium phosphate buffer (pH 7.0, 100 mM) and 3 M KHCO3 solution.

[0148] 2. Prepare enzyme solutions by dissolving the carboxylase 2,6-DHBD and dioxygenase 1HNDO prepared in Example 2 in potassium phosphate buffer (pH 7.0, 100mM).

[0149] 3. Premix the reaction components to prepare a reaction solution, as shown in Table 4.

[0150] Table 4. Coupling reaction solution (1 mL)

[0151]

[0152]

[0153] Note: The concentrations of each substance in the table are the final concentrations in the reaction solution.

[0154] Reaction conditions: 200 rpm, 30℃ for 12 h.

[0155] The reaction results were detected by high performance liquid chromatography (the specific detection method is the same as in Example 3).

[0156] The results are as follows Figure 4 As shown, comparison Figure 2 It can be seen that only a portion of the substrate 1-naphthol is converted into the product 2-carboxybenzopyruvic acid.

[0157] Example 5: Carbon fixation via coupling reaction of SAD and 1HNDO

[0158] 1. Weigh 1-naphthol and dissolve it in N,N-dimethylformamide (DMF) to prepare a 500 mM solution. Prepare potassium phosphate buffer (pH 7.0, 100 mM) and 3 M KHCO3 solution.

[0159] 2. Prepare enzyme solutions by dissolving the carboxylase SAD and dioxygenase 1HNDO prepared in Example 2 in potassium phosphate buffer (pH 7.0, 100mM).

[0160] 3. Premix the reaction components to prepare a reaction solution, as shown in Table 5.

[0161] Table 5. Coupling reaction solution (1 mL)

[0162] 1-Naphthol 15mM <![CDATA[KHCO3]]> 45mM SAD carboxylase 0.14 U / mL Dioxygenase (1HNDO) 9U / mL Potassium phosphate buffer (pH 7.0, 100mM) Add 1mL

[0163] Note: The concentrations of each substance in the table are the final concentrations in the reaction solution.

[0164] Reaction conditions: 200 rpm, 30℃ for 12 h.

[0165] The reaction results were detected by high performance liquid chromatography (the specific detection method is the same as in Example 3).

[0166] The results are as follows Figure 5 As shown, comparison Figure 2 It can be seen that only a small amount of the substrate 1-naphthol is converted into the product 2-carboxybenzopyruvic acid.

[0167] Example 6: Carbon fixation of resting cells via coupling reaction of 2,3-DHBD and 1HNDO

[0168] 1. Weigh 1-naphthol and dissolve it in N,N-dimethylformamide (DMF) to prepare a 500 mM solution. Prepare potassium phosphate buffer (pH 7.0, 100 mM) and 3 M KHCO3 solution.

[0169] 2. Prepare resting cells. Resuspend the cells expressing 2,3-DHBD and 1HNDO (E. coli BL21 Gold(DE3) expressing 2,3-DHBD and 1HNDO constructed in Example 1) in potassium phosphate buffer (pH 7.0, 100mM) until homogeneous.

[0170] 3. Premix the reaction components to prepare a reaction solution, as shown in Table 6.

[0171] Table 6. Coupling reaction solution (1 mL)

[0172] 1-Naphthol 15mM <![CDATA[KHCO3]]> 45mM 2,3-DHBD cells <![CDATA[0.5g 细胞湿重 / mL]]> 1HNDO cells <![CDATA[0.5g 细胞湿重 / mL]]> Potassium phosphate buffer (pH 7.0, 100mM) Add 1mL

[0173] Note: The concentrations of each substance in the table are the final concentrations in the reaction solution.

[0174] Reaction conditions: 30℃ for 12 hours.

[0175] The reaction results were detected by high performance liquid chromatography (the specific detection method is the same as in Example 3).

[0176] The results are as follows Figure 6 As shown, comparison Figure 2 It can be seen that all of the 1-naphthol is converted into the product 2-carboxybenzopyruvic acid.

[0177] Example 7: Carbon fixation of CO2 via coupling reaction of 2,3-DHBD and 1HNDO

[0178] 1. Weigh 1-naphthol and dissolve it in N,N-dimethylformamide (DMF) to prepare a 500 mM solution. Prepare potassium phosphate buffer (pH 7.5, 100 mM).

[0179] 2. Prepare enzyme solutions by dissolving the carboxylase 2,3-DHBD and dioxygenase 1HNDO prepared in Example 2 in potassium phosphate buffer (pH 7.5, 100mM).

[0180] 3. Premix the reaction components to prepare a reaction solution, as shown in Table 7.

[0181] Table 7. Coupling reaction solution (1 mL)

[0182] 1-Naphthol 15mM Carboxylase (2,3-DHBD) 1U / mL Dioxygenase (1HNDO) 9U / mL Potassium phosphate buffer (pH 7.5, 100mM) Add 1mL

[0183] Note: The concentrations of each substance in the table are the final concentrations in the reaction solution.

[0184] Reaction conditions: 200 rpm, 30℃ for 12 h, with CO2 continuously introduced during the reaction.

[0185] The reaction results were detected by high performance liquid chromatography (the specific detection method is the same as in Example 3).

[0186] The results are as follows Figure 7 As shown, comparison Figure 2 It is evident that the substrate 1-naphthol was almost entirely converted into the product 2-carboxybenzopyruvic acid. This demonstrates that CO2 can effectively replace HCO3. - The present invention is of great significance for improving the efficiency of CO2 fixation.

[0187] Example 8: Opening of the aromatic ring of naphthalene

[0188] 1. Weigh naphthalene and dissolve it in N,N-dimethylformamide (DMF) to prepare a 500 mM solution. Prepare potassium phosphate buffer (pH 7.0, 100 mM) and 3 M KHCO3 solution.

[0189] 2. Prepare enzyme solutions by dissolving the P450 BM-3 (A74G / F87V / L188Q), 2,3-DHBD and 1HNDO prepared in Example 2 in potassium phosphate buffer (pH 7.0, 100mM).

[0190] 3. Premix the components of the reaction to prepare a reaction solution, as shown in Table 8.

[0191] Table 8. Coupling reaction solution (1 mL)

[0192] Naphthalene 15mM NADH 60mM <![CDATA[KHCO3]]> 50mM Monooxygenase (P450BM-3(A74G / F87V / L188Q)) 0.5 mg / mL Carboxylase (2,3-DHBD) 1U / mL Dioxygenase (1HNDO) 9U / mL Potassium phosphate buffer (pH 7.0, 100mM) Add 1mL

[0193] Note: The concentrations of each substance in the table are the final concentrations in the reaction solution.

[0194] Reaction conditions: 200 rpm, 30℃ for 12 h.

[0195] The reaction results were detected by high performance liquid chromatography (the specific detection method is the same as in Example 3, only the elution procedure is different; the elution procedure for this example is shown in Table 9):

[0196] Table 9. High Performance Liquid Chromatography Elution Program 2

[0197] 0.0 0.5 1.0 10.0 0.5 20.0 15.0 0.8 80.0 24.0 0.8 80.0 30.0 0.5 10.0

[0198] The results are as follows Figure 8 As shown, comparison Figure 2 It can be seen that only naphthalene and HCO3 are present. - When used as a substrate, the reaction route designed in this invention successfully detected the generation of 2-carboxybenzopyruvic acid, demonstrating the effectiveness of this route in degrading naphthalene and fixing carbon.

[0199] Example 9: Opening of the aromatic ring of naphthalene – coenzyme cycle

[0200] 1. Weigh naphthalene and dissolve it in N,N-dimethylformamide (DMF) to prepare a 500 mM solution. Prepare potassium phosphate buffer (pH 7.0, 100 mM) and 3 M KHCO3 solution.

[0201] 2. Prepare resting cells. Resuspend P450 BM-3 (A74G / F87V / L188Q), 2,3-DHBD, and 1HNDO expression cells (E. coli BL21 Gold(DE3) expressing P450 BM-3 (A74G / F87V / L188Q), 2,3-DHBD, and 1HNDO, constructed in Example 1, in potassium phosphate buffer (pH 7.0, 100mM). Prepare resting cells for NADH regeneration and recycling (recombinant bacteria obtained by inserting the ADH encoding gene into the NdeI site of the pET21b plasmid according to the method in Example 1, the gene sequence is shown in SEQ ID No. 18, and the amino acid sequence is shown in SEQ ID No. 17) after introducing them into E. coli BL21 Gold(DE3), for use in NADH regeneration and recycling (to reduce reaction costs).

[0202] 3. Premix the components of the reaction to prepare a reaction solution, as shown in Table 10.

[0203] Table 10. Coupling reaction solution (1 mL)

[0204] Naphthalene 15mM <![CDATA[NAD + ]]> 30mM Isopropanol 30mM <![CDATA[KHCO3]]> 50mM P450BM-3 (A74G / F87V / L188Q) cells <![CDATA[0.5g 细胞湿重 / mL]]> ADH cells <![CDATA[0.5g 细胞湿重 / mL]]> 2,3-DHBD cells <![CDATA[0.5g 细胞湿重 / mL]]> 1HNDO cells <![CDATA[0.5g 细胞湿重 / mL]]> Potassium phosphate buffer (pH 7.0, 100mM) Add 1mL

[0205] Note: The concentrations of each substance in the table are the final concentrations in the reaction solution.

[0206] Reaction conditions: 30℃ for 12 hours.

[0207] The reaction results were detected by high performance liquid chromatography (the specific detection method is the same as in Example 3, only the elution procedure is different, and the elution procedure of this example is shown in Table 9).

[0208] The results are as follows Figure 9 As shown, comparison Figure 2 It can be seen that only naphthalene and HCO3 are present. - When used as a substrate, oxidized coenzyme NAD + The reaction route designed in this invention successfully detected the generation of 2-carboxybenzopyruvic acid, demonstrating the effectiveness of this route in degrading naphthalene and fixing carbon, as well as the role of the coenzyme cycle system.

[0209] Example 10: Expression Host Construction

[0210] Following gene optimization, Suzhou Genewise Biotechnology Co., Ltd. was commissioned to artificially synthesize the gene encoding the aldolase (NsaE) derived from *Pseudomonas putida*, as shown in SEQ ID No. 14 (SEQ ID No. 14 encodes the protein shown in SEQ ID No. 6). This gene was then inserted into the NcoⅠ restriction site of the pRSFDuet1 vector (New England Biolabs). The resulting expression vector was named pRSFDuet1::nsae.

[0211] The gene encoding the dehydrogenase (PhdK) derived from Nocardioides sp. KP7 was synthesized as shown in SEQ ID No. 15 (SEQ ID No. 15 encodes the protein shown in SEQ ID No. 7). This gene was inserted into the NcoI restriction site of the pRSFDuet1 vector (New England Biolabs). The resulting expression vector was named pRSFDuet1::phdk.

[0212] The gene encoding NAD(P)H oxidase (Nox) from *Lactiplantibacillus pentosus*, as shown in SEQ ID No. 16 (SEQ ID No. 16 encodes the protein shown in SEQ ID No. 8), was synthesized and inserted into the NcoI restriction site of the pRSFDuet1 vector (New England Biolabs). The resulting expression vector was named pRSFDuet1::nox.

[0213] pRSFDuet1::nsae, pRSFDuet1::phdk, and pRSFDuet1::nox were transformed into *E. coli* to obtain engineered bacteria. Each constructed positive engineered bacterium was inoculated into 5 mL of LB medium and cultured at 37°C and 200 rpm for 12 h. Then, 1 mL of seed culture was transferred to 100 mL of fresh LB medium and cultured at 37°C and 220 rpm for approximately 2 h until OD (digestive activity) was reached. 600 When the expression level reached 0.4–0.6, IPTG at a final concentration of 50 μM was added to induce expression. The expression conditions were: low temperature induction at 20–30 °C, 220 r / min, and 24 h.

[0214] After expression, cells were collected by centrifugation, resuspended in binding buffer, and concentrated 20-fold. Cells were then homogenized under high pressure, centrifuged at 12,000 rpm / min, and the supernatant was collected and filtered through a 0.45 μm filter membrane. The resulting solution was then loaded onto a Ni column. Washing buffer was used to elute contaminating proteins, and elution buffer was used to elute and recover the enzyme. The enzyme was then desalted, freeze-dried, and stored at -80°C. The protein buffer used for Ni column purification is shown in Table 1 above.

[0215] The purification effects of the three enzymes involved in this embodiment are shown in [reference needed]. Figure 1 Figure B shows that NsaE, PhdK, and Nox were successfully purified.

[0216] Aldolase (NsaE): Add appropriate amounts of NsaE, 10 mM 2'-carboxybenzylmethylpyruvate, 90 mM KHCO3, and 4% (v / v) DMF to KPB (pH 7.0, 100 mM) buffer to a final volume of 1 ml. Incubate at 30 °C for 3 h. Detect the cleavage product, o-carboxybenzaldehyde, by chromatography. Definition: The enzyme activity required to generate 1 μmol of o-carboxybenzaldehyde per minute is 1 U. A reverse reaction was observed in the activity assay, making it difficult to obtain accurate results.

[0217] o-Carboxybenzaldehyde dehydrogenase (PhdK): Add appropriate amounts of PhdK, 2 mM o-carboxybenzaldehyde, and 2 mM NAD to KPB (pH 7.0, 100 mM) buffer. + Mixed with 4% (v / v) DMF, the final volume was 120 μL, and NADH (A) was detected using a microplate reader. 340 Phthalic acid is generated. Definition: The enzyme activity required to generate 1 μmol of phthalic acid per minute is 1 U. The purified enzyme activity was measured to be 24 U / mg.

[0218] NADH oxidase (Nox): Add appropriate amounts of Nox and 2mM NADH to KPB (pH 7.0, 100mM) buffer to a final volume of 120μL. NADH (Nox) is then detected using a microplate reader. 340 Consumption. Definition: The enzyme activity required to generate 1 μmol of phthalic acid per minute is 1 U. The purified enzyme activity was measured to be 6 U / mg.

[0219] Example 11: Aldolase-catalyzed cleavage of 2-carboxybenzopyruvate

[0220] 1. Weigh 2-carboxybenzopyruvic acid and dissolve it in 50mM KHCO3 solution to prepare a 50mM solution. Prepare potassium phosphate buffer (pH 7.5, 100mM).

[0221] 2. Prepare enzyme solution by dissolving the aldolase NsaE prepared in Example 10 in potassium phosphate buffer (pH 7.5, 100mM).

[0222] 3. Premix the reaction components to prepare a reaction solution, as shown in Table 11.

[0223] Table 11. Reaction Solution

[0224] 2-Carboxybenzopyruvic acid 10mM <![CDATA[KHCO3]]> 50mM Aldolase (NsaE) 0.75mg Potassium phosphate buffer (pH 7.5, 100mM) Add to a final volume of 1.05 mL.

[0225] Note: The concentrations of each substance in the table are the final concentrations in the reaction solution.

[0226] Reaction conditions: 200 rpm, 30℃ for 7 h.

[0227] The reaction results were detected by high performance liquid chromatography (the specific detection method is the same as in Example 3).

[0228] The results are as follows Figure 10 As shown in the figure, 2-carboxyphenylpyruvate is cleaved into o-carboxybenzaldehyde. According to the law of conservation of mass in chemical reactions, the products of cleavage of 2-carboxyphenylpyruvate are o-carboxybenzaldehyde and pyruvate, and the concentrations of pyruvate and o-carboxybenzaldehyde are equal.

[0229] Example 12: Co-catalytic cleavage of 2-carboxybenzopyruvate by aldolase and dehydrogenase

[0230] 1. Weigh 2-carboxybenzopyruvic acid and dissolve it in 50mM KHCO3 solution to prepare a 50mM solution. Prepare potassium phosphate buffer (pH 7.5, 100mM).

[0231] 2. Prepare enzyme solutions by dissolving the aldolase NsaE and dehydrogenase PhdK prepared in Example 10 in potassium phosphate buffer (pH 7.5, 100mM).

[0232] 3. Premix the reaction components to prepare a reaction solution, as shown in Table 12.

[0233] Table 12. Coupling Reaction Buffer

[0234]

[0235]

[0236] Note: The concentrations of each substance in the table are the final concentrations in the reaction solution.

[0237] Reaction conditions: 200 rpm, 30℃ for 5.5 h.

[0238] The reaction results were detected by ion chromatography-liquid chromatography.

[0239] The reaction results were detected by high performance liquid chromatography.

[0240] Spectroscopic column: capillary column;

[0241] Eluents: A: ddH2O, B: NaOH (5M);

[0242] Elution procedure: as shown in Table 13.

[0243] Table 13. Ion Chromatography Elution Procedure

[0244] 0.0 1.0 1.0 10.0 1.0 1.0 20.0 1.0 15.0 30.0 1.0 30.0 35.0 1.0 60.0 38.0 1.0 60.0 45.0 1.0 1.0

[0245] Detection: Conductivity detector and electrochemical detector.

[0246] The results are as follows Figure 12 As shown, comparison Figure 11 The detection of standard pyruvate and phthalic acid showed that 2-carboxybenzopyruvate was cleaved into phthalic acid and pyruvate.

[0247] Example 13: Carboxylase, dioxygenase, aldolase, dehydrogenase, and NAD(P)H oxidase synergistically catalyze the cleavage of 1-naphthol.

[0248] 1. Weigh 1-naphthol and dissolve it in N,N-dimethylformamide (DMF) to prepare a 375 mM solution. Prepare potassium phosphate buffer (pH 7.5, 100 mM).

[0249] 2. Prepare enzyme solutions by dissolving the carboxylase 2,3-DHBD, dioxygenase 1HNDO, aldolase NsaE, dehydrogenase PhdK, and NAD(P)H oxidase Nox prepared in Example 2 in potassium phosphate buffer (pH 7.5, 100 mM).

[0250] 3. Premix the reaction components to prepare a reaction solution, as shown in Table 13.

[0251] Table 13. Coupling reaction solution (1 mL)

[0252] 1-Naphthol 7.5mM Carboxylase (2,3-DHBD) 1U / mL Dioxygenase (1HNDO) 9U / mL Aldolase (NsaE) 0.75 mg / mL Dehydrogenase (PhdK) 18U / mL NAD(P)H oxidase (Nox) 4.5 U / mL <![CDATA[NAD + ]]> 0.1mM <![CDATA[KHCO3]]> 90mM Potassium phosphate buffer (pH 7.5, 100mM) Add 1mL

[0253] Note: The concentrations of each substance in the table are the final concentrations in the reaction solution.

[0254] Reaction conditions: 200 rpm, 30℃ for 12 h.

[0255] The reaction results were detected by ion-liquid chromatography (the specific detection method is the same as in Example 12).

[0256] The results are as follows Figure 13 As shown, comparison Figure 11It is evident that the substrate 1-naphthol is almost entirely converted into the product phthalic acid. Therefore, a one-pot reaction can be used to cleave 1-naphthol using CO2 fixation to synthesize phthalic acid. This invention is of great significance for CO2 fixation and phthalic acid synthesis.

[0257] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. <110> Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences <120> Methods for multi-enzyme coupling transformation of aromatic compounds <130> GNCLN221037 <160> 18 <170> PatentIn version 3.5 <210> 1 <211> 1051 <212> PRT <213> Artificial sequence <400> 1 Met Gly Met Thr Ile Lys Glu Met Pro Gln Pro Lys Thr Phe Gly Glu 1 5 10 15 Leu Lys Asn Leu Pro Leu Leu Asn Thr Asp Lys Pro Val Gln Ala Leu 20 25 30 Met Lys Ile Ala Asp Glu Leu Gly Glu Ile Phe Lys Phe Glu Ala Pro 35 40 45 Gly Arg Val Thr Arg Tyr Leu Ser Ser Gln Arg Leu Ile Lys Glu Ala 50 55 60 Cys Asp Glu Ser Arg Phe Asp Lys Asn Leu Ser Gln Gly Leu Lys Phe 65 70 75 80 Val Arg Asp Phe Ala Gly Asp Gly Leu Val Thr Ser Trp Thr His Glu 85 90 95 Lys Asn Trp Lys Lys Ala His Asn Ile Leu Leu Pro Ser Phe Ser Gln 100 105 110 Gln Ala Met Lys Gly Tyr His Ala Met Met Val Asp Ile Ala Val Gln 115 120 125 Leu Val Gln Lys Trp Glu Arg Leu Asn Ala Asp Glu His Ile Glu Val 130 135 140 Pro Glu Asp Met Thr Arg Leu Thr Leu Asp Thr Ile Gly Leu Cys Gly 145 150 155 160 Phe Asn Tyr Arg Phe Asn Ser Phe Tyr Arg Asp Gln Pro His Pro Phe 165 170 175 Ile Thr Ser Met Val Arg Ala Leu Asp Glu Ala Met Asn Lys Gln Gln 180 185 190 Arg Ala Asn Pro Asp Asp Pro Ala Tyr Asp Glu Asn Lys Arg Gln Phe 195 200 205 Gln Glu Asp Ile Lys Val Met Asn Asp Leu Val Asp Lys Ile Ile Ala 210 215 220 Asp Arg Lys Ala Ser Gly Glu Gln Ser Asp Asp Leu Leu Thr His Met 225 230 235 240 Leu Asn Gly Lys Asp Pro Glu Thr Gly Glu Pro Leu Asp Asp Glu Asn 245 250 255 Ile Arg Tyr Gln Ile Ile Thr Phe Leu Ile Ala Gly His Glu Thr Thr 260 265 270 Ser Gly Leu Leu Ser Phe Ala Leu Tyr Phe Leu Val Lys Asn Pro His 275 280 285 Val Leu Gln Lys Ala Ala Glu Glu Ala Ala Arg Val Leu Val Asp Pro 290 295 300 Val Pro Ser Tyr Lys Gln Val Lys Gln Leu Lys Tyr Val Gly Met Val 305 310 315 320 Leu Asn Glu Ala Leu Arg Leu Trp Pro Thr Ala Pro Ala Phe Ser Leu 325 330 335 Tyr Ala Lys Glu Asp Thr Val Leu Gly Gly Glu Tyr Pro Leu Glu Lys 340 345 350 Gly Asp Glu Leu Met Val Leu Ile Pro Gln Leu His Arg Asp Lys Thr 355 360 365 Ile Trp Gly Asp Asp Val Glu Glu Phe Arg Pro Glu Arg Phe Glu Asn 370 375 380 Pro Ser Ala Ile Pro Gln His Ala Phe Lys Pro Phe Gly Asn Gly Gln 385 390 395 400 Arg Ala Cys Ile Gly Gln Gln Phe Ala Leu His Glu Ala Thr Leu Val 405 410 415 Leu Gly Met Met Leu Lys His Phe Asp Phe Glu Asp His Thr Asn Tyr 420 425 430 Glu Leu Asp Ile Lys Glu Thr Leu Thr Leu Lys Pro Glu Gly Phe Val 435 440 445 Val Lys Ala Lys Ser Lys Lys Ile Pro Leu Gly Gly Ile Pro Ser Pro 450 455 460 Ser Thr Glu Gln Ser Ala Lys Lys Val Arg Lys Lys Ala Glu Asn Ala 465 470 475 480 His Asn Thr Pro Leu Leu Val Leu Tyr Gly Ser Asn Met Gly Thr Ala 485 490 495 Glu Gly Thr Ala Arg Asp Leu Ala Asp Ile Ala Met Ser Lys Gly Phe 500 505 510 Ala Pro Gln Val Ala Thr Leu Asp Ser His Ala Gly Asn Leu Pro Arg 515 520 525 Glu Gly Ala Val Leu Ile Val Thr Ala Ser Tyr Asn Gly His Pro Pro 530 535 540 Asp Asn Ala Lys Gln Phe Val Asp Trp Leu Asp Gln Ala Ser Ala Asp 545 550 555 560 Glu Val Lys Gly Val Arg Tyr Ser Val Phe Gly Cys Gly Asp Lys Asn 565 570 575 Trp Ala Thr Thr Tyr Gln Lys Val Pro Ala Phe Ile Asp Glu Thr Leu 580 585 590 Ala Ala Lys Gly Ala Glu Asn Ile Ala Asp Arg Gly Glu Ala Asp Ala 595 600 605 Ser Asp Asp Phe Glu Gly Thr Tyr Glu Glu Trp Arg Glu His Met Trp 610 615 620 Ser Asp Val Ala Ala Tyr Phe Asn Leu Asp Ile Glu Asn Ser Glu Asp 625 630 635 640 Asn Lys Ser Thr Leu Ser Leu Gln Phe Val Asp Ser Ala Ala Asp Met 645 650 655 Pro Leu Ala Lys Met His Gly Ala Phe Ser Thr Asn Val Val Ala Ser 660 665 670 Lys Glu Leu Gln Gln Pro Gly Ser Ala Arg Ser Thr Arg His Leu Glu 675 680 685 Ile Glu Leu Pro Lys Glu Ala Ser Tyr Gln Glu Gly Asp His Leu Gly 690 695 700 Val Ile Pro Arg Asn Tyr Glu Gly Ile Val Asn Arg Val Thr Ala Arg 705 710 715 720 Phe Gly Leu Asp Ala Ser Gln Gln Ile Arg Leu Glu Ala Glu Glu Glu 725 730 735 Lys Leu Ala His Leu Pro Leu Ala Lys Thr Val Ser Val Glu Glu Leu 740 745 750 Leu Gln Tyr Val Glu Leu Gln Asp Pro Val Thr Arg Thr Gln Leu Arg 755 760 765 Ala Met Ala Ala Lys Thr Val Cys Pro Pro His Lys Val Glu Leu Glu 770 775 780 Ala Leu Leu Glu Lys Gln Ala Tyr Lys Glu Gln Val Leu Ala Lys Arg 785 790 795 800 Leu Thr Met Leu Glu Leu Leu Glu Lys Tyr Pro Ala Cys Glu Met Lys 805 810 815 Phe Ser Glu Phe Ile Ala Leu Leu Pro Ser Ile Arg Pro Arg Tyr Tyr 820 825 830 Ser Ile Ser Ser Ser Pro Arg Val Asp Glu Lys Gln Ala Ser Ile Thr 835 840 845 Val Ser Val Val Ser Gly Glu Ala Trp Ser Gly Tyr Gly Glu Tyr Lys 850 855 860 Gly Ile Ala Ser Asn Tyr Leu Ala Glu Leu Gln Glu Gly Asp Thr Ile 865 870 875 880 Thr Cys Phe Ile Ser Thr Pro Gln Ser Glu Phe Thr Leu Pro Lys Asp 885 890 895 Pro Glu Thr Pro Leu Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro 900 905 910 Phe Arg Gly Phe Val Gln Ala Arg Lys Gln Leu Lys Glu Gln Gly Gln 915 920 925 Ser Leu Gly Glu Ala His Leu Tyr Phe Gly Cys Arg Ser Pro His Glu 930 935 940 Asp Tyr Leu Tyr Gln Glu Glu Leu Glu Asn Ala Gln Ser Glu Gly Ile 945 950 955 960 Ile Thr Leu His Thr Ala Phe Ser Arg Met Pro Asn Gln Pro Lys Thr 965 970 975 Tyr Val Gln His Val Met Glu Gln Asp Gly Lys Lys Leu Ile Glu Leu 980 985 990 Leu Asp Gln Gly Ala His Phe Tyr Ile Cys Gly Asp Gly Ser Gln Met 995 1000 1005 Ala Pro Ala Val Glu Ala Thr Leu Met Lys Ser Tyr Ala Asp Val 1010 1015 1020 His Gln Val Ser Glu Ala Asp Ala Arg Leu Trp Leu Gln Gln Leu 1025 1030 1035 Glu Glu Lys Gly Arg Tyr Ala Lys Asp Val Trp Ala Gly 1040 1045 1050 <210> 2 <211> 338 <212> PRT <213> Artificial sequence <400> 2 Met Leu Gly Lys Ile Ala Leu Glu Glu Ala Phe Ala Leu Pro Arg Phe 1 5 10 15 Glu Glu Lys Thr Arg Trp Trp Ala Ser Leu Phe Ser Thr Asp Ala Glu 20 25 30 Thr His Val Lys Glu Ile Thr Asp Ile Asn Lys Ile Arg Ile Glu His 35 40 45 Ala Asp Lys His Gly Val Gly Tyr Gln Ile Leu Ser Tyr Thr Ala Pro 50 55 60 Gly Val Gln Asp Ile Trp Asp Pro Val Glu Ala Gln Ala Leu Ala Val 65 70 75 80 Glu Ile Asn Asp Tyr Ile Ala Glu Gln Val Arg Val Asn Pro Asp Arg 85 90 95 Phe Gly Ala Phe Ala Thr Leu Ser Met His Asn Pro Lys Glu Ala Ala 100 105 110 Asp Glu Leu Arg Arg Cys Val Glu Lys Tyr Gly Phe Lys Gly Ala Leu 115 120 125 Val Asn Asp Thr Gln Arg Ala Gly Pro Asp Gly Asp Asp Met Ile Phe 130 135 140 Tyr Asp Asn Ala Asp Trp Asp Ile Phe Trp Gln Thr Cys Thr Glu Leu 145 150 155 160 Asp Val Pro Phe Tyr Met His Pro Arg Asn Pro Thr Gly Thr Ile Tyr 165 170 175 Glu Lys Leu Trp Ala Asp Arg Lys Trp Leu Val Gly Pro Pro Leu Ser 180 185 190 Phe Ala His Gly Val Ser Leu His Val Leu Gly Met Val Thr Asn Gly 195 200 205 Val Phe Asp Arg His Pro Lys Leu Gln Ile Ile Met Gly His Leu Gly 210 215 220 Glu His Val Pro Phe Asp Met Trp Arg Ile Asn His Trp Phe Glu Asp 225 230 235 240 Arg Lys Lys Leu Leu Gly Leu Ala Glu Thr Cys Lys Lys Thr Ile Arg 245 250 255 Asp Tyr Phe Ala Glu Asn Ile Trp Ile Thr Thr Ser Gly His Phe Ser 260 265 270 Thr Thr Thr Leu Asn Phe Cys Met Ala Glu Val Gly Ser Asp Arg Ile 275 280 285 Leu Phe Ser Ile Asp Tyr Pro Phe Glu Thr Phe Ser Asp Ala Cys Glu 290 295 300 Trp Phe Asp Asn Ala Glu Leu Asn Gly Thr Asp Arg Leu Lys Ile Gly 305 310 315 320 Arg Glu Asn Ala Lys Lys Leu Phe Lys Leu Asp Ser Tyr Lys Asp Ser 325 330 335 Ser Ala <210> 3 <211> 327 <212> PRT <213> Artificial sequence <400> 3 Met Gln Gly Lys Val Ala Leu Glu Glu His Phe Ala Ile Pro Glu Thr 1 5 10 15 Leu Gln Asp Ser Ala Gly Phe Val Pro Gly Asp Tyr Trp Lys Glu Leu 20 25 30 Gln His Arg Leu Leu Asp Ile Gln Asp Thr Arg Leu Lys Leu Met Asp 35 40 45 Ala His Gly Ile Glu Thr Met Ile Leu Ser Leu Asn Ala Pro Ala Val 50 55 60 Gln Ala Ile Pro Asp Arg Arg Lys Ala Ile Glu Ile Ala Arg Arg Ala 65 70 75 80 Asn Asp Val Leu Ala Glu Glu Cys Ala Lys Arg Pro Asp Arg Phe Leu 85 90 95 Ala Phe Ala Ala Leu Pro Leu Gln Asp Pro Asp Ala Ala Thr Glu Glu 100 105 110 Leu Gln Arg Cys Val Asn Asp Leu Gly Phe Val Gly Ala Leu Val Asn 115 120 125 Gly Phe Ser Gln Glu Gly Asp Gly Gln Thr Pro Leu Tyr Tyr Asp Leu 130 135 140 Pro Gln Tyr Arg Pro Phe Trp Gly Glu Val Glu Lys Leu Asp Val Pro 145 150 155 160 Phe Tyr Leu His Pro Arg Asn Pro Leu Pro Gln Asp Ser Arg Ile Tyr 165 170 175 Asp Gly His Pro Trp Leu Leu Gly Pro Thr Trp Ala Phe Ala Gln Glu 180 185 190 Thr Ala Val His Ala Leu Arg Leu Met Ala Ser Gly Leu Phe Asp Glu 195 200 205 His Pro Arg Leu Asn Ile Ile Leu Gly His Met Gly Glu Gly Leu Pro 210 215 220 Tyr Met Met Trp Arg Ile Asp His Arg Asn Ala Trp Val Lys Leu Pro 225 230 235 240 Pro Arg Tyr Pro Ala Lys Arg Arg Phe Met Asp Tyr Phe Asn Glu Asn 245 250 255 Phe His Ile Thr Thr Ser Gly Asn Phe Arg Thr Gln Thr Leu Ile Asp 260 265 270 Ala Ile Leu Glu Ile Gly Ala Asp Arg Ile Leu Phe Ser Thr Asp Trp 275 280 285 Pro Phe Glu Asn Ile Asp His Ala Ser Asp Trp Phe Asn Ala Thr Ser 290 295 300 Ile Ala Glu Ala Asp Arg Val Lys Ile Gly Arg Thr Asn Ala Arg Arg 305 310 315 320 Leu Phe Lys Leu Asp Gly Ala 325 <210> 4 <211> 350 <212> PRT <213> Artificial sequence <400> 4 Met Arg Gly Lys Val Ser Leu Glu Glu Ala Phe Glu Leu Pro Lys Phe 1 5 10 15 Ala Ala Gln Thr Lys Glu Lys Ala Glu Leu Tyr Ile Ala Pro Asn Asn 20 25 30 Arg Asp Arg Tyr Phe Glu Glu Ile Leu Asn Pro Cys Gly Asn Arg Leu 35 40 45 Glu Leu Ser Asn Lys His Gly Ile Gly Tyr Thr Ile Tyr Ser Ile Tyr 50 55 60 Ser Pro Gly Pro Gln Gly Trp Thr Glu Arg Ala Glu Cys Glu Glu Tyr 65 70 75 80 Ala Arg Glu Cys Asn Asp Tyr Ile Ser Gly Glu Ile Ala Asn His Lys 85 90 95 Asp Arg Met Gly Ala Phe Ala Ala Leu Ser Met His Asp Pro Lys Gln 100 105 110 Ala Ser Glu Glu Leu Thr Arg Cys Val Lys Glu Leu Gly Phe Leu Gly 115 120 125 Ala Leu Val Asn Asp Val Gln His Ala Gly Pro Glu Gly Glu Thr His 130 135 140 Ile Phe Tyr Asp Gln Pro Glu Trp Asp Ile Phe Trp Gln Thr Cys Val 145 150 155 160 Asp Leu Asp Val Pro Phe Tyr Leu His Pro Glu Pro Pro Phe Gly Ser 165 170 175 Tyr Leu Arg Asn Gln Tyr Glu Gly Arg Lys Tyr Leu Ile Gly Pro Pro 180 185 190 Val Ser Phe Ala Asn Gly Val Ser Leu His Val Leu Gly Met Ile Val 195 200 205 Asn Gly Val Phe Asp Arg Phe Pro Lys Leu Lys Val Ile Leu Gly His 210 215 220 Leu Gly Glu His Ile Pro Gly Asp Phe Trp Arg Ile Glu His Trp Phe 225 230 235 240 Glu His Cys Ser Arg Pro Leu Ala Lys Ser Arg Gly Asp Val Phe Ala 245 250 255 Glu Lys Pro Leu Leu His Tyr Phe Arg Asn Asn Ile Trp Leu Thr Thr 260 265 270 Ser Gly Asn Phe Ser Thr Glu Thr Leu Lys Phe Cys Val Glu His Val 275 280 285 Gly Ala Glu Arg Ile Leu Phe Ser Val Asp Ser Pro Tyr Glu His Ile 290 295 300 Asp Val Gly Cys Gly Trp Tyr Asp Asp Asn Ala Lys Ala Ile Met Glu 305 310 315 320 Ala Val Gly Gly Glu Lys Ala Tyr Lys Asp Ile Gly Arg Asp Asn Ala 325 330 335 Lys Lys Leu Phe Lys Leu Gly Lys Phe Tyr Asp Ser Glu Ala 340 345 350 <210> 5 <211> 361 <212> PRT <213> Artificial sequence <400> 5 Met Ser Thr Ala Glu Ser Ser Glu Leu Arg Glu Phe Asp Val Glu Leu 1 5 10 15 Glu Ala Ala Asn Leu Arg Gly Gln Trp Ile Tyr Asp Asp Met Leu Glu 20 25 30 Ser Val Val Gly Gly Pro Lys Pro Ala Gly Val Pro Phe Leu Trp Arg 35 40 45 Trp His Asp Val Tyr Ala Lys Leu Leu Lys Ser Cys Asp Val Met Pro 50 55 60 Glu Ser Leu Thr Ala Arg Arg Asn Leu Ser Phe Ile Asn Pro Asp Ala 65 70 75 80 Arg Gly Thr Thr His Thr Ile Asn Met Gly Met Gln Met Leu Lys Pro 85 90 95 Gly Glu Ile Ala Tyr Ala His Arg His Thr Met Ala Ala Leu Arg Phe 100 105 110 Ala Ile Gln Gly Gly Pro Gly Leu Val Thr Val Val Asp Gly Glu Pro 115 120 125 Cys Gln Met Asp Thr Tyr Asp Leu Val Leu Thr Pro Arg Trp Thr Trp 130 135 140 His Asp His Glu Asn Ala Thr Ser Glu Asn Val Val Trp Leu Asp Val 145 150 155 160 Leu Asp Ile Gly Leu Val Leu Gly Leu Asn Val Pro Phe Tyr Glu Pro 165 170 175 Tyr Gly Glu Met Arg Gln Pro Gln Arg Glu Asp Pro Gly Glu His Leu 180 185 190 Ala Asp Arg Gly Gly Met Leu Arg Pro Ala Trp Glu Gln Val Lys Ala 195 200 205 Ala Asn Phe Pro Tyr Arg Tyr Pro Trp Arg Asp Val Glu Arg Gln Leu 210 215 220 Gln Arg Met Ala Gly Leu Ala Gly Ser Pro Tyr Asp Gly Val Val Leu 225 230 235 240 Arg Tyr Ala Asn Pro Val Thr Gly Gly Ser Thr Met Pro Thr Leu Asp 245 250 255 Cys Trp Val Gln Leu Leu Arg Pro Gly Gln Gln Thr Glu Ala His Arg 260 265 270 His Thr Ser Ser Ala Val Tyr Phe Val Val Arg Gly Glu Gly Thr Thr 275 280 285 Val Val Asp Gly Val Glu Leu Asp Trp Gly Pro His Asp Ser Phe Val 290 295 300 Val Pro Asn Trp Ser Thr His His Phe Val Asn Arg Ser Ala Glu Asn 305 310 315 320 Ala Leu Leu Phe Ser Val Asn Asp Ile Pro Thr Leu Lys Ala Leu Asp 325 330 335 Leu Tyr Tyr Glu Glu Pro Glu Leu Ser Leu Gly Thr Gln Pro Phe Pro 340 345 350 Pro Val Pro Ala Asn Leu Arg Ala Arg 355 360 <210> 6 <211> 334 <212> PRT <213> Artificial sequence <400> 6 Met Ser Asn Lys Ile Met Lys Thr Ser Arg Leu Thr Ala Glu Asp Ile 1 5 10 15 Asn Gly Ala Trp Thr Ile Met Pro Thr Pro Ser Thr Pro Asp Ala Ser 20 25 30 Asp Trp Arg Ser Thr Ala Thr Val Asp Leu Glu Glu Thr Ala Arg Ile 35 40 45 Val Glu Glu Leu Ile Ala Ala Gly Val Asn Gly Ile Leu Ser Met Gly 50 55 60 Thr Phe Gly Glu Cys Ala Thr Leu Thr Trp Asp Glu Lys Arg Asp Tyr 65 70 75 80 Val Ser Thr Ile Val Glu Thr Ile Arg Gly Arg Val Pro Tyr Phe Cys 85 90 95 Gly Thr Thr Ala Leu Asn Thr Arg Glu Val Ile Arg Gln Thr Arg Glu 100 105 110 Leu Ile Asp Ile Gly Ala Asn Gly Thr Met Leu Gly Val Pro Met Trp 115 120 125 Val Lys Met Asp Leu Pro Thr Ala Val Gln Phe Tyr Arg Asp Val Ala 130 135 140 Asp Ala Val Pro Glu Ala Ala Ile Ala Ile Tyr Ala Asn Pro Glu Ala 145 150 155 160 Phe Lys Phe Asp Phe Pro Arg Pro Phe Trp Ala Glu Met Ser Lys Ile 165 170 175 Pro Gln Val Val Thr Ala Lys Tyr Leu Gly Ile Gly Met Leu Asp Leu 180 185 190 Asp Leu Arg Leu Ala Pro Asn Ile Arg Phe Leu Pro His Glu Asp Asp 195 200 205 Tyr Tyr Ala Ala Ala Arg Ile Asn Pro Glu Arg Ile Thr Ala Phe Trp 210 215 220 Ser Ser Gly Ala Met Cys Gly Pro Ala Thr Ala Ile Met Leu Arg Asp 225 230 235 240 Glu Val Val Arg Ala Lys Ser Thr Gly Asp Trp Ala Lys Ala Lys Ala 245 250 255 Ile Ser Asp Asp Met Arg Ala Ala Asp Ser Thr Leu Phe Pro Arg Gly 260 265 270 Asp Phe Ser Glu Phe Ser Lys Tyr Asn Ile Gly Leu Glu Lys Ala Arg 275 280 285 Met Asp Ala Ala Gly Trp Leu Lys Ala Gly Pro Cys Arg Pro Pro Tyr 290 295 300 Asn Leu Val Pro Glu Asp Tyr Leu Ala Gly Ala Gln Lys Ser Gly Lys 305 310 315 320 Ala Trp Ala Ala Leu His Ala Lys Tyr Ser Asn Glu Leu Lys 325 330 <210> 7 <211> 485 <212> PRT <213> Artificial sequence <400> 7 Met Thr Thr Pro Arg Lys Phe Asp Glu Tyr Arg Trp Asn Val Leu Val 1 5 10 15 Asp Gly Val Pro Leu Asn Val Glu Ser Arg Tyr Pro Ile Ser Asp Pro 20 25 30 Ser Thr Gly Arg Tyr Leu Thr Gln Val Pro Asp Cys Ala Glu Ala Asp 35 40 45 Val Asp Arg Ala Val Gln Ala Ser Arg Gln Ala Gln Ala Glu Trp Gly 50 55 60 Ala Leu Pro Pro Arg Ala Arg Ala Ala Lys Leu Arg Glu Leu Ile Thr 65 70 75 80 Leu Leu Arg Glu His Arg Glu Glu Phe Ala Met Leu Asp Ala Ile Asp 85 90 95 Gly Gly Phe Pro Ile Ser Met Met Arg Asn Asp Val Asp Ala Ala Leu 100 105 110 Glu Leu Met Asp Ile Phe Ala Asp Met Ala Leu Asp Leu Gly Gly Lys 115 120 125 Thr Ile Pro Val Ser Thr Asn Leu His Phe Thr Thr His Glu Pro Phe 130 135 140 Gly Val Val Ala Arg Ile Gly Ala Phe Asn His Pro Phe Phe Phe Ala 145 150 155 160 Ala Ser Lys Val Ala Ala Pro Leu Met Ala Gly Asn Ser Val Ile Leu 165 170 175 Lys Ala Pro Asp Gln Thr Pro Leu Ser Ser Leu Arg Leu Ala Glu Val 180 185 190 Ala Ala Glu Val Leu Pro Gln Asn Leu Leu Ile Thr Ile Ser Gly Arg 195 200 205 Gly Arg Val Ala Gly Arg Ala Ile Val Arg His Pro Gln Ile Lys Arg 210 215 220 Ile Gly Phe Ile Gly Ser Thr Asp Thr Gly Arg Ser Ile Gln Arg Asp 225 230 235 240 Ala Ala Glu Val Ala Val Lys His Ile Ser Leu Glu Leu Gly Gly Lys 245 250 255 Asn Ala Gln Ile Val Phe Ala Asp Ala Asp Leu Glu Gln Ala Ala Leu 260 265 270 Gly Ala Val Asn Gly Met Asn Phe Thr Trp Thr Ala Gly Gln Ser Cys 275 280 285 Gly Ser Thr Ser Arg Leu Leu Val His Glu Ser Val Ala Asp Gln Val 290 295 300 Ile Ala Arg Val Val Glu Leu Val Ser Ala Ile Ala Val Gly Pro Pro 305 310 315 320 Leu Asp Glu Asn Ala Gln Met Gly Pro Leu Val Ser Gln Ala Gln Tyr 325 330 335 Asp Lys Ser Val His Ala Ile Gly Glu Gly Ile Arg Glu Gly Ala Lys 340 345 350 Val Val Ala Gly Gly Gly Arg Pro Glu Gly Val Gly Glu Gly Gly Trp 355 360 365 Tyr Leu Ala Pro Thr Val Leu Ala Asp Val Arg Pro Gly Ser Phe Ile 370 375 380 Glu Gln Asn Glu Ile Phe Gly Pro Val Leu Ser Val Ile Ile Phe Ala 385 390 395 400 Thr Asp Asp Glu Ala Val Ala Ile Ala Asn Gly Val Glu Tyr Gly Leu 405 410 415 Thr Ala Ser Val Trp Thr Ser Asp Ile Thr Arg Ala His Leu Ile Ala 420 425 430 Arg Arg Val Glu Ala Gly Tyr Val Leu Val Asn Gly Gly Ser Arg His 435 440 445 Tyr Trp Gly Leu Pro Phe Gly Gly Val Lys Ser Ser Gly Val Gly Ser 450 455 460 Glu Glu Ser Met Glu Glu Leu Ile Ser Tyr Thr Glu Thr Lys Thr Thr 465 470 475 480 Thr Val Val Leu Gly 485 <210> 8 <211> 450 <212> PRT <213> Artificial sequence <400> 8 Met Lys Val Ile Val Ile Gly Cys Thr His Ala Gly Thr Ala Ala Val 1 5 10 15 Asn Gln Ile Leu Ala Ser Asn Pro Glu Thr Asp Val Thr Ile Tyr Glu 20 25 30 Arg Asn Asp Asn Val Ser Phe Leu Ser Cys Gly Ile Ala Leu Tyr Leu 35 40 45 Gly Gly Glu Val Ala Asp Pro Gln Gly Leu Phe Tyr Ser Ser Pro Glu 50 55 60 Gln Leu Ala Lys Leu Gly Ala Asn Val His Met Gln His Asp Val Thr 65 70 75 80 Asp Val Asp Thr Glu Asn His Glu Ile Thr Val Thr Asp Leu Lys Thr 85 90 95 Gly Glu Ser Lys Lys Asp Tyr Tyr Asp Lys Leu Val Val Thr Thr Gly 100 105 110 Ser Trp Pro Val Ile Pro Pro Ile Asp Gly Ile Asp Ser Pro Asn Val 115 120 125 Tyr Leu Cys Lys Asn Trp Thr His Ala Gln Ser Leu Trp Glu Ala Ala 130 135 140 Lys Pro Ala Lys Arg Val Ile Val Ile Gly Gly Gly Tyr Ile Gly Thr 145 150 155 160 Glu Leu Val Glu Ala Tyr Gln Lys Gln Gly Lys Glu Val Thr Leu Ile 165 170 175 Asp Gly Leu Pro Arg Ile Leu Asn Lys Tyr Leu Asp Lys Gly Phe Thr 180 185 190 Asp Arg Val Glu Lys Asp Phe Val Asp His Gly Ile Lys Met Ala Leu 195 200 205 Asn Gln Met Val Lys Gly Phe Ser Asp Asp Gly Lys Glu Val Thr Val 210 215 220 Lys Thr Asp Lys Gly Ser Tyr Thr Ala Asp Met Ala Ile Leu Cys Val 225 230 235 240 Gly Phe Arg Pro Asn Thr Ser Leu Leu Lys Gly Lys Val Asp Met Asn 245 250 255 Pro Asn Gly Ser Ile Lys Thr Asn Asp Tyr Met Gln Thr Ser Asp Pro 260 265 270 Asp Ile Tyr Gly Ala Gly Asp Ser Val Ala Val His Tyr Asn Pro Thr 275 280 285 Lys Lys Asp Ala Tyr Ile Pro Leu Ala Thr Asn Ala Val Arg Gln Gly 290 295 300 Thr Leu Val Gly Leu Asn Ile Phe Lys Pro Thr Arg Lys Tyr Met Gly 305 310 315 320 Thr Gln Ser Thr Ser Gly Leu Met Leu Phe Gly Lys Thr Ile Val Ser 325 330 335 Ser Gly Met Thr Leu Glu His Ala Gln Ala Glu Lys Val Pro Ala Glu 340 345 350 Ala Val Thr Phe Glu Asp Asn Tyr Arg Pro Glu Phe Met Pro Thr Thr 355 360 365 Lys Pro Val Leu Met Gln Leu Val Tyr Asn Pro Glu Thr Arg Glu Ile 370 375 380 Leu Gly Ala Gln Phe Met Ser Glu His Asp Val Ser Gln Ser Ala Asn 385 390 395 400 Val Ile Ser Val Met Ile Gln Asn His Asn Thr Ile Asp Asp Leu Gly 405 410 415 Phe Val Asp Met Phe Phe Gln Pro Ile Tyr Asp Arg Pro Phe Asn Tyr 420 425 430 Leu Asn Leu Leu Gly Gln Ala Ala Ile Ala His Ala Ala Glu Lys Val 435 440 445 Thr Glu 450 <210> 9 <211> 3156 <212> DNA <213> Artificial sequence <400> 9 atgggcatga caattaaaga aatgcctcag ccaaaaacgt ttggagagct taaaaatta 60 ccgttattaa acacagataa accggttcaa gctttgatga aaattgcgga tgaattagga 120 gaaatcttta aattcgaggc gcctggtcgt gtaacgcgct acttatcaag tcagcgtcta 180 attaaagaag catgcgatga atcacgcttt gataaaaact taagtcaagg tcttaaattt 240 gtacgtgatt ttgcaggaga cgggttagtg acaagctgga cgcatgaaaa aaattggaaa 300 aaagcgcata atatcttact tccaagcttc agtcagcagg caatgaaagg ctatcatgcg 360 atgatggtcg atatcgccgt gcagcttgtt caaaagtggg agcgtctaaa tgcagatgag 420 catattgaag taccggaaga catgacacgt ttaacgcttg atacaattgg tctttgcggc 480 tttaactatc gctttaacag cttttaccga gatcagcctc atccatttat tacaagtatg 540 gtccgtgcac tggatgaagc aatgaacaag cagcagcgag caaatccaga cgacccagct 600 tatgatgaaa acaagcgcca gtttcaagaa gatatcaagg tgatgaacga cctagtagat 660 aaaattatg cagatcgcaa agcaagcggt gaacaaagcg atgatttat aacgdatag 720 ctaaacggaa aagatccaga aacgggtgag ccgcttgatg acgagaacat tcgctatcaa 780 attattacat tcttaattgc gggacacgaa acaacaagtg gtcttttatc atttgcgctg 840 tatttcttag tgaaaaatcc acatgtatta caaaaagcag cagaagaagc agcacgagtt 900 ctagtagatc ctgttccaag ctacaaacaa gtcaaacagc ttaaatatgt cggcatggtc 960 ttaaacgaag cgctgcgctt atggccaact gctcctgcgt ttccctata tgcaaaagaa 1020 gatacggtgc ttggaggaga ataccttta gaaaaaggcg acgaactaat ggttctgatt 1080 cctcagcttc accgtgataa aacaatttgg ggagacgatg tggaagagtt ccgtccagag 1140 cgttttgaaa atccaagtgc gattccgcag catgcgttta aaccgtttgg aaacggtcag 1200 cgtgcgtgta tcggtcagca gttcgctctt catgaagcaa cgctggtact tggtatgatg 1260 ctaaaacact ttgactttga agatcataca aactacgagc tggatattaa agaaacttta 1320 acgttaaaac ctgaaggctt tgtggtaaaa gcaaaatcga aaaaaattc gcttggcggt 1380 attccttcac ctagcactga acagtctgct aaaaaagtac gcaaaaagc agaaaacgct 1440 cataatacgc cgctgcttgt gctatacggt tcaaatatgg gaacagctga aggaacggcg 1500 cgtgatttag cagatattgc aatgagcaaa ggatttgcac cgcaggtcgc aacgcttgat 1560 tcacacgccg gaaatcttcc gcgcgaagga gctgtattaa ttgtaacggc gtcttataac 1620 ggtcatccgc ctgataacgc aaagcaattt gtcgactggt tagaccaagc gtctgctgat 1680 gaagtaaaag gcgttcgcta ctccgtattt ggatgcggcg ataaaaactg ggctactacg 1740 tatcaaaaag tgcctgcttt tatcgatgaa acgcttgccg ctaaaggggc agaaaacatc 1800 gctgaccgcg gtgaagcaga tgcaagcgac gactttgaag gcacatatga agaatggcgt 1860 gaacatatgt ggagtgacgt agcagcctac tttaacctcg acattgaaaa cagtgaagat 1920 aataaatcta ctctttcact tcaatttgtc gacagcgccg cggatatgcc gcttgcgaaa 1980 atgcacggtg cgttttcaac gaacgtcgta gcaagcaaag aacttcaaca gccaggcagt 2040 gcacgaagca cgcgacatct tgaaattgaa cttccaaaag aagcttctta tcaagaagga 2100 gatcatttag gtgttattcc tcgcaactat gaaggaatag taaaccgtgt aacagcaagg 2160 ttcggcctag atgcatcaca gcaaatccgt ctggaagcag aaagaaaaa attagctcat 2220 ttgccactcg ctaaaacagt atccgtagaa gagcttctgc aatacgtgga gcttcaagat 2280 cctgttacgc gcacgcagct tcgcgcaatg gctgctaaaa cggtctgccc gccgcataaa 2340 gtagagcttg aagccttgct tgaaaagcaa gcctacaaag aacaagtgct ggcaaaacgt 2400 ttaaaatgc ttgaactgct tgaaaaatac ccggcgtgtg aaatgaaatt cagcgaattt 2460 atcgcccttc tgccaagcat acgcccgcgc tattactcga tttcttcatc acctcgtgtc 2520 gatgaaaaac aagcaagcat cacggtcagc gttgtctcag gagaagcgtg gagcggatat 2580 ggagaatata aaagaattgc gtcgaactat cttgccgagc tgcaagaagg agatacgatt 2640 acgtgcttta tttccacacc gcagtcagaa tttacgctgc caaaagaccc tgaaacgccg 2700 cttatcatgg tcggaccggg aacaggcgtc gcgccgttta gaggctttgt gcaggcgcgc 2760 aaaagctaa aagaaagg acagtcactt ggagaagcac atttatactt cggctgccgt 2820 tcacctcatg aagactatct gtatcaagaa gagcttgaaa acgcccaaag cgaaggcatc 2880 attacgcttc ataccgcttt ttctcgcatg ccaaatcagc cgaaaacata cgttcagcac 2940 gtaatggaac aagacggcaa gaaattgatt gaacttcttg atcaaggagc gcacttctat 3000 atttgcggag acggaagcca aatggcacct gccgttgaag caacgcttat gaaaagctat 3060 gctgacgtc accaagtgag tgaagcagac gctcgcttat ggctgcagca gctagaagaa 3120 aaaggccgat acgcaaaaga cgtgtgggct gggtaa 3156 <210> 10 <211> 1017 <212> DNA <213> Artificial sequence <400> 10 atgctgggta aaatcgctct ggaagaagct ttcgctctgc cgcgtttcga agaaaaacc 60 cgtggtggg cttctctgtt ctctaccgac gctgaaaccc acgttaaaga aatcaccgac 120 atcaacaaaa tccgtatcga acacgctgac aaacacggtg ttggttacca gatcctgtct 180 tacaccgctc cgggtgttca ggacatctgg gacccggttg aagctcaggc tctggctgtt 240 gaaatcaacg actacatcgc tgaacaggtt cgtgttaacc cggaccgttt cggtgctttc 300 gctaccctgt ctatgcacaa cccgaaagaa gctgctgacg aactgcgtcg ttgcgttgaa 360 aaatacggtt tcaaaggtgc tctggttaac gacacccagc gtgctggtcc ggacggtgac 420 gacatgatct tctacgacaa cgctgactgg gacatcttct ggcagacctg caccgaactg 480 gacgttccgt tctacatgca cccgcgtaac ccgaccggta ccatctacga aaaactgtgg 540 gctgaccgta aatggctggt tggtccgccg ctgtctttcg ctcacggtgt ttctctgcac 600 gttctgggta tggttaccaa cggtgttttc gaccgtcacc cgaaactgca gatcatcatg 660 ggtcacctgg gtgaacacgt tccgttcgac atgtggcgta tcaaccactg gttcgaagac 720 cgtaaaaaac tgctgggtct ggctgaaacc tgcaaaaaaa ccatccgtga ctacttcgct 780 gaaaacatct ggatcaccac ctctggtcac ttctctacca ccaccctgaa cttctgcatg 840 gctgaagttg gttctgaccg tatcctgttc tctatcgact acccgttcga aaccttctct 900 gacgcttgcg aatggttcga caacgctgaa ctgaacggta ccgaccgtct gaaaatcggt 960 cgtgaaaacg ctaaaaaact gttcaaactg gactcttaca aagactcttc tgcttaa 1017 <210> 11 <211> 984 <212> DNA <213> Artificial sequence <400> 11 atgcagggta aagttgctct ggaagaacac ttcgctatcc cggaaaccct gcaggactct 60 gctggtttcg ttccgggtga ctactggaaa gaactgcagc accgtctgct ggacatccag 120 gacacccgtc tgaaactgat ggacgctcac ggtatcgaaa ccatgatcct gtctctgaac 180 gctccggctg ttcaggctat cccggaccgt cgtaaagcta tcgaaatcgc tcgtcgtgct 240 aacgacgttc tggctgaaga atgcgctaaa cgtccggacc gtttcctggc tttcgctgct 300 ctgccgctgc aggacccgga cgctgctacc gaagaactgc agcgttgcgt taacgacctg 360 ggtttcgttg gtgctctggt taacggtttc tctcaggaag gtgacggtca gaccccgctg 420 tactacgacc tgccgcagta ccgtccgttc tggggtgaag ttgaaaaact ggacgttccg 480 ttctacctgc acccgcgtaa cccgctgccg caggactctc gtatctacga cggtcacccg 540 tggctgctgg gtccgacctg ggctttcgct caggaaaccg ctgttcacgc tctgcgtctg 600 atggcttctg gtctgttcga cgaacacccg cgtctgaaca tcatcctggg tcacatgggt 660 gaaggtctgc cgtacatgat gtggcgtatc gaccaccgta acgcttgggt taaactgccg 720 ccgcgttacc cggctaaacg tcgtttcatg gactacttca acgaaaactt ccacatcacc 780 acctctggta acttccgtac ccagaccctg atcgacgcta tcctggaaat cggtgctgac 840 cgtatcctgt tctctaccga ctggccgttc gaaaacatcg accacgcttc tgactggttc 900 aacgctacct ctatcgctga agctgaccgt gttaaaatcg gtcgtaccaa cgctcgtcgt 960 ctgttcaaac tggacggtgc ttaa 984 <210> 12 <211> 1053 <212> DNA <213> Artificial sequence <400> 12 atgcgtggta aagtttctct ggaagaagct ttcgaactgc cgaaattcgc tgctcagacc 60 aaagaaaaag ctgaactgta catcgctccg aacaaccgtg accgttactt cgaagaaatc 120 ctgaacccgt gcggtaaccg tctggaactg tctaacaaac acggtatcgg ttacaccatc 180 tactctatct actctccggg tccgcagggt tggaccgaac gtgctgaatg cgaagaatac 240 gctcgtgaat gcaacgacta catctctggt gaaatcgcta accacaaaga ccgtatgggt 300 gctttcgctg ctctgtctat gcacgacccg aaacaggctt ctgaagaact gacccgttgc 360 gttaaagaac tgggtttcct gggtgctctg gttaacgacg ttcagcacgc tggtccggaa 420 ggtgaaaccc acatcttcta cgaccagccg gaatgggaca tcttctggca gacctgcgtt 480 gacctggacg ttccgttcta cctgcacccg gaaccgccgt tcggttctta cctgcgtaac 540 cagtacgaag gtcgtaaata cctgatcggt ccgccggttt ctttcgctaa cggtgtttct 600 ctgcacgttc tgggtatgat cgttaacggt gttttcgacc gtttcccgaa actgaaagtt 660 atcctgggtc acctgggtga acacatcccg ggtgacttct ggcgtatcga acactggttc 720 gaacactgct ctcgtccgct ggctaaatct cgtggtgacg ttttcgctga aaaaccgctg 780 ctgcactact tccgtaacaa catctggctg accacctctg gtaacttctc taccgaaacc 840 ctgaaattct gcgttgaaca cgttggtgct gaacgtatcc tgttctctgt tgactctccg 900 tacgaacaca tcgacgttgg ttgcggttgg tacgacgaca acgctaaagc tatcatggaa 960 gctgttggtg gtgaaaaagc ttacaaagac atcggtcgtg acaacgctaa aaaactgttc 1020 aaactgggta aattctacga ctctgaagct taa 1053 <210> 13 <211> 1086 <212> DNA <213> Artificial sequence <400> 13 atgtctaccg ctgaatcttc tgaactgcgt gaattcgacg ttgaactgga agctgctaac 60 ctgcgtggtc agtggatcta cgacgacatg ctggaatctg ttgttggtgg tccgaaaccg 120 gctggtgttc cgttcctgtg gcgttggcac gacgtttacg ctaaactgct gaaatcttgc 180 gacgttatgc cggaatctct gaccgctcgt cgtaacctgt ctttcatcaa cccggacgct 240 cgtggtacca cccacaccat caacatgggt atgcagatgc tgaaaccggg tgaaatcgct 300 tacgctcacc gtcacaccat ggctgctctg cgtttcgcta tccagggtgg tccgggtctg 360 gttaccgttg ttgacggtga accgtgccag atggacacct acgacctggt tctgaccccg 420 cgttggacct ggcacgacca cgaaaacgct acctctgaaa acgttgtttg gctggacgtt 480 ctggacatcg gtctggttct gggtctgaac gttccgttct acgaaccgta cggtgaaatg 540 cgtcagccgc agcgtgaaga cccgggtgaa cacctggctg accgtggtgg tatgctgcgt 600 ccggcttggg aacaggttaa agctgctaac ttcccgtacc gttacccgtg gcgtgacgtt 660 gaacgtcagc tgcagcgtat ggctggtctg gctggttctc cgtacgacgg tgttgttctg 720 cgttacgcta acccggttac cggtggttct accatgccga ccctggactg ctgggttcag 780 ctgctgcgtc cgggtcagca gaccgaagct caccgtcaca cctcttctgc tgtttacttc 840 gttgttcgtg gtgaaggtac caccgttgtt gacggtgttg aactggactg gggtccgcac 900 gactctttcg ttgttccgaa ctggtctacc caccacttcg ttaaccgttc tgctgaaaac 960 gctctgctgt tctctgttaa cgacatcccg accctgaaag ctctggacct gtactacgaa 1020 gaaccggaac tgtctctggg tacccagccg ttcccgccgg ttccggctaa cctgcgtgct 1080 cgttaa 1086 <210> 14 <211> 1005 <212> DNA <213> Artificial sequence <400> 14 atgtctaaca aaatcatgaa aacctctcgt ctgaccgctg aagacatcaa cggtgcttgg 60 accatcatgc cgaccccgtc taccccggac gcttctgact ggcgttctac cgctaccgtt 120 gacctggaag aaaccgctcg tatcgttgaa gaactgatcg ctgctggtgt taacggtatc 180 ctgtctatgg gtaccttcgg tgaatgcgct accctgacct gggacgaaaa acgtgactac 240 gtttctacca tcgttgaaac catccgtggt cgtgttccgt acttctgcgg taccaccgct 300 ctgaacaccc gtgaagttat ccgtcagacc cgtgaactga tcgacatcgg tgctaacggt 360 accatgctgg gtgttccgat gtgggttaaa atggacctgc cgaccgctgt tcagttctac 420 cgtgacgttg ctgacgctgt tccggaagct gctatcgcta tctacgctaa cccggaagct 480 ttcaaattcg acttcccgcg tccgttctgg gctgaaatgt ctaaaatccc gcaggttgtt 540 accgctaaat acctgggtat cggtatgctg gacctggacc tgcgtctggc tccgaacatc 600 cgtttcctgc cgcacgaaga cgactactac gctgctgctc gtatcaaccc ggaacgtatc 660 accgctttct ggtcttctgg tgctatgtgc ggtccggcta ccgctatcat gctgcgtgac 720 gaagttgttc gtgctaaatc taccggtgac tgggctaaag ctaaagctat ctctgacgac 780 atgcgtgctg ctgactctac cctgttcccg cgtggtgact tctctgaatt ctctaaatac 840 aacatcggtc tggaaaaagc tcgtatggac gctgctggtt ggctgaaagc tggtccgtgc 900 cgtccgccgt acaacctggt tccggaagac tacctggctg gtgctcagaa atctggtaaa 960 gcttgggctg ctctgcacgc taaatactct aacgaactga aataa 1005 <210> 15 <211> 1458 <212> DNA <213> Artificial sequence <400> 15 atgaccaccc cgcgtaaatt cgacgaatac cgttggaacg ttctggttga cggtgttccg 60 ctgaacgttg aatctcgtta cccgatctct gacccgtcta ccggtcgtta cctgacccag 120 gttccggact gcgctgaagc tgacgttgac cgtgctgttc aggcttctcg tcaggctcag 180 gctgaatggg gtgctctgcc gccgcgtgct cgtgctgcta aactgcgtga actgatcacc 240 ctgctgcgtg aacaccgtga agaattcgct atgctggacg ctatcgacgg tggtttcccg 300 atctctatga tgcgtaacga cgttgacgct gctctggaac tgatggacat cttcgctgac 360 atggctctgg acctgggtgg taaaactata ccggtaagca ccaacctgca cttcaccacc 420 cacgaaccgt tcggtgttgt tgctcgtatc ggtgctttca accacccgtt cttcttcgct 480 gcttctaaag ttgctgctcc gctgatggct ggtaactctg ttatcctgaa agctccggac 540 cagaccccgc tgtcttctct gcgtctggct gaagttgctg ctgaagttct gccgcagaac 600 ctgctgatca ccatctctgg tcgtggtcgt gttgctggtc gtgctatcgt tcgtcacccg 660 cagatcaaac gtatcggttt catcggttct accgacaccg gtcgttctat ccagcgtgac 720 gctgctgaag ttgctgttaa acacatctct ctggaactgg gtggtaaaaa tgcgcagatc 780 gtattcgcgg acgctgacct ggaacaggct gctctgggtg ctgttaacgg tatgaacttc 840 acctggaccg ctggtcagtc ttgcggttct acctctcgtc tgctggttca cgaatctgtt 900 gctgaccagg ttatcgctcg tgttgttgaa ctggtttctg ctatcgctgt tggtccgccg 960 ctggacgaaa acgctcagat gggtccgctg gtttctcagg ctcagtacga caaatctgtt 1020 cacgctatcg gtgaaggtat ccgtgaaggt gctaaagttg ttgctggtgg tggtcgtccg 1080 gaaggtgttg gtgaaggtgg ttggtacctg gctccgaccg ttctggctga cgttagacca 1140 ggtagcttca tcgaacagaa tgaaatcttc ggtccggttc tgtctgttat catcttcgct 1200 accgacgacg aagctgttgc tatcgctaac ggtgttgaat acggtctgac cgcttctgtt 1260 tggacctctg acatcacccg tgctcacctg atcgctcgtc gtgttgaagc tggttacgtt 1320 ctggttaacg gtggttctcg tcactactgg ggtctgccgt tcggtggtgt taaatcttct 1380 ggtgttggtt ctgaagaatc tatggaagaa ctgatctctt acaccgaaac caaaaccacc 1440 accgttgttc tgggttaa 1458 <210> 16 <211> 1353 <212> DNA <213> Artificial sequence <400> 16 atgaaagtta tcgtaattgg ttgtactcat gccggaactg ctgctgtaaa tcaaatcttg 60 gcgtcaaatc cagaaacaga cgtcacgatt tatgaacgga atgacaatgt gtcatttctc 120 tcctgtggga ttgccctcta tcttggtggc gaagttgccg atccacaagg gctcttctat 180 tccagtccag aacaattagc caaattaggc gcgaatgttc atatgcaaca tgatgtgacc 240 gacgtggata ccgaaaatca tgaaattacc gttactgatt tgaagaccgg cgaatccaag 300 aaagattatt acgacaaatt agttgtcaca actggttcat ggcctgtaat tccaccaatc 360 gatggtatcg acagcccgaa cgtttacctc tgcaagaact ggacgcatgc ccaaagttta 420 tgggaagctg ccaagccagc taagcgcgtc atcgttatcg gtggggcta cattgggact 480 gaattagtcg aagcttatca gaagcaaggt aaaggaagtta ccttaattga tggcttacca 540 cggattttaa acaagtattt agaacaaggc ttcactgacc gggtcgaaaa agacttcgtt 600 gaccatggca tcaagatggc cttaaatcag atggttaaag gcttcagtga tgatggcaag 660 720 ggtttccggc caaacaccag cctattaaag ggcaaagttg acatgaaccc gaacggctct 780 attaagacaa atgactacat gcaaacatct gaccctgata tctacggtgc tggtgattcc 840 gttgcggttc actacaaccc aactagaag gatgcctaca ttccattagc cactaacgcg 900 gttcgccaag ggactttagt tggtttgaac atcttcaagc caacccggaa gtacatgggg 960 acgcaatcaa cttctggttt aatgttattc ggcaagacga tcgtttcttc tgggatgacc 1020 ttggaacatg ctcaagctga aaaggtacct gcagaagccg ttacctttga agataactac 1080 cgtccagaat ttatgccaac cacgaaacca gttctgatgc aattggttta caacccagag 1140 acgcgtgaaa tcttaggggc ccaattcatg agtgaacatg acgtttcaca atcggctaac 1200 gtgatctcag tgatgattca aaatcacaac acgatcgatg acttaggctt tgttgacatg 1260 ttcttccagc caatctatga ccgtccattc aactacttga acttattagg ccaagcagcc 1320 atcgctcatg cggctgaaaa agtgactgaa taa 1353 <210> 17 <211> 363 <212> PRT <213> Artificial sequence <400> 17 Met Thr Thr Ser Thr Thr Gln Lys Val Ala Thr Met Lys Thr Phe Val 1 5 10 15 Met Lys Gln Ile Gly Glu Thr Ala Trp Ile Asp Lys Glu Lys Pro Glu 20 25 30 Ala Gly Pro Arg Asp Ala Ile Leu Arg Pro Ile Ala Ile Ala Pro Cys 35 40 45 Thr Ser Asp Ile His Thr Val Tyr Glu Gly Gly Ile Gly Glu Arg Gln 50 55 60 Asn Leu Val Leu Gly His Glu Ala Val Gly Glu Val Ile Glu Val Gly 65 70 75 80 Ser Lys Val Glu Asp Phe Arg Ser Gly Asp Arg Val Ile Val Pro Ala 85 90 95 Ile Thr Pro Asp Trp Tyr Asn Thr Asp Ile Gln Asp Asn Tyr His Gln 100 105 110 His Ser Asn Gly Met Leu Phe Gly Phe Gln Phe Ala Asn Leu Lys Asp 115 120 125 Gly Val Phe Ser Glu Tyr Phe His Val Asn Asp Ala Asp Leu Asn Leu 130 135 140 Ala His Leu Pro Asp Glu Ile Ser Pro Glu Ala Ala Val Met Leu Thr 145 150 155 160 Asp Met Val Thr Thr Gly Leu His Gly Ala Glu Leu Ala Asp Ile Glu 165 170 175 Phe Gly Asp Ser Val Ala Val Ile Gly Ile Gly Pro Val Gly Leu Met 180 185 190 Ala Ile Ala Gly Ala Lys Leu Arg Gly Ala Ser Arg Leu Phe Gly Ala 195 200 205 Gly Ser Arg Glu Val Cys Ala Glu Val Ala Ser Asp Phe Gly Met Thr 210 215 220 Asp Gln Ile Asn Tyr Lys Glu Val Pro Ile Ser Glu Gln Ile Asp Ser 225 230 235 240 Leu Thr Tyr Gly Lys Gly Val Asp Ala Thr Ile Ile Ala Gly Gly Asp 245 250 255 Ser Asp Val Leu Thr Thr Ala Val Glu Ile Thr Lys Pro Gly Gly Asn 260 265 270 Ile Ser Asn Ile Asn Tyr Phe Ser Ile Gly Glu Ser Leu Pro Ile Pro 275 280 285 Arg Leu Ala Trp Gly Asn Gly Met Ala His Lys Thr Ile Lys Gly Gly 290 295 300 Leu Cys Pro Gly Gly Arg Ile Arg Met Glu Arg Leu Ala Asn Leu Val 305 310 315 320 Thr Thr Gly Arg Leu Asn Pro Glu Lys Leu Ile Thr His His Tyr Asn 325 330 335 Lys Phe Glu Asp Ile Glu Glu Ala Phe Lys Leu Met Lys Asp Lys Pro 340 345 350 Arg Asp Leu Ile Lys Pro Val Val Thr Ile Asp 355 360 <210> 18 <211> 1092 <212> DNA <213> Artificial sequence <400> 18 atgacaacat ctacaacaca aaaagtagct acaatgaaaa cattcgtcat gaaacagatc 60 ggtgaaacag cttggataga caaagaaaag ccagaagcag gaccaagaga tgccattctt 120 cgccccatag ctatagctcc atgcacctca gacattcata ccgtctatga aggtggtata 180 240 agcaaagttg aagattttag atcaggagac agagttatag ttccggccat cacaccggat 300 tggtacaata ctgatattca ggataactat catcaacact ccaatggcat gctatttggc 360 tttcaatttg ccaacttgaa ggatggtgtt ttttctgagt attttcacgt taacgatgca 420 gatcttaacc tggctcacct tccagatgag atcagtcctg aagccgctgt aatgttaaca 480 gatatggtta ccacaggctt acacggtgca gagttggctg atatagaatt tggtgacagt 540 gttgcagtaa taggaattgg tccagtagga ctaatggcaa tagctggagc caaattacgt 600 ggtgcatcta gactatttgg agctggcagt agagaggttt gtgctgaggt tgctagcgat 660 720 ttaacttatg gtaaaggcgt tgatgctact atcattgccg gtggcgacag cgatgtacta 780 acaactgcag ttgaaataac aaaacccgga ggcaatattt caaatattaa ttacttcagc 840 attggagaat cacttcccat accacgttta gcatggggaa atggaatggc ccacaaaaca 900 atcaagggcg gactatgtcc cgggggacgc attagaatgg aaagattagc aaatctagtt 960 acaacaggca ggttaaatcc agaaaagctt atcactcatc attacaataa gtttgaagat 1020 attgaagaag catttaagtt aatgaaagat aaaccacgag acttaatcaa accagtagta 1080 actagatact ag 1092

Claims

1. A method for degrading naphthalene and / or fixing CO2, comprising the following steps: (a1) Naphthalene reacts with monooxygenase to produce 1-naphthol; (a2) 1-Naphthol reacts with carboxylase to produce 1-hydroxy-2-benzoic acid; (a3) 1-Hydroxy-2-benzoic acid reacts with dioxygenase to produce 2-carboxybenzopyruvic acid; (a4) 2-Carboxybenzopyruvate reacts with aldolase to produce o-carboxybenzaldehyde; (a5) o-Carboxybenzaldehyde reacts with dehydrogenase to produce phthalic acid.

2. The method according to claim 1, characterized in that: In step (a1), NAD(P)H provides the reducing power.

3. The method according to claim 2, characterized in that: The reducing power provided by NAD(P)H is achieved through either of the following methods: 1) directly adding NAD(P)H to the reaction system; or 2) forming the coenzyme NAD(P)H / NAD(P) in the reaction system. + cycle.

4. The method according to claim 3, characterized in that: The coenzyme NAD(P)H / NAD(P) is formed in the reaction system. + The cycle can be achieved by either of the following methods: 1) adding alcohol dehydrogenase ADH and NAD(P) to the reaction system. + ;2) Add cells that express alcohol dehydrogenase ADH and NAD(P) to the reaction system. + ; 3) Add alcohol dehydrogenase ADH and NAD(P)H to the reaction system; 4) Add cells capable of expressing alcohol dehydrogenase ADH and NAD(P)H to the reaction system; 5) When steps (a1) and (a5) are completed in the same reaction system, add NAD(P)H or NAD(P)H to the reaction system. + .

5. The method according to claim 1, characterized in that: In step (a2), HCO3 - Alternatively, CO2 can be used as another substrate.

6. The method according to claim 1, characterized in that: In the method, the catalytic reactions of various enzymes are carried out in any of the following ways: 1) the corresponding enzyme is directly added to the reaction system; 2) cells capable of expressing the corresponding enzyme are added to the reaction system.

7. The method according to claim 1, characterized in that: In step (a5), the reaction requires NAD(P). + .

8. The method according to claim 7, characterized in that: NAD(P) in the reaction + It can be introduced in any of the following ways: 1) Directly adding NAD(P) to the reaction system. + ;2) Coenzyme NAD(P)H / NAD(P) is formed in the reaction system. + cycle.

9. The method according to claim 8, characterized in that: The coenzyme NAD(P)H / NAD(P) is formed in the reaction system. + The cycle is achieved by: 1) adding NAD(P)H oxidase and NAD(P) to the reaction system. + 2) Add NAD(P)H oxidase and NAD(P)H to the reaction system; 3) When steps (a1) and (a5) are completed in the same reaction system, add NAD(P)H or NAD(P) to the reaction system. + .

10. The method according to claim 1, characterized in that: Steps (a1)-(a3) are completed in one step in the same reaction system, which is referred to as reaction system I.

11. The method according to claim 10, characterized in that: The reaction system I contains 1) naphthalene, 2) a monooxygenase or a cell capable of expressing the monooxygenase, 3) a carboxylase or a cell capable of expressing the carboxylase, 4) a dioxygenase or a cell capable of expressing the dioxygenase, 5) NAD(P)H, and 6) HCO3-. - Or CO2, 7) reaction buffer I.

12. The method according to claim 11, characterized in that: In the reaction system I, the final concentration of naphthalene is 15 mM, the final concentration of NAD(P)H is 60 mM, and the final concentration of HCO3 is... - The final concentration is 50 mM or CO2 is continuously introduced into the reaction system I during the reaction process.

13. The method according to claim 11, characterized in that: The pH of the reaction buffer I is 6.5-8.

0.

14. The method according to claim 10, characterized in that: The reaction temperature is 25-35℃, and the reaction time is 3-12h.

15. The method according to claim 1, characterized in that: Steps (a4)-(a5) are completed in one step in the same reaction system, which is referred to as reaction system II.

16. The method according to claim 15, characterized in that: The reaction system II contains: 1) 2-carboxybenzopyruvate, 2) aldolase or cells capable of expressing the aldolase, 3) dehydrogenase or cells capable of expressing the dehydrogenase, and 4) NAD(P). + 5) KHCO3, 6) Reaction buffer II.

17. The method according to claim 16, characterized in that: In the reaction system II, the final concentration of 2-carboxybenzopyruvic acid is 10 mM, the final concentration of NAD(P)H is 15 mM, and the final concentration of KHCO3 is 50 mM.

18. The method according to claim 16, characterized in that: The pH of the reaction buffer II is 6.5-8.

0.

19. The method according to claim 15, characterized in that: The reaction temperature is 25-35℃, and the reaction time is 3-12h.

20. The method according to claim 1, characterized in that: Steps (a2)-(a5) are completed in one step in the same reaction system, which is referred to as reaction system III.

21. The method according to claim 20, characterized in that: The reaction system III contains 1) 1-naphthol, 2) a carboxylase or a cell capable of expressing the carboxylase, 3) a dioxygenase or a cell capable of expressing the dioxygenase, 4) an aldolase or a cell capable of expressing the aldolase, 5) a dehydrogenase or a cell capable of expressing the dehydrogenase, 6) NAD(P)H oxidase or a cell capable of expressing the NAD(P)H oxidase, and 7) NAD(P) + 8) KHCO3, 9) Reaction buffer III.

22. The method according to claim 21, characterized in that: In reaction system III, the final concentration of 1-naphthol is 7.5 mM, and the concentration of NAD(P) is... + The final concentration of KHCO3 is 0.5 mM and the final concentration of KHCO3 is 90 mM.

23. The method according to claim 21, characterized in that: The pH of the reaction buffer III is 6.5-8.

0.

24. The method according to claim 20, characterized in that: The reaction temperature is 25-35℃, and the reaction time is 3-12h.

25. The method according to any one of claims 1-24, characterized in that: The monooxygenase is derived from Bacillus megaterium (Bacillus megaterium). Bacillus megaterium Monooxygenases.

26. The method according to claim 25, characterized in that: The source is Bacillus megaterium ( Bacillus megaterium The amino acid sequence of the monooxygenase is shown in SEQ ID No.

1.

27. The method according to any one of claims 1-24, characterized in that: The carboxylase is derived from Aspergillus oryzae ( Aspergillus oryzae The carboxylase of ) originates from rhizobia ( Rhizobium Carboxylases from *Candida sp.* or derived from *Candida sp.* (sp.) Trichosporon moniliiforme ) carboxylase.

28. The method according to claim 27, characterized in that: The source is Aspergillus oryzae ( Aspergillus oryzae The amino acid sequence of the carboxylase derived from Rhizobium is shown in SEQ ID No. 2; Rhizobium The amino acid sequence of the carboxylase derived from *Candida sp.* is shown in SEQ ID No. 3; the carboxylase derived from *Candida sp.* is shown in SEQ ID No.

3. Trichosporon moniliiforme The amino acid sequence of the carboxylase is shown in SEQ ID No.

4.

29. The method according to any one of claims 1-24, characterized in that: The dioxygenase is derived from Mycobacterium van Barenella ( Mycobacterium vanbaalenii PYR-1 is a dioxygenase.

30. The method according to claim 29, characterized in that: The bacteria derived from Mycobacterium van Barenella ( Mycobacterium vanbaalenii The amino acid sequence of the dioxygenase of PYR-1 is shown in SEQ ID No.

5.

31. The method according to any one of claims 1-24, characterized in that: The aldolase is derived from *Pseudomonas putida* (…). Pseudomonas putida Aldolase.

32. The method according to claim 31, characterized in that: The bacteria derived from *Pseudomonas putida* ( Pseudomonas putida The amino acid sequence of the aldolase is shown in SEQ ID No.

6.

33. The method according to any one of claims 1-24, characterized in that: The dehydrogenase is derived from Nocardia ( Nocardioides sp. KP7 dehydrogenase.

34. The method according to claim 33, characterized in that: The source is Nocardia ( Nocardioides sp. The amino acid sequence of the dehydrogenase KP7 is shown in SEQ ID No.

7.

35. The method according to claim 9 or 21, characterized in that: The NAD(P)H oxidase is derived from Lactobacillus parasiticus (… Lactiplantibacillus pentosus NAD(P)H oxidase.

36. The method according to claim 35, characterized in that: The source is Lactobacillus parasiticus ( Lactiplantibacillus pentosus The amino acid sequence of NAD(P)H oxidase is shown in SEQ ID No.

8.

37. The method according to claim 4, characterized in that: The amino acid sequence of the alcohol dehydrogenase ADH is shown in SEQ ID No.

17.

38. A method for generating o-carboxybenzaldehyde from naphthalene as a substrate, comprising steps (a1)-(a4) of the method according to any one of claims 1-37.

39. A method for generating phthalic acid from naphthalene as a substrate, comprising steps (a1)-(a5) of the method according to any one of claims 1-37.

40. A method for generating o-carboxybenzaldehyde from 1-naphthol as a substrate, comprising steps (a2)-(a4) of the method according to any one of claims 1-37.

41. A method for generating phthalic acid from 1-naphthol as a substrate, comprising steps (a2)-(a5) of the method according to any one of claims 1-37.

42. A set of enzymes or cells in the degradation of naphthalene and / or fixation of CO2; The complete set of enzymes is either (B1) or (B2): (B1) is composed of carboxylase, dioxygenase, aldolase, dehydrogenase and NAD(P)H oxidase; (B2) is composed of monooxygenase, alcohol dehydrogenase ADH, carboxylase, dioxygenase, aldolase, dehydrogenase and NAD(P)H oxidase; The assembled cells are as follows (C1) or (C2): (C1) is composed of cells capable of expressing the carboxylase, cells capable of expressing the dioxygenase, cells capable of expressing the aldolase, cells capable of expressing the dehydrogenase, and cells capable of expressing the NAD(P)H oxidase. (C2) is composed of cells capable of expressing the monooxygenase, cells capable of expressing the alcohol dehydrogenase ADH, cells capable of expressing the carboxylase, cells capable of expressing the dioxygenase, cells capable of expressing the aldolase, cells capable of expressing the dehydrogenase, and cells capable of expressing the NAD(P)H oxidase. The monooxygenase is derived from Bacillus megaterium (Bacillus megaterium). Bacillus megaterium Monooxygenases derived from Bacillus megaterium (Beta tumefaciens) Bacillus megaterium The amino acid sequence of the monooxygenase is shown in SEQ ID No. 1; The carboxylase is derived from Aspergillus oryzae ( Aspergillus oryzae The carboxylase of ) originates from rhizobia ( Rhizobium Carboxylases from *Candida sp.* or derived from *Candida sp.* (sp.) Trichosporon moniliiforme The carboxylase derived from Aspergillus oryzae ( Aspergillus oryzae The amino acid sequence of the carboxylase derived from Rhizobium is shown in SEQ ID No. 2; Rhizobium The amino acid sequence of the carboxylase derived from *Candida sp.* is shown in SEQ ID No. 3; the carboxylase derived from *Candida sp.* is shown in SEQ ID No.

3. Trichosporon moniliiforme The amino acid sequence of the carboxylase of ) is shown in SEQ ID No. 4; The dioxygenase is derived from Mycobacterium van Barenella ( Mycobacterium vanbaalenii PYR-1) dioxygenase; the one derived from Mycobacterium van Barenella ( Mycobacterium vanbaalenii The amino acid sequence of the dioxygenase of PYR-1 is shown in SEQ ID No. 5; The aldolase is derived from *Pseudomonas putida* (…). Pseudomonas putida Aldolases derived from *Pseudomonas putida* ( Pseudomonas putida The amino acid sequence of the aldolase is shown in SEQ ID No. 6; The dehydrogenase is derived from Nocardia ( Nocardioides sp. The dehydrogenase derived from KP7; Nocardioides sp. The amino acid sequence of the dehydrogenase of KP7 is shown in SEQ ID No. 7; The NAD(P)H oxidase is derived from Lactobacillus parasiticus (… Lactiplantibacillus pentosus NAD(P)H oxidase derived from Lactobacillus parasiticus ( Lactiplantibacillus pentosus The amino acid sequence of NAD(P)H oxidase is shown in SEQ ID No. 8; The amino acid sequence of the alcohol dehydrogenase ADH is shown in SEQ ID No.

17.

43. Application of complete enzyme sets or complete cell sets in the preparation of o-carboxybenzaldehyde; The complete set of enzymes is as follows (D1) or (D2): (D1) is composed of carboxylase, dioxygenase, and aldolase; (D2) is composed of monooxygenase, alcohol dehydrogenase ADH, the carboxylase, the dioxygenase and the aldolase; The assembled cells are either (E1) or (E2): (E1) consists of cells capable of expressing the carboxylase, cells capable of expressing the dioxygenase, and cells capable of expressing the aldolase; (E2) consists of cells capable of expressing the monooxygenase, cells capable of expressing the alcohol dehydrogenase ADH, cells capable of expressing the carboxylase, cells capable of expressing the dioxygenase, and cells capable of expressing the aldolase; The monooxygenase is derived from Bacillus megaterium (Bacillus megaterium). Bacillus megaterium Monooxygenases derived from Bacillus megaterium (Beta tumefaciens) Bacillus megaterium The amino acid sequence of the monooxygenase is shown in SEQ ID No. 1; The carboxylase is derived from Aspergillus oryzae ( Aspergillus oryzae The carboxylase of ) originates from rhizobia ( Rhizobium Carboxylases from *Candida sp.* or derived from *Candida sp.* (sp.) Trichosporon moniliiforme The carboxylase derived from Aspergillus oryzae ( Aspergillus oryzae The amino acid sequence of the carboxylase derived from Rhizobium is shown in SEQ ID No. 2; Rhizobium The amino acid sequence of the carboxylase derived from *Candida sp.* is shown in SEQ ID No. 3; the carboxylase derived from *Candida sp.* is shown in SEQ ID No.

3. Trichosporon moniliiforme The amino acid sequence of the carboxylase of ) is shown in SEQ ID No. 4; The dioxygenase is derived from Mycobacterium van Barenella ( Mycobacterium vanbaalenii PYR-1) dioxygenase; the one derived from Mycobacterium van Barenella ( Mycobacterium vanbaalenii The amino acid sequence of the dioxygenase of PYR-1 is shown in SEQ ID No. 5; The aldolase is derived from *Pseudomonas putida* (…). Pseudomonas putida Aldolases derived from *Pseudomonas putida* ( Pseudomonas putida The amino acid sequence of the aldolase is shown in SEQ ID No. 6; The amino acid sequence of the alcohol dehydrogenase ADH is shown in SEQ ID No.

17.

44. Application of complete enzyme or complete cell sets in the preparation of phthalic acid; The complete set of enzymes is either (B1) or (B2): (B1) is composed of carboxylase, dioxygenase, aldolase, dehydrogenase and NAD(P)H oxidase; (B2) is composed of monooxygenase, alcohol dehydrogenase ADH, carboxylase, dioxygenase, aldolase, dehydrogenase and NAD(P)H oxidase; The assembled cells are as follows (C1) or (C2): (C1) is composed of cells capable of expressing the carboxylase, cells capable of expressing the dioxygenase, cells capable of expressing the aldolase, cells capable of expressing the dehydrogenase, and cells capable of expressing the NAD(P)H oxidase. (C2) is composed of cells capable of expressing the monooxygenase, cells capable of expressing the alcohol dehydrogenase ADH, cells capable of expressing the carboxylase, cells capable of expressing the dioxygenase, cells capable of expressing the aldolase, cells capable of expressing the dehydrogenase, and cells capable of expressing the NAD(P)H oxidase. The monooxygenase is derived from Bacillus megaterium (Bacillus megaterium). Bacillus megaterium Monooxygenases derived from Bacillus megaterium (Beta tumefaciens) Bacillus megaterium The amino acid sequence of the monooxygenase is shown in SEQ ID No. 1; The carboxylase is derived from Aspergillus oryzae ( Aspergillus oryzae The carboxylase of ) originates from rhizobia ( Rhizobium Carboxylases from *Candida sp.* or derived from *Candida sp.* (sp.) Trichosporon moniliiforme The carboxylase derived from Aspergillus oryzae ( Aspergillus oryzae The amino acid sequence of the carboxylase derived from Rhizobium is shown in SEQ ID No. 2; Rhizobium The amino acid sequence of the carboxylase derived from *Candida sp.* is shown in SEQ ID No. 3; the carboxylase derived from *Candida sp.* is shown in SEQ ID No.

3. Trichosporon moniliiforme The amino acid sequence of the carboxylase of ) is shown in SEQ ID No. 4; The dioxygenase is derived from Mycobacterium van Barenella ( Mycobacterium vanbaalenii PYR-1) dioxygenase; the one derived from Mycobacterium van Barenella ( Mycobacterium vanbaalenii The amino acid sequence of the dioxygenase of PYR-1 is shown in SEQ ID No. 5; The aldolase is derived from *Pseudomonas putida* (…). Pseudomonas putida Aldolases derived from *Pseudomonas putida* ( Pseudomonas putida The amino acid sequence of the aldolase is shown in SEQ ID No. 6; The dehydrogenase is derived from Nocardia ( Nocardioides sp. The dehydrogenase derived from KP7; Nocardioides sp. The amino acid sequence of the dehydrogenase of KP7 is shown in SEQ ID No. 7; The NAD(P)H oxidase is derived from Lactobacillus parasiticus (… Lactiplantibacillus pentosus NAD(P)H oxidase derived from Lactobacillus parasiticus ( Lactiplantibacillus pentosus The amino acid sequence of NAD(P)H oxidase is shown in SEQ ID No. 8; The amino acid sequence of the alcohol dehydrogenase ADH is shown in SEQ ID No. 17.

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