CN117660392A - Application of a coenzyme self-sufficient chassis cell in biosynthesis - Google Patents

Abstract

The invention discloses application of a coenzyme self-contained chassis cell in biosynthesis, wherein the coenzyme self-contained chassis cell carries a recombinant expression vector of an NAD kinase with an amino acid sequence shown as SEQ ID NO.1 and a coding gene of a mutant thereof, and the NAD kinase mutant is obtained by single-point mutation or multi-point superposition mutation of positions 110, 170 and 259 of the amino acid sequence shown as SEQ ID NO. 1. The coenzyme self-contained chassis cell can be used for biosynthesis of pranlukast intermediate 3AHAP, ursodeoxycholic acid UDCA and Ruimegem intermediate compound II, the coenzyme self-contained chassis cell is introduced into a multienzyme system to convert NAD+ into NADP+ and is reduced by glucose dehydrogenase GDH to generate coenzyme NADPH, and the coenzyme NADPH provides reducing force for catalyzing and synthesizing a medical intermediate, a pesticide intermediate and the like by the multienzyme system.

Description

Application of a coenzyme self-sufficient chassis cell in biosynthesis

Technical Field

The present invention relates to the field of biotechnology; in particular, to NAD kinase and its mutants, as well as coenzyme self-sufficient chassis cells containing NAD kinase and its mutants, and applications in biosynthesis of pharmaceutical intermediates.

Background Art

In genetic engineering, "host cells" usually refer to cells used as carriers in genetic engineering experiments. These cells are used to carry plasmids or foreign genes, and through genetic recombination methods, they are able to express and produce specific proteins. Host cells can be transformed or modified to enable them to accept, replicate and express foreign genes.

In genetic engineering, the target gene is usually inserted into the genome of a plasmid vector or chassis cell, making it the host of the target gene. The chassis cell will use its own biosynthetic machinery to produce the protein encoded by the inserted gene. This technology is widely used in the fields of biopharmaceuticals, gene therapy, agricultural biotechnology, etc. to produce various biological products such as drugs, growth factors, enzymes, hormones, antibodies, etc.

In genetic engineering research, it is critical to select the right chassis cells, because different cell types may have different effects on gene expression and protein production. Some commonly used chassis cells include Escherichia coli, yeast (Saccharomyces cerevisiae), mammalian cells (such as CHO cells, HEK293 cells, etc.), etc. Selecting the right chassis cells is an important step in genetic engineering research, which can affect production efficiency and product quality.

3-Amino-2-hydroxyacetophenone, also known as 1-(3-Amino-2-hydroxyphenyl)ethanone (3AHAP for short), is a key intermediate in the synthesis of Pranlukast. Pranlukast is an oral drug for the treatment of bronchial asthma. It is a leukotriene (CysLTs) receptor antagonist that can effectively inhibit the LTD4 receptor to treat asthma. Compared with other leukotriene antagonists, Pranlukast has the characteristics of strong selective inhibition, wide applicability, and few side effects. It has broad market prospects and great research value.

Ursodeoxycholic acid (UDCA) is an endogenous bile acid that has been shown to dissolve gallstones and show better efficacy than other endogenous bile acids in treating gallbladder and liver-related diseases. To date, UDCA is the only drug approved by the U.S. Food and Drug Administration (FDA) for the treatment of primary biliary cirrhosis.

Rimegepant is the first small molecule CGRP receptor antagonist with an orally rapidly disintegrating tablet (ODT) formulation, which treats migraine by blocking CGRP receptors. In 2020, the U.S. FDA approved Biohaven's CGRP receptor inhibitor Nurtec ODT orally disintegrating tablets for the treatment of acute migraine in adults.

The synthesis method of the pranlukast intermediate 3AHAP is mainly chemical synthesis, which can be obtained through a four-step reaction pathway. The second step of this pathway involves Fries rearrangement, which involves the rearrangement of phenolic esters to o- or p-acylphenols under the catalysis of Lewis or Bronsted acids. However, this step has a high risk of environmental pollution due to the use of organic solvents such as chloroform and dichloromethane. In addition, the catalysts used in the reaction system, such as AlCl3, BF3 and TiCl4, may release toxic gases, which are harmful to the environment.

Ursodeoxycholic acid (UDCA) is currently prepared by chemical or biosynthetic pathways: the chemical pathway produces UDCA through a seven-step synthesis, using cholic acid (CA) or chenodeoxycholic acid (CDCA) as the starting substrate, and this route requires the use of toxic and dangerous hydrazine, CrO3 and pyridine reagents, and produces a large amount of waste, in addition to the overall yield of only about 30%. UDCA produced by chemical synthesis is still far from meeting market demand in terms of quantity and quality.

The existing synthesis methods of remegipam intermediates are mainly chemical methods, including the route using 3-amino-2-chloropyridine and 1-CBZ-4-piperidone as starting substrates, which requires multiple steps of reaction, has high pollution, low yield, and complex operation, and does not meet the conditions for industrial production. Therefore, it is urgent to find a green, environmentally friendly, high-yield, and easy-to-operate preparation method.

Compared with chemical synthesis, biosynthesis is more environmentally friendly, efficient and safe. The biosynthesis of pranlukast intermediate 3AHAP, ursodeoxycholic acid UDCA and remegipam intermediate is gradually developing. Its biosynthesis is mainly free enzyme catalytic synthesis or whole cell catalysis. Whole cell catalysis or free enzyme catalytic synthesis uses m-nitroacetophenone 3NAP, 7-ketolithocholic acid 7-KLCA or pyridine-2,3-diamine and 4-oxopiperidone hydrochloride as substrates to generate pranlukast intermediate 3AHAP, ursodeoxycholic acid UDCA and remegipam intermediate through multi-enzyme cascade reaction.

The biosynthesis of the intermediates 3AHAP of Pranlukast, UDCA of ursodeoxycholic acid and remegipam of sedative is catalyzed by a multi-enzyme system, and the catalytic process requires NADPH as an electron donor. The catalysis of 1 mol of substrate functional groups requires the consumption of an equal amount of NADPH cofactor, so the concentration of NADPH cofactor has a significant impact on its catalytic activity. However, the NADPH cofactor is expensive and is not allowed to be added in large quantities in actual industrial applications. Glucose dehydrogenase (GDH) can oxidize glucose to produce gluconic acid and reduce NAD(P) ⁺ to NAD(P)H at the same time. It is a key enzyme in the coenzyme regeneration system in redox biosynthesis. CN116426578A discloses a method for biosynthesizing Prunster intermediates, which utilizes glucose dehydrogenase GDH to catalyze glucose to provide the required NADPH for the reaction pathway, without adding pure NADPH to the reaction system. However, the amount of NADP contained in the cell is limited, and the NADPH that can be generated is also less, which cannot meet the needs of the reduction reaction. Generally speaking, in order to improve the reaction efficiency, additional NADPH still needs to be added to the reaction system, which will lead to increased costs. The price of NADP cofactor is much lower than that of NADPH, and the price of NAD is much lower than that of NADP. The phosphorylation of NAD+ by NAD kinase (NADK) is the only known mechanism for the de novo generation of NADP. Nicotinamide adenine dinucleotide (NAD+) phosphate (NADP (H)) plays a vital role in redox homeostasis and metabolism. If the redox equilibrium state in the cell can be regulated by introducing the overexpression of NAD (H) kinase gene, the yield of the target product may be increased.

NAD kinase and NADH kinase use ATP or inorganic polyphosphate as phosphorylation donors to specifically catalyze the 2'-hydroxy phosphorylation of the adenosine ribose moiety of NAD(H) to generate NADP(H). The intracellular NAD(H) content of E. coli is much higher than that of NADP(H). NAD kinase (NAD(H) Kinase) catalyzes the phosphorylation of NAD(H) and converts it into NADP(H), which is the direct source and key step of NADP(H) synthesis in microorganisms.

Introducing the constructed coenzyme self-sufficient chassis cells containing NAD kinase NADK into the above-mentioned multi-enzyme system reaction, catalyzing NAD to generate NADP to supply the multi-enzyme system reaction to generate expensive cofactor NADPH, can effectively improve the catalytic efficiency and reduce production costs: by modifying the key amino acid residues of NAD kinase NADK, the specific enzyme activity of the key enzyme can be improved, which can effectively reduce the use of cells. This is of great significance for improving atomic economy, reducing use costs, and alleviating environmental pressure, so screening high-yield NAD kinase mutants is particularly important.

Summary of the invention

The technical problem to be solved by the present invention is to overcome the defects of low enzyme activity of NAD kinase in the prior art. The purpose of the present invention is to construct a coenzyme self-sufficient chassis cell, select Escherichia coli BL21 (DE3) as the chassis cell, and construct NAD kinase NAD or its mutant derived from Corynebacterium glutamicum on a plasmid vector, transform it into Escherichia coli BL21 (DE3) to form a coenzyme self-sufficient chassis cell, and apply it to a multi-enzyme system to produce pharmaceutical intermediates such as pranlukast intermediate 3-amino-2-hydroxyacetophenone, ursodeoxycholic acid, and remedipam intermediate.

The invention provides a NAD kinase derived from Corynebacterium glutamicum, the amino acid sequence of which is shown in SEQ ID NO.1.

The present invention also provides a NAD kinase mutant, which is obtained by performing single-point mutation or multi-point superimposed mutation at positions 110, 170 and 259 of the amino acid sequence shown in SEQ ID NO.1.

The NAD kinase of the amino acid sequence shown in SEQ ID NO.1 may be referred to as the wild-type enzyme of NAD kinase in this application. It is an amino acid sequence annotated as NAD kinase (CgNADK) derived from Corynebacterium glutamicum, which is obtained by optimization of the present invention. The nucleotide sequence of the wild-type enzyme may be the nucleotide sequence shown in SEQ ID NO.4.

The NAD kinase or a mutant thereof described in the present application has NAD kinase activity, ie, an activity of converting NAD into NADP. In particular, the NAD kinase mutant described in the present application has improved activity of converting NAD into NADP.

Furthermore, the NAD kinase mutant preferably has one or more of the following mutations:

(1) mutating the amino acid residue G at position 170 of the amino acid sequence shown in SEQ ID NO.1 to R, K, A, H or P;

(2) mutating the amino acid residue F at position 259 of the amino acid sequence shown in SEQ ID NO.1 to G, A or H;

(3) The amino acid residue G at position 110 of the amino acid sequence shown in SEQ ID NO.1 is mutated to W, E or R.

More preferably, the NAD kinase mutant is one of the following:

(a) the amino acid residue G at position 170 of the amino acid sequence shown in SEQ ID NO.1 is mutated to R, and the amino acid residue F at position 259 is mutated to A;

(b) the amino acid residue G at position 170 of the amino acid sequence shown in SEQ ID NO.1 is mutated to R, and the amino acid residue G at position 110 is mutated to W;

(c) the amino acid residue G at position 170 of the amino acid sequence shown in SEQ ID NO.1 is mutated to R, and the amino acid residue G at position 110 is mutated to E;

(d) The amino acid residue G at position 170 of the amino acid sequence shown in SEQ ID NO.1 is mutated to R, and the amino acid residue G at position 110 is mutated to R.

More preferably, the NAD kinase mutant is (a) wherein the amino acid residue G at position 170 of the amino acid sequence shown in SEQ ID NO.1 is mutated to R, and the amino acid residue F at position 259 is mutated to A, and the amino acid sequence is shown in SEQ ID NO.3.

Due to the particularity of the amino acid sequence, any fragment or variant of the peptide protein containing the amino acid sequence shown in the present invention, such as its conservative variant, biologically active fragment or derivative, as long as the fragment or variant of the peptide protein has a homology of more than 90% with the aforementioned amino acid sequence, belongs to the protection scope of the present invention. Specifically, the changes include the deletion, insertion or replacement of amino acids in the amino acid sequence; wherein, for the conservative changes of the variant, the replaced amino acid has a similar structure or chemical property to the original amino acid, such as replacing leucine with isoleucine, and the variant may also have non-conservative changes, such as replacing alanine with glycine.

Furthermore, the amino acid sequence in which the 170th G of the amino acid sequence shown in SEQ ID NO.1 is mutated to R is shown in SEQ ID NO.2.

The amino acid sequence shown in SEQ ID NO.1, in which the 170th amino acid residue G is mutated to R, and the 259th amino acid residue F is mutated to A, is shown in SEQ ID NO.3.

The present invention also provides a gene encoding the NAD kinase or a mutant thereof.

Among them, the nucleotide sequence of the coding gene corresponding to the amino acid sequence shown in SEQ ID NO.1 is shown in SEQ ID NO.4.

Due to the particularity of the nucleotide sequence, any variant of the polynucleotide shown in the present invention, as long as it has more than 90% homology with the aforementioned polynucleotide, belongs to the scope of protection of the present invention. The variant of the polynucleotide refers to a polynucleotide sequence with one or more nucleotide changes. The variant of this polynucleotide can be a natural variant or a non-natural variant, including a substitution variant, a deletion variant and an insertion variant. As known in the art, an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion or insertion of a polynucleotide, but will not substantially change the function of the peptide protein encoded by it.

The present invention also provides a recombinant expression vector containing a gene encoding NAD kinase or a mutant thereof, and a coenzyme self-sufficient chassis cell containing the gene encoding NAD kinase or a mutant thereof constructed using the recombinant expression vector. The coenzyme self-sufficient chassis cell is an Escherichia coli chassis cell, and the host cell is usually Escherichia coli BL21 (DE3).

The present invention also provides the use of the coenzyme self-sufficient chassis cell in the biosynthesis of pranlukast intermediate 3AHAP, ursodeoxycholic acid UDCA or remegipam intermediate compound II.

The coenzyme self-sufficient chassis cell comprises NAD kinase or a mutant thereof.

Furthermore, the application method is: introducing the coenzyme self-sufficient chassis cells into a multi-enzyme reaction system, converting NAD+ into NADP+, and glucose dehydrogenase GDH reducing NADP+ to generate coenzyme NADPH, and coenzyme NADPH provides electrons for the biosynthetic pharmaceutical intermediate pranlukast intermediate 3AHAP, ursodeoxycholic acid UDCA or remegipan intermediate compound II, that is, providing reducing ability.

Specifically, the present invention provides the application of coenzyme self-sufficient chassis cells in the biosynthesis of pranlukast intermediate 3AHAP, and the application method is: introducing the coenzyme self-sufficient chassis cells into a multi-enzyme reaction system, converting NAD+ into NADP+, glucose dehydrogenase GDH reducing NADP+ to generate coenzyme NADPH, supplying nitroreductase coupled hydroxylamine benzene mutase, and bioconverting the substrate m-nitroacetophenone into the pranlukast intermediate 3-amino-2-hydroxyacetophenone.

The reaction process is as follows: NAD kinase or its mutant in the coenzyme self-sufficient chassis cell converts NAD+ into NADP+, glucose dehydrogenase GDH reduces NADP+ to generate coenzyme NADPH, and under the action of electrons provided by NADPH, nitrobenzene reductase nbzA catalyzes m-nitroacetophenone 3NAP to generate 3-hydroxyaminoacetophenone 3HAAP; hydroxylamine benzene variant enzyme habA catalyzes 3-hydroxyaminoacetophenone 3HAAP to generate 3-amino-2-hydroxyacetophenone 3AHAP.

The reaction formula is shown below:

Specifically, the preferred application method is as follows:

An enzyme mixed system is formed with nitrobenzene reductase nbzA, hydroxylamine benzene variant enzyme habA, glucose dehydrogenase GDH, NAD kinase or its mutant contained in coenzyme self-sufficient chassis cells as catalysts, m-nitroacetophenone 3NAP is used as a substrate, glucose, coenzyme NAD and ATP are added to construct a reaction system to synthesize 3-amino-2-hydroxyacetophenone 3AHAP.

Furthermore, in the reaction system, the concentration of the substrate m-nitroacetophenone 3NAP is 1-50 g/L, and the concentration of glucose is 20-80 g/L.

Furthermore, the solvent of the reaction system is a pH 6.5-8 buffer solution, preferably a pH 7-8, 20-50 mM phosphate buffer solution.

The reaction temperature is 25 to 50° C., preferably 30 to 35° C. The reaction time is 1 to 5 hours.

In the reaction system, the concentration of coenzyme NAD is 1-15 mM, and the concentration of ATP is 1-15 mM.

After the reaction is completed, the reaction solution is separated and purified to obtain pure 3-amino-2-hydroxyacetophenone 3AHAP.

The coenzyme self-sufficient chassis cells can be added to the reaction system in the form of bacterial cells, or in the form of crude enzyme liquid after cell crushing or pure enzyme after purification.

The nitrobenzene reductase nbzA, hydroxylamine benzene variant enzyme habA, and glucose dehydrogenase can be in the form of enzymes or in the form of bacteria. The enzyme forms include free enzymes, immobilized enzymes, including purified enzymes, crude enzymes, fermentation broths, carrier-immobilized enzymes, cell fragments, etc.; the bacteria forms include living bacteria cells and/or dead bacteria cells.

When nitrobenzene reductase nbzA, hydroxylamine benzene variant enzyme habA, and glucose dehydrogenase are in the form of crude enzyme solutions, in the reaction system, the mass concentration of the crude enzyme solution of nitrobenzene reductase nbzA is 2-10 g/L, the mass concentration of the crude enzyme solution of hydroxylamine benzene variant enzyme habA is 20-50 g/L, and the mass concentration of the crude enzyme solution of glucose dehydrogenase GDH is 40-80 g/L.

The mass concentration of the crude enzyme solution of NAD kinase or its mutant obtained after the coenzyme self-sufficient chassis cells are broken is 10-30 g/L.

Furthermore, the nitrobenzene reductase nbzA, hydroxylamine benzene variant enzyme habA, glucose dehydrogenase GDH, and coenzyme self-sufficient chassis cells containing NAD kinase or its mutants can be prepared according to the following method: the seed liquid of recombinant genetically engineered bacteria containing genes encoding the nitrobenzene reductase nbzA, hydroxylamine benzene variant enzyme habA, glucose dehydrogenase GDH or NAD kinase or its mutants is respectively inoculated into LB culture medium containing kanamycin or streptomycin, and cultured at 35-37°C until OD600 reaches 0.8, and the inducer IPTG with a final concentration of 0.5-1mM is added to the fermentation broth, and the culture is induced at 28-30°C, and the obtained culture is centrifuged, and the bacterial precipitate is collected to obtain the nitrobenzene reductase nbzA, hydroxylamine benzene variant enzyme habA, glucose dehydrogenase GDH, and coenzyme self-sufficient chassis cells containing NAD kinase or its mutants.

The crude enzyme solution can be prepared as follows: nitrobenzene reductase nbzA, hydroxylamine benzene variant enzyme habA, glucose dehydrogenase GDH or coenzyme self-sufficient chassis cells containing NAD kinase or its mutant are resuspended in 50mM pH8.0 phosphate buffer, homogenized and broken, and the broken liquid is centrifuged to remove the precipitate to obtain a crude enzyme solution containing nitrobenzene reductase nbzA, hydroxylamine benzene variant enzyme habA, glucose dehydrogenase GDH or NAD kinase or its mutant. The crude enzyme solution can be further purified by nickel column and dialyzed for desalting to obtain pure protein of nitrobenzene reductase nbzA, hydroxylamine benzene variant enzyme habA, glucose dehydrogenase GDH or NAD kinase or its mutant.

The gene encoding the nitrobenzene reductase nbzA, hydroxylamine benzene variant enzyme habA and glucose dehydrogenase GDH is prepared by inserting the coding genes of the nitrobenzene reductase nbzA, hydroxylamine benzene variant enzyme habA and glucose dehydrogenase GDH into a recombinant expression vector and then transferring the vector into a host bacterium for transformation.

The coenzyme self-sufficient chassis cell containing NAD kinase or its mutant is prepared by inserting the coding gene of NAD kinase or its mutant into a recombinant expression vector and then transferring the vector into a host bacterium for transformation.

Nitrobenzene reductase nbzA, hydroxylamine benzene variant enzyme habA, and glucose dehydrogenase GDH can be obtained by referring to CN116426578A, and the encoding genes are disclosed in Chinese patent CN116426578A. According to CN116426578A, recombinant genetic engineering bacteria containing genes encoding the nitrobenzene reductase nbzA, hydroxylamine benzene variant enzyme habA, and glucose dehydrogenase GDH can be obtained.

The present invention also provides the use of a coenzyme self-sufficient chassis cell containing NAD kinase or a mutant thereof in the biosynthesis of ursodeoxycholic acid (UDCA). The method of the use is as follows: the coenzyme self-sufficient chassis cell is introduced into a multi-enzyme reaction system, NAD ⁺ is converted into NADP ⁺ , glucose dehydrogenase GDH reduces NADP+ to generate coenzyme NADPH, which is supplied to hydroxysteroid dehydrogenase 7b-HSDH to bioconvert substrate 7-ketolithocholic acid (7-KLCA) into product ursodeoxycholic acid (UDCA).

The reaction formula is shown below:

Specifically, the preferred application method is as follows:

An enzyme mixed system is formed using hydroxysteroid dehydrogenase 7b-HSDH, glucose dehydrogenase GDH, NAD kinase or its mutant contained in coenzyme self-sufficient chassis cells as catalysts, 7-ketolithocholic acid 7-KLCA is used as a substrate, glucose, coenzyme NAD and ATP are added to construct a reaction system to synthesize ursodeoxycholic acid UDCA.

Furthermore, in the reaction system, the concentration of the substrate 7-ketolithocholic acid 7-KLCA is 10-40 g/L, and the mass fraction of glucose is 2-5%.

Furthermore, the solvent of the reaction system is a pH 6.5-8 buffer solution, preferably a pH 7-8, 50-100 mM phosphate buffer solution.

The reaction temperature is 25 to 50° C., preferably 30 to 35° C. The reaction time is 0.25 to 1 hour.

In the reaction system, the concentration of coenzyme NAD is 1-15 mM, and the concentration of ATP is 1-15 mM.

After the reaction is completed, the reaction solution is separated and purified to obtain pure ursodeoxycholic acid UDCA.

The catalyst includes hydroxysteroid dehydrogenase 7b-HSDH and glucose dehydrogenase GDH, and the catalyst can be in the form of an enzyme or a bacterial cell. The enzyme includes free enzymes, immobilized enzymes, including purified enzymes, crude enzymes, fermentation broths, carrier-immobilized enzymes, cell fragments, etc. The bacterial cell includes living bacterial cells and/or dead bacterial cells.

The coenzyme self-sufficient chassis cells can be added to the reaction system in the form of bacterial cells, or in the form of crude enzyme liquid after cell crushing or pure enzyme after purification.

The catalyst is preferably in the form of wet bacteria or crude enzyme solution. When the catalyst is in the form of wet bacteria, in the reaction system, the mass concentration of hydroxysteroid dehydrogenase 7b-HSDH wet bacteria is 5-15 g/L, the mass concentration of glucose dehydrogenase GDH wet bacteria is 2-10 g/L, and the mass concentration of coenzyme self-sufficient chassis cells is 5-15 g/L.

Furthermore, the mass ratio of hydroxysteroid dehydrogenase 7b-HSDH wet bacteria, glucose dehydrogenase GDH wet bacteria, and coenzyme self-sufficient chassis cells is preferably 2:1:2.

The concentration of the substrate 7-ketolithocholic acid 7-KLCA is 10-40 g/L, so that it is in a supersaturated state in the reaction system.

Hydroxysteroid dehydrogenase 7b-HSDH can be obtained with reference to patent CN 109182284 A or other public documents or patents, and is a wild type or mutant. The recombinant genetic engineering bacteria containing the coding gene of the hydroxysteroid dehydrogenase 7b-HSDH are cultured, and the expression of the hydroxysteroid dehydrogenase 7b-HSDH is induced to obtain wet bacteria of the hydroxysteroid dehydrogenase 7b-HSDH, and further obtain wet enzyme liquid, or separate and purify to obtain pure protein.

The present invention also provides the use of a coenzyme self-sufficient chassis cell containing NAD kinase or a mutant thereof in the biosynthesis of a remedipam intermediate. The method of the use is as follows: the coenzyme self-sufficient chassis cell is introduced into a multi-enzyme reaction system, NAD+ is converted into NADP+, glucose dehydrogenase GDH reduces NADP+ to generate a coenzyme NADPH, which is supplied to an imine reductase IRED, and substrates pyridine-2,3-diamine and 4-oxopiperidone hydrochloride are reduced to generate a remedipam intermediate precursor compound II.

The intermediate precursor compound II of Remigipam further reacts with N,N'-carbonyldiimidazole CDI to generate compound II.

The reaction formula is shown below:

In the above reaction process, the introduction of NAD kinase and its mutants converts a large amount of NAD+ in the cell that is not related to the reaction into NADP+, providing sufficient and necessary NADP(H) for the synthesis route and providing energy for the reaction. It not only overcomes the problem of additional addition of NADPH in the biosynthetic pathway, consumption of pure NADPH, and high production cost, but also improves the catalytic efficiency.

Specifically, the preferred application method is as follows:

An enzyme mixed system is formed using imine reductase IRED, glucose dehydrogenase GDH, NAD kinase or its mutant contained in coenzyme self-sufficient chassis cells as catalysts, pyridine-2,3-diamine and 4-oxopiperidone hydrochloride are used as substrates, glucose, coenzyme NAD and ATP are added to construct a reaction system, and a reduction reaction is carried out to generate a remegipan intermediate precursor compound II.

Furthermore, in the reaction system, the concentration of substrate pyridine-2,3-diamine is 3 to 20 g/L, the concentration of 4-oxopiperidone hydrochloride is 1 to 10 g/L, and the concentration of glucose is 3 to 20 g/L.

Preferably, the molar ratio of pyridine-2,3-diamine to 4-oxopiperidone hydrochloride is 2 to 10:1.

Furthermore, the solvent of the reaction system is a pH 6.5-8 buffer solution containing 5% by volume of DMAO, preferably a pH 7-8, 50-100 mM phosphate buffer solution containing 5% by volume of DMAO.

The reaction temperature is 25 to 50° C., preferably 25 to 30° C. The reaction time is 10 to 15 hours.

In the reaction system, the concentration of coenzyme NAD is 1-15 mM, and the concentration of ATP is 1-15 mM.

After the reaction is completed, the reaction solution is separated and purified to obtain a pure product of the intermediate precursor compound II of Remegipam.

The catalyst includes imine reductase IRED and glucose dehydrogenase GDH, and the catalyst can be in the form of enzyme or bacterial cell. The enzyme includes free enzyme, immobilized enzyme, including purified enzyme, crude enzyme, fermentation broth, carrier-immobilized enzyme, cell fragments, etc.; the bacterial cell includes living bacterial cells and/or dead bacterial cells.

The coenzyme self-sufficient chassis cells can be added to the reaction system in the form of bacterial cells, or in the form of crude enzyme liquid after cell crushing or pure enzyme after purification.

The catalyst is preferably in the form of wet bacteria or crude enzyme solution. When the catalyst is in the form of wet bacteria, in the reaction system, the mass concentration of the imine reductase IRED wet bacteria is 10-40 g/L, and the mass concentration of the coenzyme self-sufficient chassis cells is 5-15 g/L. Glucose dehydrogenase GDH is added in the form of dry powder with a mass concentration of 0.5-1 g/L.

Imine reductase IRED can be obtained by referring to patent CN116813612A. The recombinant genetic engineering bacteria containing the coding gene of the imine reductase IRED are cultured, and the expression of the imine reductase IRED is induced to obtain wet bacteria of the imine reductase IRED, which are further crushed to obtain wet enzyme liquid, or separated and purified to obtain pure protein.

The beneficial effects of the present invention are:

1. The present invention can improve the specific enzyme activity of key enzymes by modifying the key amino acid residues of NAD kinase NADK. The enzyme activity of the constructed NAD kinase mutant is significantly improved compared with the wild-type enzyme, which can effectively reduce the use of cells and thus reduce costs. It can be applied to the industrial production of the biosynthesis of pranlukast intermediate 3AHAP, ursodeoxycholic acid UDCA and rimeglam intermediates.

2. The present invention uses a coenzyme self-sufficient chassis cell containing NAD kinase NADK to introduce a multi-enzyme reaction system, catalyzes NAD to generate NADP to supply the multi-enzyme system to generate expensive cofactor NADPH, and provides the required NADP (H) for the multi-enzyme reaction system to catalyze the generation of pranlukast intermediate 3AHAP, ursodeoxycholic acid UDCA and remegipan intermediate, without adding pure NADPH to the reaction system, thereby reducing production costs and improving catalytic efficiency. Compared with the problems of large pollution, high cost and low reaction rate in the prior art, the biosynthetic pathway provided by the present invention is green and safe, has high atomic economy, low cost, mild and effective, high catalytic efficiency, and has great industrial value.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a reaction conversion diagram of NAD+, NADP+, NADH, and NADPH.

Figure 2 is a map of the CgNADK-pCDFDuet recombinant expression vector.

FIG. 3 is a graph showing the protein content standard curve in Example 5.

FIG4 is a bar chart comparing the relative enzyme activities of pure enzymes of NAD kinase and its mutants.

FIG5 is a bar graph comparing the conversion rates of the substrate m-nitroacetophenone in the biosynthesis of the pranlukast intermediate 3AHAP under different multi-enzyme systems.

FIG6 is a bar graph showing the conversion rate of the substrate 7-ketolithocholic acid 7-KLCA in the biosynthesis of ursodeoxycholic acid UDCA under different multi-enzyme systems.

FIG. 7 is a bar graph showing the conversion rate comparison of the intermediate compound II in the biosynthesis of Remigipam under different multi-enzyme systems.

DETAILED DESCRIPTION

The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

The enzyme activity of the present invention includes the specific enzyme activity and the characteristics of the enzyme activity.

Capital letters herein represent amino acids as are known to those skilled in the art, and according to the present application, they represent the corresponding amino acid residues.

The experimental methods in the present invention are all conventional methods unless otherwise specified. For details of gene cloning operations, please refer to "Molecular Cloning Experiment Guide" compiled by J. Sambrook et al.

The reagents and raw materials used in the present invention are commercially available.

The positive improvement effect of the present invention is that the enzyme activity of the NAD kinase mutant of the present invention is improved, which is conducive to industrial production.

Description of the sequence listing:

SEQ ID NO. 1 is the amino acid sequence of NAD kinase annotated from CgNadK.

SEQ ID NO. 2 is the amino acid sequence of NAD kinase mutant I (G170R).

SEQ ID NO. 3 is the amino acid sequence of NAD kinase mutant II (G170R/F259A).

SEQ ID NO. 4 is the nucleotide sequence of NAD kinase (CgNadK).

The NADP (H) detection kit used in the embodiment was purchased from EnzyChromTM, coenzyme NADP and NAD, ATP, chromatographic grade methanol and acetonitrile, o-phthalaldehyde, and N-acetyl-L-cysteine were all purchased from J&K Chemical Technology Co., Ltd.; streptomycin, kanamycin, and IPTG were purchased from Shanghai Shengong; yeast powder and peptone were purchased from Oxoid; glucose-6 phosphate and glucose-6 phosphate dehydrogenase were purchased from Shanghai Ruji Biotechnology Development Co., Ltd.; plasmid extraction kit and DNA purification and recovery kit were purchased from Hangzhou Qingke Zixi Biotechnology Co., Ltd.; one-step cloning kit was purchased from Novozyme Co., Ltd.; E. coli BL21 (DE3), plasmid pET-28a (+), pCDFDuet-1 and whole gene synthesis were completed by Shengong Biotechnology (Shanghai) Co., Ltd.; DNA markers, low molecular weight standard proteins, and protein precast gels were purchased from Beijing GenStar Co., Ltd.; ClonExpress II OneStep Cloning Kit seamless cloning kit was purchased from Nanjing Novozyme Biotechnology Co., Ltd.; pfu DNA polymerase and DpnI endonuclease were purchased from Thermo Fisher Scientific (China) Co., Ltd.; primer synthesis and sequence sequencing were completed by Hangzhou Qingke Zixi Biotechnology Co., Ltd. The use of the above reagents refers to the product instructions.

The reagents used in the downstream catalytic process, including m-nitroacetophenone, 3-amino-2-hydroxyacetophenone, 7-ketolithocholic acid 7-KLCA, ursodeoxycholic acid UDCA, pyridine-2,3-diamine, and 4-oxopiperidone hydrochloride, were purchased from Aladdin Reagent (Shanghai, China), and other commonly used reagents were purchased from Sinopharm Chemical Reagent Co., Ltd.

The following examples detect the products of the reactions by high performance liquid chromatography (HPLC) and analyze the products.

The HPLC analysis method of the product 3-amino-2-hydroxyacetophenone is: chromatographic column/ phenyl; column temperature/40℃; flow rate/1mL/min; detection wavelength/235nm; mobile phase: 13.5mM trifluoroacetic acid: pure acetonitrile = 75:25.

The HPLC analysis method of ursodeoxycholic acid UDCA is as follows: chromatographic column/QSC18, 5μm, 4.6×250mm; column temperature/30°C; flow rate/1mL/min; detection wavelength/210nm; mobile phase: 20mM phosphate buffer: pure acetonitrile = 40:60.

Isolation and purification method of Remigipam intermediate precursor compound II: After the reaction is completed, the pH is adjusted to 10 with a saturated Na ₂ CO ₃ solution, and extracted with ethyl acetate (3*1 mL). Then dried with anhydrous Na ₂ SO ₄ , the organic solvent is removed by vacuum distillation, and the crude product is purified by silica gel column chromatography to obtain the calculated mass of compound II.

The HPCL analysis method of NADP(H) is as follows: chromatographic column/QSC18, 5μm, 4.6×250mm; column temperature/30℃; flow rate/1mL/min; detection wavelength/261nm; injection volume is 10μL; mobile phase: the gradient mobile phase contains two solvents (PBS buffer with a pH of 6.8 and a concentration of 50mM and chromatographic grade methanol), and the program setting of the gradient mobile phase is shown in Table 1. The method for preparing the mobile phase of PBS buffer with a pH of 6.8 and a concentration of 50mM is as follows: prepare 1L of 50mM Na2HPO4 aqueous solution (alkaline) and NaH2PO4 (acidic) aqueous solution respectively, then slowly add Na2HPO4 aqueous solution (alkaline) to 1L NaH2PO4 aqueous solution (acidic), and put the pH meter detection probe into the beaker at the same time until the pH is 6.8. After the buffer is prepared, filter it through the water film, put it into a 2L blue-mouth bottle, remove bubbles by ultrasonic for 30min, let it stand to room temperature, and set aside.

Table 1

High-throughput NADP(H) detection method:

Inoculate the transformant into a 96-well plate and culture it in a 37°C constant temperature shaker for 12-16 hours at a shaker speed of 200 rpm. Then transfer the seed culture solution of the 96-well plate to the 96-well plate fermentation medium, add IPTG inducer when OD600 = 0.4-0.7, and culture it in a 28°C constant temperature shaker for 12-16 hours at a shaker speed of 200 rpm. Centrifuge the cultured 96-well fermentation solution at 4000 rpm for 10 minutes, discard the supernatant, and collect the bacteria. Resuspend the collected bacteria with pH 7.5 phosphate buffer and iATPSnFR1.1 bacterial solution to make a 50 g/L suspension. Weigh a small amount of ATP powder into a 1.5 mL EP tube, and add pH 7.5 phosphate buffer to make a 100 mM ATP solution. Take 90 μL of the prepared 50 g/L iATPSnFR1.1 suspension and add it to a black, flat 96-well plate with an opaque bottom, add 10 μL of 100 mM ATP solution and react for 3 minutes, and measure the fluorescence intensity at an excitation wavelength of 485 nm and an emission wavelength of 515 nm.

Example 1: Construction of recombinant coenzyme self-sufficient chassis cells

Escherichia coli BL21 (DE3) was used as a chassis cell, a plasmid pCDFDuet was selected, and a restriction endonuclease was used to cut the plasmid and the wild-type NAD kinase CgNADK derived from Corynebacterium glutamicum (GenBank No.: WP_011014345, the amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is SEQ ID NO.4), so that the pCDFDuet plasmid and the NAD kinase nucleotide sequence produce complementary sticky ends at specific cutting sites, and a DNA ligase was used to connect the sticky ends of the two to form a CgNADK-pCDFDuet recombinant expression vector. The ligated recombinant expression vector was transformed into Escherichia coli BL21 (DE3) chassis cells. The specific method was as follows: 10 μL of the ligated CgNADK-pCDFDuet recombinant expression vector was added to the competent Escherichia coli BL21 (DE3) cells, ice-bathed for 30 minutes, and then placed in a 42°C water bath for heat shock for 90 seconds. Then, 600 μL of LB culture medium was added, and the cells were cultured in a 37°C constant temperature shaker for 1 hour. After low-speed centrifugation, 500 μL of supernatant was removed, and the cells were mixed with a pipette and evenly spread on the culture dish. The cells were cultured for 14 hours, and the positive colonies were picked and cultured in a streptomycin-resistant liquid LB culture medium for 8 hours, and then the nucleic acid sequence was sequenced. The sequencing was verified to be correct to obtain the recombinant coenzyme self-sufficient chassis cells for the subsequent expression of the recombinase.

Example 2: Preparation of genetically engineered bacteria

The above-constructed coenzyme self-sufficient chassis cells containing NAD kinase CgNADK and wild-type nitroreductase (nbzA), hydroxylamine benzene mutase (habA), hydroxysteroid dehydrogenase (7b-HSDH), imine reductase (IRED) and glucose dehydrogenase (GDH) preserved in this laboratory were activated by streaking and sequenced for subsequent expression of recombinant enzymes. The coding genes of wild-type nitroreductase (nbzA), hydroxylamine benzene mutase (habA) and glucose dehydrogenase (GDH) are disclosed in Chinese patent CN116426578A. According to the method of CN116426578A, the coding gene is inserted into a recombinant expression vector and then transferred into the host bacterium E.coli BL21 (DE3) for transformation to prepare a recombinant genetic engineering bacterium containing the genes encoding the nitrobenzene reductase nbzA, hydroxylamine benzene mutase habA and glucose dehydrogenase GDH.

Hydroxysteroid dehydrogenase (7b-HSDH) was obtained according to patent CN 109182284 A and was wild type. Imine reductase (IRED) was obtained according to patent CN116813612A.

LB liquid culture medium composition: peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, dissolved in water and fixed to volume, sterilized at 121℃ for 20min, and set aside.

After the engineered bacteria with correct sequencing were activated by streaking on a plate, a single colony was inoculated into 10 ml LB liquid culture medium containing 50 μg/ml kanamycin or streptomycin, and cultured at 37°C for 10-12 hours. The inoculation amount was transferred to 100 ml of fresh LB liquid culture medium containing 50 μg/ml kanamycin or streptomycin at 2%, and the culture was shaken at 37°C until OD600 reached about 0.8, then cooled to 30°C, IPTG was added to a final concentration of 0.5 mM, and induced culture was carried out for 16 hours. After the culture was completed, the culture solution was centrifuged at 8000 rpm for 10 minutes, the supernatant was discarded, the bacteria were collected, and stored in a -20°C refrigerator for standby use. The bacteria collected after the culture was washed twice with 50 mM pH 8.0 phosphate buffer, then resuspended in 50 mL pH 8.0 phosphate buffer, homogenized and broken, and the broken solution was centrifuged to remove the precipitate to obtain a crude enzyme solution containing recombinant NADK enzyme for protein purification and subsequent reactions.

Example 3 Construction of NAD kinase mutants (positions 110, 170, and 259)

Based on the wild-type CgNadK sequence described in Example 1, positions 110, 170, and 259 were mutated. The PCR primer sequences were designed for the mutants with mutations at positions 110, 170, and 259 of the mutated NAD kinase sequence, and the design was performed according to the mutation order of the mutation sites as shown in Tables 2 to 4:

Table 2

Table 3

Serial number Primer name Primer sequences 1 170AF CCTGAATCGCCGCGCAGTTTTGGATGCAACG 2 170RF CCTGAATCGCCGCCGAGTTTTGGATGCAACG 3 170NF CCTGAATCGCCGCAATGTTTTGGATGCAACG 4 170DF CCTGAATCGCCGCGATGTTTTGGATGCAACG 5 170CF CCTGAATCGCCGCTGTGTTTTGGATGCAACG 6 170QF CCTGAATCGCCGCCAGGTTTTGGATGCAACG 7 170EF CCTGAATCGCCGGAAGTTTTTGGATGCAACG 8 170GF CCTGAATCGCCGCGGTTTTGGATGCAACG 9 170HF CCTGAATCGCCGCCATGTTTTGGATGCAACG 10 170IF CCTGAATCGCCGCATTGTTTTGGATGCAACG 11 170LF CCTGAATCGCCGCCTGGTTTTGGATGCAACG 12 170KF CCTGAATCGCCGCAAAGTTTTGGATGCAACG 13 170MF CCTGAATCGCCGCATGGTTTTGGATGCAACG 14 170PF CCTGAATCGCCGCCCGGTTTTGGATGCAACG 15 170SF CCTGAATCGCCGCAGCGTTTTGGATGCAACG 16 170TF CCTGAATCGCCGCACCGTTTTGGATGCAACG 17 170WF CCTGAATCGCCGCTGGGTTTTGGATGCAACG 18 170YF CCTGAATCGCCGCTATGTTTTGGATGCAACG 19 170VF CCTGAATCGCCGCGTTGTTTTGGATGCAACG 20 170R GCGGCGATTCAGGTTTTCGATGCTCACTTCG

Table 4

The PCR (25 μL) amplification system is:

25 μL of 2×PCR buffer, 1.5 μL of upstream and downstream primers, 1 μL of template plasmid, 1 μL of dNTP, 1 μL of high-fidelity enzyme, add ddH ₂ O to make up to 50 μL.

The PCR amplification program is:

(1) Pre-denaturation at 95°C for 5 min, (2) Denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 5 min, repeat 30 cycles, (3) Extension at 72°C for 10 min, (4) Storage at 4°C.

After PCR, 5 μL of the amplified product was analyzed by nucleic acid gel electrophoresis. The PCR product with clear target band was added with 2 μL Dpn I endonuclease and digested at 37°C for 1 hour. After the reaction was completed, the clean up was transformed into BL21 competent cells, spread on LB solid medium containing 50 μg/mL kanamycin, cultured at 37°C overnight, and the bacteria were harvested to obtain transformants containing mutants.

Example 4 High-throughput screening of mutant libraries

The screening was carried out according to the following experimental steps:

The transformants obtained in Example 2 were inoculated into a 96-well plate and cultured in a 37°C constant temperature shaker for 12-16 hours at a shaker speed of 200 rpm. The seed culture solution of the 96-well plate was then transferred to a 96-well plate fermentation medium, and IPTG inducer was added at OD ₆₀₀ = 0.4-0.7, and cultured in a 28°C constant temperature shaker for 12-16 hours at a shaker speed of 200 rpm. The cultured 96-well fermentation solution was centrifuged at 4000 rpm for 10 minutes, the supernatant was discarded, and the bacteria were collected. The collected bacteria were resuspended with a pH 7.5 phosphate buffer and iATPSnFR1.1 bacterial solution to form a 50 g/L suspension. A small amount of ATP powder was weighed and poured into a 1.5 mL EP tube, and a pH 7.5 phosphate buffer was added to form a 100 mM ATP solution. Take 90 μL of the prepared 50 g/L iATPSnFR1.1 suspension and add it to a black, flat 96-well plate with an opaque bottom. Add 10 μL of 100 mM ATP solution and react for 3 minutes. Measure the fluorescence intensity at an excitation wavelength of 485 nm and an emission wavelength of 515 nm to screen positive clones.

The positive clones obtained from the primary screening were cultured and used for secondary screening. The positive mutants were obtained according to the method described in Example 1. The collected recombinant bacteria of NAD kinase and its mutants were washed twice with 50 mM pH 8.0 phosphate buffer, then resuspended in 50 mL pH 8.0 phosphate buffer, homogenized and disrupted, and the disrupted liquid was centrifuged to remove the precipitate to obtain a crude enzyme solution containing NAD kinase and its mutants for protein purification.

Example 5 Protein purification of NAD kinase and its mutants

(1) Reagent preparation: Ni column equilibration buffer (1 L): 50 mM pH 7 Tris-HCL, 200 mM NaCl, 50 mM imidazole, 2 mM 2-mercaptoethanol; eluent (1 L): 50 mM pH 7 Tris-HCL, 200 mM NaCl, 250 mM imidazole, 2 mM 2-mercaptoethanol; 20% (v/v) ethanol (200 mL).

(2) Sample processing: The crude enzyme solution of NAD kinase and its mutants obtained in Example 5 was taken as a sample for protein purification.

(3) Rinse the protein purifier tubing with ultrapure water at a flow rate of 4 mL/min for 10 min.

(4) Connect the Ni column to the instrument and flush it with the balance solution at a flow rate of 4 mL/min until the baseline is stable. (5) Load the sample at a flow rate of 2 mL/min, with a sample volume of about 20 mL per time. Observe the remaining sample volume to avoid inhaling air.

(6) After loading the sample, continue flushing with the equilibration solution at a flow rate of 2 mL/min until the baseline is stable.

(7) After the baseline is stable, rinse with eluent at a flow rate of 2 mL/min and observe the changes in UV value. When the UV value shows a rising peak, collect the protein in a pre-cooled 10 mL centrifuge tube after 90 seconds and place it on ice. (8) Continue to rinse with eluent until the baseline is stable.

(9) Rinse with the equilibration solution to stabilize the baseline again and continue with the next sample loading and purification.

(10) After completion, flush the column with 20% ethanol at a flow rate of 5 mL/min for 20 min to preserve the column. The entire purification process must be kept at a low temperature to avoid enzyme inactivation.

Example 6 Comparison of Enzyme Activities of NAD Kinase and Mutants

The positive clones (G170R; G110W; G110E; G110R; F259G; F259A; F259H); (G170R/F259H, G170R/F259G; G170R/F259A; G170R/G110W; G170R/G110E; G170R/G110R) obtained by the initial screening in Example 4 and purified by the protein in Example 5 were rescreened, and the catalytic efficiency of the mutants was detected by HPLC.

The protein concentration of the purified NAD kinase and its positive clones was determined using a BCA kit. The principle is that Cu ²⁺ is reduced to Cu ²⁺ in an alkaline environment, which can form a blue complex with the BCA reagent and have a specific absorption value at 562nm. The protein content can be calculated by referring to the standard curve. The specific operation method is as follows:

1. Drawing of the standard curve.

(1) Add the reagents according to Table 5 to a clean standard 96-well transparent plate.

Table 5

Hole No. 1 2 3 4 5 6 7 8 Protein standard solution (μL) 0 1 2 4 8 12 16 20 Deionized water (μL) 20 19 18 16 12 8 4 0 Corresponding protein content (ug) 0 0.5 1.0 2.0 4.0 6.0 8.0 10.0

(2) Preparation of BCA working solution

Add BCA reagent A and BCA reagent B in a volume ratio of 50:1 into a 2 mL EP tube, mix well, and set aside.

(3) Add the prepared working solution to the first 8 holes, immediately place in the microplate reader, shake slightly and keep at 37°C for 30 minutes, and then measure the absorbance at 562nm. A standard curve can be drawn based on the absorbance corresponding to the standard protein concentration, as shown in Figure 2. The standard curve equation is y = 0.19397x + 0.03427.

The catalytic efficiency of NAD kinase and its mutants was compared by measuring the amount of NADP produced by HPLC. Enzyme activity definition: The amount of enzyme required to catalyze the production of 1 μmol NADP from NAD and ATP per minute at 35°C and pH 7.5 is 1 enzyme activity unit (U).

Specific enzyme activity (U/g): the amount of enzyme activity contained in each gram of pure enzyme.

Reaction system (1 ml): 200 mM pH 7.5 Tris-HCL, 2 mM NAD, 10 mM ATP, 10 mM MgCl2, 0.1 g/L pure enzyme of NAD kinase and its mutants. After 10 min of mixing and reaction, the amount of NADP generated was detected in the liquid phase to calculate the enzyme activity. The protein concentration of NAD kinase and its mutants was obtained according to the standard curve (Figure 2), and the specific enzyme activity of NAD kinase and its mutants was calculated. Each group of samples was carried out three times in parallel. The specific enzyme activity results are shown in Figure 4 and Table 6.

Table 6

As shown in Figure 4 and Table 6, the specific enzyme activity of the mutant pure enzymes is greatly improved compared with the wild-type NAD kinase. Among them, the G170R/F259A mutant No. 10 has the highest specific enzyme activity. G170R/G110W, G170R/G110E, and G170R/G110R also have high specific enzyme activities.

Example 7 Verification of the catalytic efficiency of adding NAD kinase

(1) The catalytic efficiency of the reaction with and without NAD kinase was compared by measuring the amount of 3-amino-2-hydroxyacetophenone produced. The 2 ml reaction system includes: 20 g/L substrate m-nitroacetophenone, 10 mM ATP, 8 mM NAD, 50 mM pH 8.0 phosphate buffer, 50 g/L glucose, 5 g/L nitroreductase crude enzyme solution, 35 g/L hydroxylamine benzyl mutase crude enzyme solution and 60 g/L glucose dehydrogenase crude enzyme solution, 15 g/L CgNADK kinase or the optimal mutant (G170R/F259A mutant No. 10) coenzyme self-sufficient chassis cells (for the control nbzA-habA-GDH, an equal amount of PBS buffer was added), the reaction temperature was set at 30°C, and after reacting for 2 hours, a sample of the reaction solution was taken for treatment, and the concentration of 3-amino-2-hydroxyacetophenone was determined by HPLC and the 3AHAP yield was calculated. The yield and conversion rate results are shown in Table 7 and Figure 5.

Table 7

It can be seen from the results in Table 7 and Figure 5 that the catalytic efficiency was greatly improved after the introduction of NAD kinase CgNADK in the multi-enzyme system. After 2 hours of reaction, the yield increased from the original 1.59 g/L to 2.52 g/L, and the substrate conversion rate increased from 7.9% to 12.6%; and after the introduction of the CgNADK mutant (G170R/G259A) in the multi-enzyme system, the yield increased from the original 1.59 g/L to 5.79 g/L, and the substrate conversion rate reached the highest 29%.

(2) The catalytic efficiency of the reaction with and without NAD kinase was compared by measuring the amount of UDCA produced. The 10 ml reaction system included: 10 g/L substrate 7-ketolithocholic acid 7-KLCA, 100 mM pH 8.0 phosphate buffer, 20 g/L glucose, 10 mM ATP, 8 mM NAD, 10 g/L hydroxysteroid dehydrogenase 7b-HSDH bacteria, 5 g/L glucose dehydrogenase bacteria and 10 g/L coenzyme self-sufficient chassis cells containing CgNADK kinase or its optimal mutant (G170R/F259A mutant No. 10) (the control 7b-HSDH-GDH was added with an equal amount of PBS buffer), the reaction temperature was set at 30°C, and after 15 min of reaction, the reaction solution was sampled for treatment, and the concentration of UDCA was determined by HPLC and the UDCA yield was calculated. The yield and conversion rate results are shown in Table 8 and Figure 6.

Table 8

It can be seen from the results in Table 8 and Figure 6 that the catalytic efficiency was greatly improved after the coenzyme self-sufficient chassis cells containing NAD kinase CgNADK were introduced into the multi-enzyme system. After 15 minutes of reaction, the yield increased from the original 0.46 g/L to 5.05 g/L, and the substrate conversion rate increased from 4.6% to 50.5%; and after the CgNADK mutant (G170R/G259A) was introduced into the multi-enzyme system, the yield increased from the original 0.46 g/L to 9.06 g/L, and the substrate conversion rate reached the highest 90.6%.

(3) The catalytic efficiency of the reaction with and without NAD kinase was compared by measuring the amount of the intermediate compound II produced. The 10 ml reaction system includes: 5.46 g/L pyridine-2,3-diamine, 1.36 g/L 4-oxopiperidone hydrochloride, 100 mM pH 7.5 phosphate buffer, 5% (v/v) DMSO, 3.6 g/L glucose, 10 mM ATP, 8 mM NAD, 0.39 g imine reductase bacteria IRED and 5 mg glucose dehydrogenase powder, 0.15 g coenzyme self-sufficient chassis cells containing CgNADK kinase or its optimal mutant (G170R/F259A mutant No. 10) (for the control IRED-GDH, an equal amount of PBS buffer and 1 mg NADP were added, and NAD and ATP were not added). The reaction temperature was set to 25° C. After the reaction for 12 hours, the reaction solution sample was separated and purified to calculate the product mass, yield and conversion rate. The results are shown in Table 9 and Figure 7.

Table 9

It can be seen from the results in Table 9 and Figure 7 that the catalytic efficiency was improved after the coenzyme self-sufficient chassis cells containing NAD kinase CgNADK were introduced into the multi-enzyme system. After 12 hours of reaction, the yield increased from the original 1.45 g/L to 1.67 g/L, and the substrate conversion rate increased from 21.3% to 24.5%; and after the CgNADK mutant (G170R/G259A) was introduced into the multi-enzyme system, the yield increased from the original 1.45 g/L to 2.54 g/L, and the substrate conversion rate reached the highest 37.2%.

Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the protection scope of the invention.

Claims (10)

1. An NAD kinase derived from Corynebacterium glutamicum, the amino acid sequence of which is shown in SEQ ID NO. 1.

2. An NAD kinase mutant is characterized in that the NAD kinase mutant is obtained by single-point mutation or multi-point superposition mutation at 110 th, 170 th and 259 th of an amino acid sequence shown in SEQ ID NO. 1.

3. The NAD kinase mutant according to claim 2, characterized in that the mutation is a point mutation of one or more than two of the following:

(1) Mutating glycine at position 170 of the amino acid sequence shown in SEQ ID NO.1 into arginine, lysine, alanine, histidine or proline;

(2) Mutating phenylalanine at 259 th position of an amino acid sequence shown in SEQ ID NO.1 into glycine, alanine or histidine;

(3) The 110 th glycine of the amino acid sequence shown in SEQ ID NO.1 is mutated into tryptophan, glutamic acid or arginine.

4. The NAD kinase mutant according to claim 2, characterized in that the NAD kinase mutant is one of the following:

(a) Mutation of glycine 170 to arginine and mutation of phenylalanine 259 to alanine in the amino acid sequence shown in SEQ ID NO. 1;

(b) Mutating the 170 th glycine of the amino acid sequence shown in SEQ ID NO.1 into arginine, and mutating the 110 th glycine into tryptophan;

(c) Mutating the 170 th glycine of the amino acid sequence shown in SEQ ID NO.1 into arginine, and mutating the 110 th glycine into glutamic acid;

(d) The 170 rd glycine of the amino acid sequence shown in SEQ ID NO.1 is mutated into arginine, and the 110 th glycine is mutated into arginine.

5. A gene encoding the NAD kinase or a mutant thereof according to any one of claims 1-4.

6. A recombinant expression vector comprising a gene encoding the NAD kinase or a mutant thereof according to claim 4.

7. The coenzyme self-contained chassis cell constructed by the recombinant expression vector according to claim 5, which contains the gene encoding the NAD kinase or the mutant thereof.

8. Use of the coenzyme self-sufficient basal cell of claim 7 in biosynthesis of pranlukast intermediate 3AHAP, ursodeoxycholic acid UDCA or ramelteon intermediate compound II.

9. The application according to claim 8, characterized in that the method of application is: the coenzyme self-sufficient chassis cells are introduced into a multienzyme reaction system, NAD+ is converted into NADP+, glucose dehydrogenase GDH reduces NADP+ to generate coenzyme NADPH, and the coenzyme NADPH provides reducing capability for synthesizing a pharmaceutical intermediate pranlukast intermediate 3AHAP, ursodeoxycholic acid UDCA or a Ruimepam intermediate compound II.

10. The application according to claim 9, characterized in that the method of application is one of the following:

introducing a multienzyme reaction system into a coenzyme self-contained chassis cell, converting NAD+ into NADP+, reducing NADP+ by glucose dehydrogenase GDH to generate coenzyme NADPH, supplying nitroreductase to couple hydroxylamine phenylmutase, and biologically converting substrate m-nitroacetophenone into a pranlukast intermediate 3-amino-2-hydroxyacetophenone;

the reaction formula is shown as follows:

(II) introducing coenzyme self-sufficient type chassis cells into a multienzyme reaction system, and introducing NAD ⁺ Conversion to NADP ⁺ Glucose dehydrogenase GDH reduces NADP+ to generate coenzyme NADPH, supplies hydroxysteroid dehydrogenase 7b-HSDH, and biologically converts substrate 7-ketolithocholic acid 7-KLCA into ursodeoxycholic acid UDCA;

the reaction formula is shown as follows:

Introducing coenzyme self-contained chassis cells into a multienzyme reaction system, converting NAD+ into NADP+, reducing NADP+ by glucose dehydrogenase GDH to generate coenzyme NADPH, supplying imine reductase IRED, and reducing substrates pyridine-2, 3-diamine and 4-oxo-piperidone hydrochloride to generate a precursor compound II of the remigermpam intermediate;

the reaction formula is shown as follows: