CN117143787A - Preparation method and application of transgenic microorganism and (S) -nicotine - Google Patents

Abstract

A preparation method and application of transgenic microorganism and (S) -nicotine, relating to the technical field of bioengineering; the transgenic microorganism contains an exogenous gene a and an exogenous gene b; the exogenous gene a is used for expressing dehydrogenase, and the exogenous gene b is used for expressing N-methyltransferase. The transgenic microorganism for synthesizing (S) -nicotine can convert the myosmine biological method into (S) -nicotine in the environment with glucose and SAM through transferring exogenous genes expressing dehydrogenase and N-methyltransferase in the microorganism, has high conversion efficiency and high safety, has extremely high chiral purity, does not need eschweiler-clarke methylation organic synthesis reaction, and avoids the use of high-risk chemicals such as formic acid, formaldehyde and the like.

Description

Preparation method and application of transgenic microorganism and (S) -nicotine

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a preparation method and application of transgenic microorganism and (S) -nicotine.

Background

Nicotine (1-methyl-2- (2-pyridyl) pyrrolidine) is alkaloid existing in Solanaceae plant (Solanum), and is one of main components in tobacco, wherein the average content in tobacco is 4%, and the content of alkaloid in tobacco is 95%. The high-purity nicotine is colorless oily liquid at room temperature, has unstable property, is easy to decompose by visible light and oxidize, and is black brown after oxidation. The tobacco smoke is caused by other tobacco compounds besides nicotine, and the nicotine can be used as a natural pesticide, a smoking cessation drug and a nicotinic cholinergic agonist to exert pharmacological activity in the central and peripheral nervous systems, so that the tobacco smoke is stopped and chronic pain is relieved.

The nicotine molecule has 1 chiral center, i.e. chiral nicotine comprises two configurations of (R) -nicotine and (S) -nicotine. In recent years, the demand of natural nicotine ((S) -nicotine) in the domestic and foreign markets is increasing, because nicotine is widely applied to the fields of agriculture, medical industry, tobacco industry and the like, and especially (S) -nicotine with high purity and high chiral purity is always in demand. The price of nicotine also increases exponentially with its purity, especially chiral purity. At present, nicotine in the market is mainly extracted and chemically synthesized from tobacco, most of the nicotine extracted from the tobacco is natural nicotine, but the nicotine contains harmful impurities such as nitrosamine and the like, and the raw material tobacco is also affected by climate, land resources, period and other aspects; however, most of chemically synthesized nicotine is racemic nicotine, and an additional process is needed to further separate (S) -nicotine, which is complicated in process, high in cost and unfavorable for commercial production.

The biosynthesis method has simple process, mild reaction conditions and high specificity, and generally does not need toxic chemical components, thus being an ideal synthesis direction; recent scholars have shown that Myosmine (Myosmine) can be reduced to (S) -Nornicotine by Imine Reductase (IRED) with the aid of glucose dehydrogenase (Glucose dehydrogenase, GDH), reduced coenzyme NADH, etc., and then added with formic acid, formaldehyde to undergo Eschweiler-Clarke reaction to methylate the Nornicotine to (S) -nicotine. However, because GDH and NADH enzymes have high cost, hazardous materials such as formic acid, formaldehyde and the like are also needed, and the process is complex, the method brings a plurality of inconveniences to industrial production. Chinese patent CN202010172401.0 also proposes that under the condition of the coenzyme circulation system, using coenzyme as a hydrogen donor, using imine reductase as a catalyst, catalytic reduction of myosmine to (S) -nornicotine, and methylation of the (S) -nornicotine to obtain the (S) -nicotine, wherein the methylation reaction is a mixed reaction of (S) -nornicotine, formaldehyde and formic acid, and the chemical reaction of the coenzyme circulation system and methylation is also required. At present, the company also develops a dehydrogenase mutant L283V/L286V (CN 202111369684.9), which is 6-phosphogluconate dehydrogenase (MesPDH), but has higher imine reductase and dehydrogenase activity, and can catalyze the myosmine to generate the (S) -nornicotine with high chiral purity under the condition of only adding glucose, so that the product purity and conversion rate are higher, the problem of higher GDH and NADH enzyme cost is solved, but the biological methylation reaction of the (S) -nornicotine cannot be realized to form the (S) -nicotine. N-methyltransferase (CNMT) is present in most plants, and part of fish also contains this gene, CNMT is capable of transferring methyl groups in S-adenosylmethionine (SAM) to the corresponding compounds in the presence of methyl donors such as S-adenosylmethionine. Common substrates applicable to CNMT at present are quinoline derivatives, indole derivatives and the like, but no biological methylation method of nornicotine has been reported.

Disclosure of Invention

In order to overcome the defects of the prior art, one of the purposes of the invention is to provide a transgenic microorganism for synthesizing (S) -nicotine, which can convert a myosmine biological method into (S) -nicotine, has high conversion efficiency and high safety, does not need eschweiler-clarke methylation organic synthesis reaction, and avoids the use of high-risk chemicals such as formic acid, formaldehyde and the like.

The second object of the present invention is to provide a use of the transgenic microorganism.

The invention also aims to provide a preparation method of the (S) -nicotine, which has the advantages of simple process, mild reaction conditions, high specificity and high conversion efficiency.

One of the purposes of the invention is realized by adopting the following technical scheme:

a transgenic microorganism for synthesizing (S) -nicotine, said transgenic microorganism comprising an exogenous gene a and an exogenous gene b; the exogenous gene a is used for expressing dehydrogenase, and the exogenous gene b is used for expressing N-methyltransferase.

Further, the dehydrogenase comprises an amino acid sequence shown in SEQ ID NO:2 or a homologous dehydrogenase derived from a MesPDH enzyme having a MesPDH enzyme activity and having a sequence at least 90% identical to the MesPDH enzyme by substitution, deletion or addition of one or more amino acids in the amino acid sequence defined by the MesPDH enzyme.

Further, the N-methyltransferase comprises an amino acid sequence as set forth in SEQ ID NO:4 or a second CNMT enzyme derived from a first CNMT enzyme having at least 90% identity to the first CNMT enzyme and having a first CNMT enzyme activity, which has been substituted, deleted or added with one or more amino acids in the amino acid sequence defined by the first CNMT enzyme.

Further, the nucleotide sequence of the exogenous gene a is shown as SEQ ID NO:1 is shown in the specification; the nucleotide sequence of the exogenous gene b is shown as SEQ ID NO: 3.

Further, the microorganism includes at least one of bacteria, yeast, filamentous fungi, cyanobacteria, and plant cells.

Further, the microorganism includes at least one of escherichia, salmonella, bacillus, acinetobacter, streptomyces, corynebacterium, campylobacter, methyl monad, rhodococcus, pseudomonas, rhodobacter, synechocystis, saccharomycetes, zygosaccharomyces, kluyveromyces, candida, hansenula, debaryomyces, mucor, pichia, torulopsis, aspergillus, basidiomycetes, brevibacterium, microbacterium, arthrobacter, citrobacter, escherichia, klebsiella, pantoea, salmonella, corynebacterium, clostridium and clostridium acetobutylicum.

The second purpose of the invention is realized by adopting the following technical scheme:

use of a transgenic microorganism for the synthesis of (S) -nicotine in a process for the preparation of (S) -nicotine.

The third purpose of the invention is realized by adopting the following technical scheme:

a method for preparing (S) -nicotine, comprising the steps of:

1) Obtaining said transgenic microorganism for synthesizing (S) -nicotine;

2) Taking myosmine, glucose and methyl donor as substrates, adding the transgenic microorganism in the step 1) to perform whole-cell catalytic reaction, and separating and purifying to obtain (S) -nicotine.

Further, the method for preparing the transgenic microorganism in the step 1) comprises the following steps:

loading the exogenous gene a and the exogenous gene b into the same plasmid to construct a double-gene expression plasmid, and then electrically transforming the double-gene expression plasmid into competent host microorganisms to culture to obtain the transgenic microorganisms; or alternatively, the first and second heat exchangers may be,

respectively loading the exogenous gene a and the exogenous gene b into plasmids to obtain a first recombinant plasmid and a second recombinant plasmid, and then, electrically transforming the first recombinant plasmid and the second recombinant plasmid into competent host microorganisms, and culturing to obtain the transgenic microorganisms; or alternatively, the first and second heat exchangers may be,

taking an exon of a microorganism as a knock-in target spot, synthesizing a homologous recombination gene containing exogenous gene a, exogenous gene b and an exon homology arm, and transferring the homologous recombination gene into a host microorganism to obtain the transgenic microorganism.

Further, the plasmid is a pET28a plasmid, the host microorganism is at least one of escherichia coli BL21 (DE 3), pichia pastoris and saccharomyces cerevisiae, and the exon is exon SS9.

Further, in the whole-cell catalytic reaction in the step 2), the methyl donor is SAM, the final concentration of the myosmine is 5-30mg/mL, and the mass ratio of the myosmine, the glucose and the SAM is 1: (2.7-4): (1.2-2), wherein the reaction temperature is 25-35 ℃;

the separation step in step 2) is: centrifuging the reaction solution to remove cell precipitate, regulating the pH value of the supernatant to 9-13, and adding an extractant for extraction to obtain an isolated product; the extractant is any one of dichloromethane, methyl tertiary butyl ether, normal hexane, ethyl acetate and tetrahydrofuran, and when the extractant is tetrahydrofuran, the extractant also comprises sodium chloride with the concentration of 100-300 g/L.

Compared with the prior art, the invention has the beneficial effects that:

the transgenic microorganism for synthesizing (S) -nicotine can convert the myosmine biological method into (S) -nicotine in the environment with glucose and SAM through transferring exogenous genes expressing dehydrogenase and N-methyltransferase in the microorganism, has high conversion efficiency and high safety, has extremely high chiral purity, does not need eschweiler-clarke methylation organic synthesis reaction, and avoids the use of high-risk chemicals such as formic acid, formaldehyde and the like.

The preparation method of (S) -nicotine has the advantages of simple process, mild reaction conditions, high specificity and high conversion efficiency.

Drawings

FIG. 1 is a graph showing comparison of HPLC analysis results of each reaction system in example 5 of the present invention.

FIG. 2 is an HPLC chart of the mixed standard in example 5 of the present invention.

FIG. 3 is an HPLC chart of a PDH+PCN reaction system in example 5 of the present invention.

FIG. 4 is an HPLC chart of the PDHCN-1 reaction system in example 5 of the present invention.

FIG. 5 is an HPLC chart of the PDHCN-2 reaction system in example 5 of the present invention.

FIG. 6 is an HPLC chart of DH+CN reaction system in example 5 of the present invention.

FIG. 7 is an HPLC chart of the DHCN reaction system in example 5 of the present invention.

Detailed Description

The present invention will be further described with reference to the following specific embodiments, and it should be noted that, on the premise of no conflict, new embodiments may be formed by any combination of the embodiments or technical features described below.

Example 1

A transgenic microorganism for synthesizing (S) -nicotine, said transgenic microorganism comprising an exogenous gene a and an exogenous gene b; the exogenous gene a is a dehydrogenase gene (MesPDH), and the nucleotide sequence of the exogenous gene a is shown in SEQ ID NO:1, which is used for expressing dehydrogenase, wherein the dehydrogenase is MesPDH enzyme, and the amino acid sequence of the dehydrogenase is shown as SEQ ID NO:2 is shown in the figure; the exogenous gene b is an N-methyltransferase gene (CNMT), and the nucleotide sequence of the exogenous gene b is shown in SEQ ID NO:3, for expressing an N-methyltransferase having an amino acid sequence as set forth in SEQ ID NO: 4.

The transgenic microorganism described in this example is a transgenic microorganism carrying a single plasmid, and the preparation method thereof comprises the steps of:

1) The dehydrogenase gene (MesPDH) and the N-methyltransferase gene (CNMT) were ligated into the pET28a plasmid to obtain plasmid pET32a-MesPDH containing the dehydrogenase gene, plasmid pET28a-CNMT containing the N-methyltransferase gene, and plasmid pET28a-CNMT-MesPDH containing double gene expression.

2) The pET32a-MesPDH, pET28a-CNMT and pET28a-CNMT-MesPDH plasmids are respectively transformed into BL21 (DE 3) bacteria to obtain 3 single-plasmid expressed transgenic microorganisms which are respectively named as PDH, PCN, PDHCN-1.

Example 2

The transgenic microorganism of this example was obtained by simultaneously transforming the pET32a-MesPDH plasmid and pET28a-CNMT plasmid of example 1 into BL21 (DE 3) bacteria to obtain a transgenic microorganism expressed by double plasmids, and the transgenic microorganism is named PDHCN-2.

Example 3

The present example prepares a transgenic microorganism by gene editing the genome of the microorganism, and the preparation method thereof comprises the steps of:

1) The standardized CRISPR/CAS9 technology is adopted, an exon SS9 of BL21 (DE 3) bacteria is used as a knock-in target, an N20 sequence is designed, and a general purpose organism (Anhui) Co-Ltd synthesizes plasmid pEcgRNA-N20 (SS 9), and the nucleotide sequence of the plasmid is shown as SEQ ID NO: shown at 5.

2) The following genes were synthesized by general biology (Anhui) Inc.: pMD18-T-HDR-SS9-MesPDH (the nucleotide sequence of which is shown as SEQ ID NO: 6), pMD18-T-HDR-SS9-CNMT (the nucleotide sequence of which is shown as SEQ ID NO: 7), pMD18-T-HDR-SS9-CNMT-MesPDH (the nucleotide sequence of which is shown as SEQ ID NO: 8), and then amplifying the HRD-SS9-MesPDH sequence containing the SS9 homology arm, the HRD-SS9-CNMT sequence containing the SS9 homology arm, and the CNMT sequence HRD-SS9-CNMT-MesPDH sequence containing the SS9 homology arm by using a standardized PCR method.

The upstream primer used in the PCR amplification was 5'-AGTATTGTCCGGTAAGAATCG-3' and the downstream primer was 5'-TTGCCGACGCGCTGGACAGA-3'.

3) And (2) simultaneously and electrically converting the HRD-SS9-MesPDH sequence, the HRD-SS9-CNMT sequence and the HRD-SS9-CNMT-MesPDH sequence amplified in the step (2) and pEcgRNA-N20 (SS 9) into BL21 (DE 3) electric conversion competence containing the pEsCas9 plasmid, and screening and identifying to obtain 3 transgenic microorganisms respectively carrying the MesPDH sequence, the CNMT sequence and the CNMT-MesPDH sequence, which are named as DH, CN, DHCN.

Example 4

The method for inducing the transgenic microorganism and preparing the whole-cell enzyme solution comprises the following steps:

1. the transgenic recombinant microorganisms PDH, PCN, PDHCN-1 and PDHCN-2 of example 1-2 were streaked and activated on LB plates containing Kan (100. Mu.g/mL), respectively. After being cultured overnight at 37 ℃, single colonies are picked and respectively inoculated into 5mL LB liquid medium containing Kan, and the culture is carried out for 12-16 h at 37 ℃ under 200r/min shaking. Respectively inoculating fresh seed solution into 20mL LB liquid medium containing Kan according to 1% inoculum size, and shake culturing at 37deg.C for 3-4 h to OD at 200r/min ₆₀₀ When the concentration is 0.6-0.8, IPTG with the final concentration of 0.5mM is respectively added, the culture solution is placed at 20 ℃ and is subjected to shaking culture for 16 hours at 200r/min to carry out induction expression of the protein.

2. The transgenic recombinant microorganism DH, CN, DHCN of example 3 was streaked on LB plates for activation. After being cultured overnight at 37 ℃, single colonies are picked and respectively inoculated into 5mL LB liquid culture medium, and the culture is carried out for 12-16 h under shaking at 37 ℃ and 200 r/min. Respectively inoculating fresh seed liquid into 20mL LB liquid culture medium according to 1% inoculum size, and shake culturing at 37deg.C and 200r/min for 3-4 h to OD ₆₀₀ When the concentration is 0.6-0.8, IPTG with the final concentration of 0.5mM is respectively added, the culture solution is placed at 20 ℃ and is subjected to shaking culture for 16 hours at 200r/min to carry out induction expression of the protein.

3. After the induction was completed, the cells were collected by centrifugation at 6000rpm at 4℃for 5min, washed once with phosphate buffer (pH 7.0), and then resuspended in a suitable amount of phosphate buffer (pH 7.0) based on the wet weight of the cells to give a cell suspension having a total cell concentration of 100 mg/mL. All operations in step 3 were performed on ice or in an environment at 4 ℃.

Example 5

A method for preparing (S) -nicotine by whole cell catalysis of a transgenic microorganism, comprising the steps of:

the cell suspension of 7 obtained in example 4 was used as an enzyme solution, and the reaction system was shown in Table 1.

TABLE 1 Whole-cell catalytic reaction system

The reaction system is subjected to oscillation reaction for 24 hours at 30 ℃ and the rotating speed is 200rpm. After the reaction is finished, adding absolute ethyl alcohol with the volume being 1 time to the reaction liquid to stop the reaction, and then removing the solvent by rotary evaporation of the reaction mixed liquid at the speed of 100rpm at the temperature of 65 ℃; finally, 10mL of absolute ethanol was added to redissolve the rotary vapor, and HPLC detection was performed after filtration through a 0.45 μm organic filter head.

HPLC detection of (S) -nicotine:

qualitative and quantitative analysis of (S) -nicotine was performed on the reaction products of the reaction system of table 1 by HPLC, the chromatographic conditions were as follows:

high performance liquid chromatograph: agilent 1100Series

Chromatographic column: chiralpakAD Hcolumn (250 mm. Times.4.6mm.times.5 μm)

A detector: DAD detector for detecting 254nm wavelength

Mobile phase ratio and elution conditions: the flow rate is 1mL/min; column temperature is 30 ℃; the sample injection amount is 10 mu L; the isocratic eluting mobile phase system is n-hexane: ethanol: ethylenediamine=74.9: 25.0:0.1.

liquid phase detection result: as shown in fig. 2-7.

FIG. 2 shows HPLC results for a hybrid standard wherein (S) -nicotine peak time is about 4.42 minutes, (R) -nicotine peak time is about 4.65 minutes, (R) -nornicotine peak time is about 7.92 minutes, and (S) -nornicotine peak time is about 9.29 minutes, with a Maximin peak time of about 13.62 minutes.

FIGS. 3 to 7 show HPLC results of PDH+PCN, PDHCN-1, PDHCN-2, DH+CN and DHCN, respectively, wherein all nornicotine generated by the reactions are (S) -nornicotine, all the generated nicotine is (S) -nicotine, and the generation of (R) -nornicotine and (R) -nicotine is not detected, so that the specificity of the whole-cell catalysis preparation of (S) -nicotine by the transgenic microorganism is high, and the optical purity of the obtained product is extremely high.

Counting the peak area ratio of (S) -nornicotine, myosmine and (S) -nicotine in the figures 3-7 by adopting a normalization method, and determining the relative content of the components after the reaction of the whole-cell catalytic reaction system is finished; as a result, as shown in FIG. 1, when PDHCN-1 or PDHCN-2 was used for the reaction, the amount of the residual myosmine was 0.11% to 0.12%, the amount of the residual (S) -nornicotine was 0.06% to 0.07%, and the amount of the produced (S) -nicotine was 99.82% to 99.83%; when the reaction was performed using DHCN, the S) -nicotine production was 92.61%; when the reaction is carried out using PDH+PCN, DH+CN, the (S) -nicotine production is reduced to 60.35% -69%.

The results prove that the DHCN, PDHCN-1, PDHCN-2, PDH+PCN and DH+CN transgenic microorganisms can biologically convert the myosmine into the (S) -nicotine, wherein the S) -nicotine generation amount of the catalytic reaction of the DHCN, PDHCN-1 and PDHCN-2 transgenic microorganisms is more than 92.61%, which shows that the conversion rate of the transgenic microorganisms constructed by transferring both the MesPDH gene and the CNMT gene into organisms can be greatly improved; further, the (S) -nicotine production amount is 99.82-99.83% when PDHCN-1 or PDHCN-2 reacts, which indicates that constructing the transgenic microorganism transformed by the exogenous gene expression plasmid is beneficial to the expression of exogenous gene, thereby improving the efficiency of transforming S) -nicotine.

Example 6

The component content of glucose, SAM and myosmine in the preparation method of the (S) -nicotine is optimized:

the whole cell catalytic preparation of (S) -nicotine by the transgenic microorganism of example 5 was carried out using PDHCN-1 as an experimental strain, and specific parameters are shown in tables 2 and 3.

TABLE 2

Note that: the final concentration of fixed myosmine was 10mg/mL.

TABLE 3 Table 3

Final concentration of myosmine	Reaction progress (24 h)	Reaction progress (48 h)
			5mg/mL	99.8％	99.9％
10mg/mL	99.4％	99.6％
			30mg/mL	99.5％	99.7％
50mg/mL	72.4％	91.5％
			70mg/mL	52.3％	60.2％
100mg/mL	37.3％	40.2％

Note that: the fixed glucose addition was 1.85g equivalent of Mastigmine and the SAM addition was 3.33g equivalent of Mastigmine.

As can be seen from tables 2 to 3, when PDHCN-1 was used as the experimental strain for the reaction, only the higher reaction efficiency was used at 30mg/mL of final concentration of Mastimin, the reaction progress was high, the conversion rate was high, the optimum catalytic conditions were glucose addition of 1.85g equivalent of Mastimin and SAM addition of 3.33g equivalent of Mastimin.

The preparation method comprises the following steps:

30g of myosmine, 55.5g of glucose, 100 g of SAM, 60g of PDHCN-1 in wet weight were added to a 5L stirred reaction vessel, 2L was supplemented with 0.1M phosphate buffer having a pH of 7.0, and the reaction was carried out at 30℃and 200rpm for 24 hours, which showed 56% conversion after 6 hours and more than 99% conversion after 24 hours.

After the reaction solution was centrifuged to remove the cell precipitate, the pH of the supernatant was adjusted to 10 to 12 with 12M sodium hydroxide, and 400mL of methylene chloride was added to extract for 2 times. The resulting organic layer was subjected to rotary evaporation at 65℃at 100rpm to remove the solvent and a small amount of water, yielding 72.3g of crude nicotine, and finally 61.45g of nicotine was obtained under vacuum distillation.

The purity is higher than 99.5% by HPLC, the chiral purity is high (ee is more than or equal to 99%), and the content of both the myosmine and the (S) -nornicotine is less than 0.1%.

Example 7

Optimization of extractant:

in this example, the reaction solution after the catalytic reaction in example 6 is centrifuged to obtain a supernatant, and then dichloromethane, methyl tert-butyl ether, n-hexane, ethyl acetate and tetrahydrofuran are used as extractants, and the volume ratio of the supernatant to the extractant is 4:1, extracting, and calculating the extraction efficiency, wherein the calculation formula is as follows: extraction efficiency = [1-V (volume of extracted water layer) x c (quantitative HPLC external standard method) ]x100%; the results of the extraction efficiency of each extractant are shown in Table 4.

TABLE 4 Table 4

The results in table 4 show: a. all the extractant can extract the (S) -nicotine, and the extraction efficiency is as follows in order from big to small: in addition, in the extraction process of dichloromethane, the dichloromethane is more reactive to soluble protein, and the dichloromethane and the methyl tertiary butyl ether are not easily separated from denatured protein in the lower layer during layering. b. Under the condition of no additional sodium chloride, tetrahydrofuran is not layered, but when sodium chloride with the final concentration of 200g/L to 300g/L is added, under the condition that the pH is 9 to 13, the extraction efficiency of (S) -nicotine is 93.85 to 95.67 percent, and the most preferable extraction condition is that tetrahydrofuran is adopted as an extractant and 200g/L of sodium chloride is added, and the extraction efficiency of (S) -nicotine is highest.

The above embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, but any insubstantial changes and substitutions made by those skilled in the art on the basis of the present invention are intended to be within the scope of the present invention as claimed.

Claims (10)

1. A transgenic microorganism for the synthesis of (S) -nicotine, characterized in that: the transgenic microorganism contains an exogenous gene a and an exogenous gene b; the exogenous gene a is used for expressing dehydrogenase, and the exogenous gene b is used for expressing N-methyltransferase.

2. A transgenic microorganism for the synthesis of (S) -nicotine according to claim 1, characterized in that: the dehydrogenase comprises an amino acid sequence shown as SEQ ID NO:2 or a homologous dehydrogenase derived from a MesPDH enzyme having a MesPDH enzyme activity and having a sequence at least 90% identical to the MesPDH enzyme by substitution, deletion or addition of one or more amino acids in the amino acid sequence defined by the MesPDH enzyme.

3. A transgenic microorganism for the synthesis of (S) -nicotine according to claim 1, characterized in that: the N-methyltransferase comprises an amino acid sequence shown in SEQ ID NO:4 or a second CNMT enzyme derived from a first CNMT enzyme having at least 90% identity to the first CNMT enzyme and having a first CNMT enzyme activity, which has been substituted, deleted or added with one or more amino acids in the amino acid sequence defined by the first CNMT enzyme.

4. A transgenic microorganism for the synthesis of (S) -nicotine according to any one of claims 1-3, characterized in that: the nucleotide sequence of the exogenous gene a is shown as SEQ ID NO:1 is shown in the specification; the nucleotide sequence of the exogenous gene b is shown as SEQ ID NO: 3.

5. A transgenic microorganism for the synthesis of (S) -nicotine according to any one of claims 1-3, characterized in that: the microorganism includes at least one of bacteria, yeast, filamentous fungi, cyanobacteria, and plant cells.

6. Use of a transgenic microorganism characterized in that: use of a transgenic microorganism for the synthesis of (S) -nicotine according to any one of claims 1-5 in a process for the preparation of (S) -nicotine.

7. A method for preparing (S) -nicotine, comprising the steps of:

1) Obtaining a transgenic microorganism for the synthesis of (S) -nicotine according to any one of claims 1-6;

2) Taking myosmine, glucose and methyl donor as substrates, adding the transgenic microorganism in the step 1) to perform whole-cell catalytic reaction, and separating and purifying to obtain (S) -nicotine.

8. A method for preparing (S) -nicotine according to claim 7, characterized in that the method for preparing the transgenic microorganism in step 1) comprises the steps of:

loading the exogenous gene a and the exogenous gene b into the same plasmid to construct a double-gene expression plasmid, and then electrically transforming the double-gene expression plasmid into competent host microorganisms to culture to obtain the transgenic microorganisms; or alternatively, the first and second heat exchangers may be,

respectively loading the exogenous gene a and the exogenous gene b into plasmids to obtain a first recombinant plasmid and a second recombinant plasmid, and then, electrically transforming the first recombinant plasmid and the second recombinant plasmid into competent host microorganisms, and culturing to obtain the transgenic microorganisms; or alternatively, the first and second heat exchangers may be,

taking an exon of a microorganism as a knock-in target spot, synthesizing a homologous recombination gene containing exogenous gene a, exogenous gene b and an exon homology arm, and transferring the homologous recombination gene into a host microorganism to obtain the transgenic microorganism.

9. A method of producing (S) -nicotine according to claim 8, characterized in that: the plasmid is pET28a plasmid, the host microorganism is at least one of escherichia coli BL21 (DE 3), pichia pastoris and saccharomyces cerevisiae, and the exon is exon SS9.

10. A method of producing (S) -nicotine according to claim 7, characterized in that: in the whole-cell catalytic reaction of the step 2), the methyl donor is SAM, the final concentration of the myosmine is 5-30mg/mL, and the mass ratio of the myosmine, the glucose and the SAM is 1: (2.7-4): (1.2-2), wherein the reaction temperature is 25-35 ℃;

the separation step in step 2) is: centrifuging the reaction solution to remove cell precipitate, regulating the pH value of the supernatant to 9-13, and adding an extractant for extraction to obtain an isolated product; the extractant is any one of dichloromethane, methyl tertiary butyl ether, normal hexane, ethyl acetate and tetrahydrofuran, and when the extractant is tetrahydrofuran, the extractant also comprises sodium chloride with the concentration of 100-300 g/L.