Method for bi-directionally synthesizing chiral lactam through bi-enzyme cascade-chemical catalysis

Abstract

The invention belongs to the technical field of green chemistry, and particularly relates to a method for bi-directionally synthesizing chiral lactam through bi-enzyme cascade-chemical catalysis. According to a methylpiperidone synthesis route taking 2-methyl glutaronitrile as a raw material, nitrile hydratase is subjected to iterative saturated mutation, substrate channel residues and active site residues are subjected to semi-rational engineering transformation, and 4-cyano valeramide and 4-cyano-2-methyl butyramide are generated with high selectivity. And carrying out cascade reaction on the modified nitrile hydratase and amidase, so as to directionally generate the corresponding mono-cyano carboxylic acid. Then, a Raney nickel catalyst hydrogenation reaction is adopted to treat the whole-cell catalytic solution, and two regioselectivity products can be obtained, namely 5-methyl-2-piperidone (the yield is 96.9%, and the regioselectivity is 98.3%) and 3-methyl-2-piperidone (the yield is 67.9%, and the regioselectivity is 70.4%).

Description

Technical Field

[0001] This invention belongs to the field of green chemistry technology, specifically relating to a method for bidirectional synthesis of chiral lactams via a dual-enzyme cascade-chemical catalysis. Background Technology

[0002] Methylpiperidinones, as chiral lactams, are a crucial class of pharmaceutical and chemical intermediates with wide applications in multiple industrial sectors. In the pharmaceutical industry, they play an irreplaceable role as key chiral building blocks and core structural fragments in the synthesis of blockbuster drugs such as anti-Alzheimer's drugs, antipsychotics, and analgesics. Furthermore, these compounds are used to prepare highly effective and low-toxicity insecticides and herbicides, and in the field of polymer materials, they participate as solvents or monomers in the synthesis of high-performance polymers. With the continued expansion of downstream markets, developing high-purity, low-cost, and environmentally friendly methylpiperidinone production processes has become an urgent need for the industry.

[0003] Currently, the synthesis of methylpiperidones mainly relies on traditional chemical catalysis methods, such as amination and cyclization reactions under high temperature and pressure. However, these methods have significant limitations. These processes typically require high temperature and high pressure hydrogen environments exceeding 150°C, which not only places high demands on equipment and consumes a huge amount of energy but also poses serious safety risks. Biocatalysis methods have attracted attention due to their advantages of mild reaction conditions, high selectivity, and environmental friendliness. However, existing single-enzyme catalysis methods still have significant shortcomings, such as cumbersome process flows, high costs, and the possibility that intermediates or products may inhibit enzyme activity.

[0004] Nitrile hydratases (EC 4.2.1.84, NHase) are a class of metalloenzymes that catalyze the hydration of nitrile compounds to form corresponding amides. They are tetramers composed of two α-subunits and two β-subunits. Based on the difference in the metal ion coordinated to the catalytic center, NHases can be divided into two types: Fe-type nitrile hydratases and Co-type nitrile hydratases. The metal ion is coordinated through two oxidized cysteine ​​residues—cysteine ​​sulfinic acid (Cys-SO2H) and cysteine ​​sulfonic acid (Cys-SOH)—as well as a cysteine ​​thiol group and a water molecule. Amideases (EC 3.5.1.X) are hydrolases that catalyze the cleavage of amide bonds, converting various amides into carboxylic acids and ammonia. These enzymes typically possess a catalytic triplet composed of serine-serine-lysine residues and exist primarily as homodimers or homotetramers in aqueous solution. In the early stages of this research, a nitrile hydratase (ReNHase) derived from Rhodococcus CCM2595 was discovered, exhibiting excellent regioselectivity when using dinitrile as a substrate. It selectively catalyzes the hydrolysis of one cyano group in a dicyano molecule to generate a monocyanoamide, which can then be further hydrolyzed to form a monocyanocarboxylic acid. This two-enzyme cascade reaction involves two consecutive enzymatic steps, thus providing more pathways for reaction regulation. Therefore, the nitrile hydratase-amidase cascade can achieve a one-pot generation of carboxylic acid intermediates, laying the foundation for the formation of methylpiperidone.

[0005] Therefore, there is an urgent need in this field for a breakthrough synergistic strategy that can leverage the strengths of biocatalysis and avoid their weaknesses, creatively combining the advantages of biocatalysis and chemical catalysis. By designing novel "enzyme-chemistry" processes, each enzyme can perform its most effective conversion steps in its optimal reaction environment, and by solving the technical challenge of seamless integration between these steps, it is hoped that efficient, green, and economical production of methylpiperidone can be achieved. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a method for the bidirectional synthesis of chiral lactams via a dual-enzyme cascade-chemical catalysis. This invention utilizes a methylpiperidinone synthesis route based on 2-methylglutaronitrile. Through iterative saturation mutagenesis of a nitrile hydratase, and semi-rational engineering of substrate channel and active site residues, it achieves highly selective generation of 4-cyanopentamide and 4-cyano-2-methylbutyramide. Subsequently, the modified nitrile hydratase is cascaded with an amidase to directionally generate the corresponding monocyanocarboxylic acid. Following this, whole-cell catalytic solution is treated with a Raney nickel catalyst for hydrogenation, yielding two regioselective products: 5-methyl-2-piperidinone (96.9% yield, 98.3% regioselectivity) and 3-methyl-2-piperidinone (67.9% yield, 70.4% regioselectivity). The semi-rational design and modification method of the nitrile hydratase in this invention can regulate the regioselectivity of the nitrile hydratase for nitrile compound catalytic reactions and cascade with amidase and Raney nickel catalytic steps for the bidirectional synthesis of high-value chemicals.

[0007] The technical solution of the present invention is as follows:

[0008] A method for the bidirectional synthesis of chiral lactams via a two-enzyme cascade-chemical catalysis includes the following steps:

[0009] Step 1: Two recombinant plasmids were obtained by screening for the substrate channel amino acid sites βF41 and βV52 of ReNHase-βWT:

[0010] The amino acid sequence of the template nitrile hydratase ReNHase-βWT is shown in SEQ ID NO.1. Two recombinant plasmids were obtained by screening for the substrate channel amino acid sites βF41 and βV52 of ReNHase-βWT: the amino acid sequence of the recombinant plasmid ReNHase-F41L targeting site βF41 is shown in SEQ ID NO.2, and the amino acid sequence of the recombinant plasmid ReNHase-V52P targeting site βV52 is shown in SEQ ID NO.3.

[0011] The specific screening steps are as follows: The corresponding substrate channels in the nitrile hydratase ReNHase-βWT were calculated using Caver Analyst 2.0 software and Caver Web 2.0. In the Caver calculations, the catalytic triplet Cys-Ser-Cys was specified as the initial point, and the ligand binding energy within the tunnel was calculated, with 2-methylglutaronitrile as the ligand.

[0012] This was achieved by configuring the following parameters: minimum probe radius set to 0.9 Å, shell depth to 4 Å, shell radius to 3 Å, clustering threshold to 3.5 Å, maximum distance to 3 Å, and target radius to 5 Å. The software-generated residue analysis map provided information on how residues surrounding the channel affect its profile, identifying 13 residues that could potentially influence channel properties. Based on the distance of the residues from the active site, F41 and V52 sites were selected for full-saturation mutagenesis.

[0013] Screening was performed based on the regioselectivity of 2-cyanopentamide and 4-cyano-2-methylbutyramide. The treated bacterial culture (template ReNHase-βWT and all recombinant plasmids with full-saturation mutations) was mixed with an equal volume of 2-methylglutaronitrile solution, and the mixture was subjected to whole-cell reaction with continuous shaking under light at 30°C. The reaction solution was then rotary evaporated under reduced pressure to remove volatile components. After cooling to room temperature, deuterated dimethyl sulfoxide (DMSO-d6, 99.9% D purity) was added as a solvent. The mixture was vigorously vortexed until the residue was completely dissolved. ¹H NMR spectroscopy analysis was used to qualitatively identify the target product and its relative content by utilizing the characteristic chemical shift difference of the methyl peaks of the substrate and product. NMR analysis was performed on a Bruker 500MHz Avance III HD high-resolution NMR spectrometer. Samples were measured at a test temperature of 25°C, with the following acquisition parameters: spectral width 20 ppm, relaxation delay 1 second, and multiple scans were accumulated to ensure a sufficient signal-to-noise ratio. Based on NMR analysis, positive recombinant plasmids ReNHase-F41L and ReNHase-V52P with enhanced regioselectivity were identified. The mutation at the F41 site improved the selectivity for 4-cyano-2-methylbutyramide, while the mutation at the V52 site improved the selectivity for 4-cyanopentamide.

[0014] SEQ ID NO.1:

[0015] MDGVHDLAGVQGFGKVPHTVNADIGPTFHAEWEHLPYSLMFAGVAELGAFSVDEVRYVVERMEPRHYMMTPYYERYVIGVAALMVEKGILTQEELESLAGGPFPLS RPSESEGRPARVDTTTTFEVGQRVRVRDEYVPGHIRMPAYCRGRVGTIAHRTTEKWPFPDAIGHGRNDAGEEPTYHVTFAAEELFGSDTDGGSVVVDLFEGYLEPAA

[0016] SEQ ID NO.2:

[0017] MDGVHDLAGVQGFGKVPHTVNADIGPTFHAEWEHLPYSLMLAGVAELGAFSVDEVRYVVERMEPRHYMMTPYYERYVIGVAALMVEKGILTQEELESLAGGPFPLS RPSESEGRPARVDTTTTFEVGQRVRVRDEYVPGHIRMPAYCRGRVGTIAHRTTEKWPFPDAIGHGRNDAGEEPTYHVTFAAEELFGSDTDGGSVVVDLFEGYLEPAA

[0018] SEQ ID NO.3:

[0019] MDGVHDLAGVQGFGKVPHTVNADIGPTFHAEWEHLPYSLMFAGVAELGAFSPDEVRYVVERMEPRHYMMTPYYERYVIGVAALMVEKGILTQEELESLAGGPFPLS RPSESEGRPARVDTTTTFEVGQRVRVRDEYVPGHIRMPAYCRGRVGTIAHRTTEKWPFPDAIGHGRNDAGEEPTYHVTFAAEELFGSDTDGGSVVVDLFEGYLEPAA

[0020] Step 2: Iterative mutation screening of key amino acids in the active site of ReNHase-F41L yielded one recombinant plasmid; iterative mutation screening of key amino acids in the active site of ReNHase-V52P yielded two recombinant plasmids:

[0021] The amino acid sequence of the recombinant plasmid ReNHase-F41L / M40F targeting site βM40 of the template recombinant plasmid ReNHase-F41L is shown in SEQ ID NO.4; the amino acid sequence of the recombinant plasmid ReNHase-V52P / Y37L targeting site βY37 of the template recombinant plasmid ReNHase-V52P is shown in SEQ ID NO.5; and the amino acid sequence of the recombinant plasmid ReNHase-V52P / Y72S targeting site βY72 is shown in SEQ ID NO.6.

[0022] In this invention, the key amino acid sites of the active site were obtained through molecular docking. All molecular docking calculations were performed using AutoDock Vina software and visualized using Pymol and ChimeraX. A total of 100 molecular docking simulations were conducted for each enzyme and small molecule pair, and the highest-ranking conformation was selected as the optimal binding conformation. Conserved residues located near the active site of nitrile hydratase and at the key α-β subunit interface are crucial to the enzyme's catalytic performance. Mutations in these residues can significantly reduce enzyme activity. Therefore, amino acid conservation analysis was performed using the Clustal W website (https: / / www.genome.jp / tools-bin / clustalw). After excluding conserved amino acids, iterative double mutations were performed based on the substrate channel amino acid mutants obtained from the above screening. Similar to step 1, screening was performed based on regional selectivity. Whole-cell reaction was carried out under continuous shaking at 30°C and light conditions. Positive recombinant plasmids ReNHase-F41L / M40F, ReNHase-V52P / Y37L, and ReNHase-V52P / Y72S were screened by nuclear magnetic resonance analysis.

[0023] SEQ ID NO.4:

[0024] MDGVHDLAGVQGFGKVPHTVNADIGPTFHAEWEHLPYSLFLAGVAELGAFSVDEVRYVVERMEPRHYMMTPYYERYVIGVAALMVEKGILTQEELESLAGGPFPL SRPSESEGRPARVDTTTFEVGQRVRVRDEYVPGHIRMPAYCRGRVGTIAHRTTEKWPFPDAIGHGRNDAGEEPTYHVTFAAEELFGSDTDGGSVVVDLFEGYSEQ ID NO.5:

[0025] MDGVHDLAGVQGFGKVPHTVNADIGPTFHAEWEHLPLSLMFAGVAELGAFSPDEVRYVVERMEPRHYMMTPYYERYVIGVAALMVEKGILTQEELESLAGGPFPLS RPSESEGRPARVDTTTTFEVGQRVRVRDEYVPGHIRMPAYCRGRVGTIAHRTTEKWPFPDAIGHGRNDAGEEPTYHVTFAAEELFGSDTDGGSVVVDLFEGYLEPAA

[0026] SEQ ID NO.6:

[0027] MDGVHDLAGVQGFGKVPHTVNADIGPTFHAEWEHLPYSLMFAGVAELGAFSPDEVRYVVERMEPRHYMMTPSYERYVIGVAALMVEKGILTQEELESLAGGPFPLS RPSESEGRPARVDTTTTFEVGQRVRVRDEYVPGHIRMPAYCRGRVGTIAHRTTEKWPFPDAIGHGRNDAGEEPTYHVTFAAEELFGSDTDGGSVVVDLFEGYLEPAA

[0028] Step 3: Construct nitrile hydratase mutant:

[0029] All recombinant plasmids in this invention are constructed using gene synthesis methods. Plasmid transformation is performed by introducing recombinant plasmids encoding nitrile hydratase into the host organism using competent Escherichia coli ArcticExpress (DE3) cells to construct nitrile hydratase mutants. The specific steps are as follows: First, competent cells stored at -80℃ or in liquid nitrogen are immediately thawed on ice, and then the plasmid is added to the cell culture medium. The cell-plasmid mixture is gently tapped at the bottom of the centrifuge tube to ensure uniform mixing, and then incubated in an ice bath for 20-30 min. The sample is then subjected to heat shock treatment in a 42℃ water bath for 45-90 s, and immediately transferred to an ice bath to cool for 3 min before being added to LB medium. After thorough mixing, the cells are cultured at 37℃ with shaking at 200 rpm for 60-100 min to promote cell recovery. Finally, 100-200 μl of bacterial suspension is evenly spread on LB solid medium containing the kanamycin resistance gene, and the culture plate is inverted and cultured overnight at 37℃.

[0030] Step 4: Using a one-pot reaction of nitrile hydratase mutant and amidase, 4-cyano-2-methylbutyric acid and 4-cyanopentanoic acid are generated, and an aqueous whole-cell reaction solution is obtained.

[0031] This invention aims to achieve a one-pot production of monocyanocarboxylic acid intermediates. First, a nitrile hydratase mutant strain is prepared as competent cells. The steps are as follows: BT medium is dissolved in ultrapure water (0.037 g / ml), followed by autoclaving. Simultaneously, an E. coli strain producing the nitrile hydratase mutant is aseptically picked using a sterile inoculation loop and inoculated into sterile test tubes. The tubes are then cultured at 37°C and 250 rpm on a shaker for 12-16 h to reach the logarithmic growth phase. All operations during the preparation of competent cells must be performed under sterile conditions. The bacterial culture medium must be cooled in an ice bath, and vigorous shaking should be avoided during operation. Initially, an appropriate amount of pre-cultured bacterial solution is transferred to sterilized BT medium. Subsequently, the cells are cultured on a shaker at 37°C and 250 rpm until OD (occurrence limit) is reached. 600The bacterial concentration reached 0.5-0.6. The bacterial suspension was placed on ice for 5-10 min. Then, the bacterial pellet was collected by centrifugation at 4°C and 5000 rpm. After centrifugation, the pellet was thoroughly resuspended in BT Buffer A. Centrifugation at 4°C and 5000 rpm was repeated to collect the bacterial pellet again. Finally, the pellet was resuspended in BT Buffer B (BT Buffer A to BT Buffer B volume ratio 4:1) to obtain competent cells, which were then aliquoted into centrifuge tubes and stored at -80°C or liquid nitrogen for later use.

[0032] Then, using transformed competent cells, a recombinant plasmid encoding amidase was introduced into the host organism to achieve co-expression of nitrile hydratase and amidase in *E. coli*. The steps are as follows: First, competent cells stored at -80℃ or in liquid nitrogen were immediately thawed on ice. Then, the recombinant plasmid encoding amidase was added to the cells. The cell-plasmid mixture was gently tapped at the bottom of the centrifuge tube to ensure uniform mixing, and incubated on ice for 20-30 min. The sample was then subjected to heat shock at 42℃ for 45-90 s, followed by rapid transfer to ice for 3 min. LB medium preheated to 37℃ was then added. After thorough mixing, the cells were cultured at 37℃ with shaking at 200 rpm for 60-100 min to promote cell recovery. Finally, 100-200 μl of bacterial suspension was evenly spread on LB solid medium (preheated to 37℃) containing kanamycin and ampicillin resistance genes, and the culture plate was inverted and incubated overnight at 37℃. An engineered dual-plasmid strain was used to carry out a whole-cell catalytic biotransformation reaction under continuous oscillating light conditions to directly convert the substrate 2-methylglutaronitrile into 4-cyano-2-methylbutyric acid and 4-cyanovaleric acid without intermediates. The final aqueous whole-cell reaction solution was used as the reaction precursor in step 5. Step 5: Hydrogenation with Raney nickel catalyst to obtain regioselective products: 5-methyl-2-piperidinone and 3-methyl-2-piperidinone

[0033] The aqueous whole-cell reaction solution obtained above was subjected to catalytic hydrogenation under H2 atmosphere using a Raney nickel catalyst (10-20% wt%) to produce 5-methyl-2-piperidinone and 3-methyl-2-piperidinone. However, due to mechanical disruption or enzymatic degradation of cells, intracellular proteins or small molecules are released from the reaction solution. These substances can clog the catalyst's active sites and reduce the reaction rate. To address this issue, a pretreatment process was implemented, and a detoxification method was developed to pretreat the aqueous whole-cell reaction solution: the aqueous whole-cell reaction solution was subjected to thermal inactivation and denaturation of proteins at 100°C for 10-20 min, followed by filtration through a 0.22 μm membrane to remove large molecular fragments. Subsequently, ammonia gas was introduced under ice bath conditions to convert free carboxylic acids into ammonium salt complexes, while the pre-reduction of the Raney nickel catalyst further reduced toxic impurities. This comprehensive method restored the catalytic efficiency to 97% of the theoretical maximum value, and the hydrogenation rate was significantly increased by 5.8 times compared to the untreated control group.

[0034] The beneficial effects of this invention are:

[0035] (1) The nitrile hydratase modified by iterative saturation mutation can significantly regulate the regioselectivity of the catalytic reaction of 2-methylglutaronitrile in both directions. ReNHase-F41L / M40F changed the regioselectivity from 4-cyanopentamide to 4-cyano-2-methylbutanamide, while ReNHase-V52P / Y37L and ReNHase-V52P / Y72S further improved the regioselectivity of 4-cyanopentamide, realizing the bidirectional synthesis from the same substrate to different products. This has important reference significance for the study of regioselectivity regulation in catalytic reactions.

[0036] (2) The system constructed in this invention, which co-expresses nitrile hydratase and amidase in Escherichia coli, enables efficient one-pot generation of carboxylic acid intermediates without intermediate purification, laying the foundation for the generation of methylpiperidone;

[0037] (3) This invention realizes the hydrogenation reaction of aqueous system under mild conditions under the catalytic conditions of Raney nickel catalyst to produce 5-methyl-2-piperidinone and 3-methyl-2-piperidinone, which fully meets the requirements of green chemical synthesis, provides a new synthetic route, and also provides a theoretical basis for its industrial application. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the synthesis route of the method of the present invention.

[0039] Figure 2 This is a schematic diagram showing the C2 / C5 region selectivity of ReNHase-βWT and single-point mutants for 4-cyano-2-methylbutyramide and 4-cyanopentamide.

[0040] Figure 3 This is a schematic diagram of the key amino acid catalytic residues in the active sites of ReNHase-F41L (a) and ReNHase-V52P (b) predicted by molecular docking.

[0041] Figure 4 This is a schematic diagram illustrating the time-process analysis of the transformation efficiency of each strain under optimal conditions.

[0042] Figure 5 This is a schematic diagram illustrating the optimal operating range of key factors determined by response surface methodology in the synthesis of 5-methyl-2-piperidinone. Detailed Implementation

[0043] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings and technical solutions.

[0044] Example 1: Preparation of 3-methyl-2-piperidinone:

[0045] (1) For amino acid iterative mutations:

[0046] Iterative full-saturation mutagenesis was performed on key amino acids of the substrate channel and key amino acids of the active site. The resulting mutant plasmids were transformed into competent cells. Recombinant *E. coli* cells were cultured in LB medium containing kanamycin (50 μg / mL) (1% inoculum) at 220 rpm and 37 °C. The absorbance (OD) at 600 nm was measured. 600 When the cell count reached 0.6-0.8, IPTG (1% of the seed volume) at a final concentration of 0.1 mM was added to induce nitrile hydratase expression (16℃, 160 rpm, 16 hours). Cells were then collected by centrifugation at 4℃ and 6000 rpm. The collected cell pellet was washed twice with PB buffer (10 mM, pH 7.4). The washed cells were resuspended in PB buffer for subsequent reactions.

[0047] The treated bacterial culture was mixed with an equal volume of 200 mM 2-methylglutaronitrile solution, and the mixture was subjected to whole-cell reaction with continuous shaking under 30°C and light conditions. The reaction was terminated with acetonitrile. Screening was based on the regioselectivity of 4-cyano-2-methylbutyramide. For the first round of mutations based on substrate channel amino acids, ReNHase-F41L was selected as a candidate enzyme for subsequent semi-rational design. Figure 2 As shown, ReNHase-F41L improved the regioselectivity of 4-cyano-2-methylbutyramide to 63.4 ± 0.5%; a second round of iterative saturation mutagenesis based on key amino acids at the active site was performed using ReNHase-F41L, such as... Figure 3As shown in a, after excluding conserved amino acids, mutations were performed on four key amino acid sites: Y37, M40, Y72, and Y76, which further improved the regioselectivity of 4-cyano-2-methylbutyramide. ReNHase-F41L / M40F increased the regioselectivity of 4-cyano-2-methylbutyramide to 70.1 ± 1.2%.

[0048] (2) One-pot method for the preparation of 4-cyano-2-methylbutyric acid:

[0049] Engineered dual-plasmid strains were constructed by co-introducing nitrile hydratase and amidase plasmids into *E. coli*, and the optimal reaction conditions (pH, temperature, and cell concentration) were investigated. First, the optimal pH (3-11) reaction conditions were investigated by mixing the treated bacterial culture with an equal volume of 400 mM 2-methylglutaronitrile solution and performing a whole-cell reaction with continuous shaking at 40℃ under light conditions for 2 h. The optimal pH for ReNHase-F41L / M40F–R312 was found to be pH=7. Next, the optimal temperature (30-60℃) reaction conditions were investigated by mixing the treated bacterial culture with an equal volume of 400 mM 2-methylglutaronitrile solution and performing a whole-cell reaction with continuous shaking under various temperature and light conditions for 1 h. The optimal temperature for ReNHase-F41L / M40F–R312 was found to be 40℃. Finally, the optimal cell concentration for ReNHase-F41L / M40F–R312 was found to be 3.75 g DCW / L. Under the optimal conditions obtained above, a time gradient reaction was carried out ( Figure 4 The ReNHase-F41L / M40F–R312 amidase cascade reaction operated under mild conditions, achieving a conversion rate of >99% and a regioselectivity of 67.5% for 4-cyano-2-methylbutyric acid.

[0050] (3) Preparation of 3-methyl-2-piperidinone by hydrogenation with Raney nickel catalyst:

[0051] The reaction was carried out using an aqueous cell conversion solution that had undergone detoxification treatment and was saturated with ammonia. After a vacuum was created by a circulating water pump, hydrogen gas was introduced to fill the entire system. This process was repeated three times before the reaction was carried out under water bath conditions. Figure 5 The optimal operating range for achieving a conversion greater than 94% was determined using a response surface methodology design. The optimal range was defined as a catalyst concentration of 19-21%, a reaction temperature of 45-55℃, and a reaction time of 2-4 h. Within this optimal operating range, the solution generated by ReNHase-F41L / M40F–R312 achieved a conversion exceeding 96%, yielding 67.9% of 3-methyl-2-piperidinone (with a selectivity of 70.4%).

[0052] Example 2: Preparation of 5-methyl-2-piperidinone:

[0053] (1) For amino acid iterative mutations:

[0054] Iterative full-saturation mutagenesis was performed on key amino acids of the substrate channel and key amino acids of the active site. The resulting mutant plasmids were transformed into competent cells. Recombinant *E. coli* cells were cultured in LB medium containing kanamycin (50 μg / mL) (1% inoculum) at 220 rpm and 37 °C. The absorbance (OD) at 600 nm was measured. 600 When the cell count reached 0.6-0.8, IPTG (1% of the seed volume) at a final concentration of 0.1 mM was added to induce nitrile hydratase expression (16℃, 160 rpm, 16 hours). Cells were then collected by centrifugation at 4℃ and 6000 rpm. The collected cell pellet was washed twice with PB buffer (10 mM, pH 7.4). The washed cells were resuspended in PB buffer for subsequent reactions.

[0055] The treated bacterial culture was mixed with an equal volume of 200 mM 2-methylglutaronitrile solution, and the mixture was subjected to whole-cell reaction with continuous shaking under 30°C and light conditions. The reaction was terminated with acetonitrile. Screening was based on the regioselectivity of 4-cyanopentamide; for the first round of mutations based on substrate channel amino acids, ReNHase-V52P was selected as a candidate enzyme for subsequent semi-rational design. Figure 2 As shown, ReNHase-V52P improved the regioselectivity of 4-cyanopentamide to 70.2 ± 0.3%; a second round of iterative saturation mutagenesis based on key amino acids at the active site was performed using ReNHase-V52P, such as... Figure 3 As shown in b, after excluding conserved amino acids, mutations were performed on three key amino acid sites: Y37, Y72, and Y76, which further improved the regioselectivity of 4-cyanopentamide. The regioselectivity of ReNHase-V52P / Y37L and ReNHase-V52P / Y72S for 4-cyanopentamide was increased to 95.8 ± 0.4% and 95.3 ± 0.4%, respectively.

[0056] (2) One-pot method for the preparation of 4-cyanopentanoic acid:

[0057] Engineered dual-plasmid strains were constructed by co-introducing nitrile hydratase plasmid and amidase plasmid into Escherichia coli, and the optimal reaction conditions (pH, temperature and cell concentration) were investigated. First, the optimal pH (3-11) reaction conditions for the strains were investigated. The treated bacterial culture was mixed with an equal volume of 400 mM 2-methylglutaronitrile solution, and the whole-cell reaction was carried out with continuous shaking for 2 h under 40℃ and light conditions. The optimal pH reaction conditions for ReNHase-V52P / Y37L–R312 and ReNHase-V52P / Y72S–R312 were found to be pH=9 and pH=8, respectively. Next, the optimal temperature (30-60℃) reaction conditions for the strains were investigated. The treated bacterial culture was mixed with an equal volume of 400 mM 2-methylglutaronitrile solution, and the whole-cell reaction was carried out with continuous shaking for 1 h under various temperature and light conditions. The optimal temperature reaction condition for both ReNHase-V52P / Y37L–R312 and ReNHase-V52P / Y72S–R312 was found to be 40℃. The optimal cell concentration was then investigated. The optimal conditions for ReNHase-V52P / Y37L–R312 and ReNHase-V52P / Y72S–R312 are 1 g DCW / L and 4 g DCW / L, respectively. A time gradient reaction was carried out under the above-obtained optimal conditions. Figure 4 The ReNHase-V52P / Y37L–R312 amidase cascade showed superior performance, producing 4-cyanopentanoic acid with an ultra-high regioselectivity of 98.7% and a conversion rate exceeding 99%. Notably, the ReNHase-V52P / Y72S–R312 amidase cascade exhibited the highest regioselectivity (99.67%) for 4-cyanopentanoic acid, but the yield decreased to 76%.

[0058] (3) Preparation of 5-methyl-2-piperidinone by hydrogenation with Raney nickel catalyst:

[0059] The reaction was carried out using an aqueous cell conversion solution that had undergone detoxification treatment and was saturated with ammonia. After a vacuum was created by a circulating water pump, hydrogen gas was introduced to fill the entire system. This process was repeated three times before the reaction was carried out under water bath conditions. Figure 5 The optimal operating range for achieving a yield greater than 94% was determined using a response surface methodology design. The optimal range was defined as a catalyst concentration of 19-21%, a reaction temperature of 45-55℃, and a reaction time of 2-4 h. Within this optimal operating range, the reaction solution generated by ReNHase-V52P / Y37L–R312 achieved a substrate conversion of >98% via catalytic hydrogenation, yielding 96.9% of 5-methyl-2-piperidinone (with a selectivity of 98.3%).

Claims

1. A method for the bidirectional synthesis of chiral lactams via a dual-enzyme cascade-chemical catalysis, characterized in that, Includes the following steps: Step 1: Two recombinant plasmids were obtained by screening for the substrate channel amino acid sites βF41 and βV52 of ReNHase-βWT: The amino acid sequence of the template nitrile hydratase ReNHase-βWT is shown in SEQ ID NO.

1. Two recombinant plasmids were obtained by screening for the substrate channel amino acid sites βF41 and βV52 of ReNHase-βWT: the amino acid sequence of the recombinant plasmid ReNHase-F41L targeting site βF41 is shown in SEQ ID NO.2, and the amino acid sequence of the recombinant plasmid ReNHase-V52P targeting site βV52 is shown in SEQ ID NO.

3. Step 2: Iterative mutation screening of key amino acids in the active site of ReNHase-F41L yielded one recombinant plasmid; iterative mutation screening of key amino acids in the active site of ReNHase-V52P yielded two recombinant plasmids: The amino acid sequence of the recombinant plasmid ReNHase-F41L / M40F targeting site βM40 of the template recombinant plasmid ReNHase-F41L is shown in SEQ ID NO.4; the amino acid sequence of the recombinant plasmid ReNHase-V52P / Y37L targeting site βY37 of the template recombinant plasmid ReNHase-V52P is shown in SEQ ID NO.5; and the amino acid sequence of the recombinant plasmid ReNHase-V52P / Y72S targeting site βY72 is shown in SEQ ID NO.

6. Step 3: Construct a nitrile hydratase mutant: A recombinant plasmid encoding nitrile hydratase was introduced into a host organism using competent Escherichia coli ArcticExpress cells to construct a nitrile hydratase mutant. Step 4: Using a one-pot reaction of nitrile hydratase mutant and amidase, 4-cyano-2-methylbutyric acid and 4-cyanopentanoic acid are generated, and an aqueous whole-cell reaction solution is obtained. First, competent cells were prepared from the nitrile hydratase mutant strain. Then, the recombinant plasmid encoding amidase was introduced into the host organism using the transformed competent cells to achieve co-expression of nitrile hydratase and amidase in Escherichia coli. The engineered dual-plasmid strain was used to carry out whole-cell catalytic biotransformation under continuous oscillating light conditions to directly convert the substrate 2-methylglutaronitrile into 4-cyano-2-methylbutyric acid and 4-cyanopentanoic acid without intermediates. The final aqueous whole-cell reaction solution was used as the reaction precursor in step 5. Step 5: Hydrogenation with Raney nickel catalyst to obtain regioselective products: 5-methyl-2-piperidinone and 3-methyl-2-piperidinone The aqueous whole-cell reaction solution obtained above was subjected to catalytic hydrogenation under H2 atmosphere using Raney nickel catalyst to produce 5-methyl-2-piperidinone and 3-methyl-2-piperidinone.

2. The method for bidirectional synthesis of chiral lactams via a dual-enzyme cascade-chemical catalysis according to claim 1, characterized in that, The specific screening steps in Step 1 are as follows: The corresponding substrate channels in the nitrile hydratase ReNHase-βWT were calculated using Caver Analyst 2.0 software and Caver Web 2.0; in the Caver calculation, the catalytic triple Cys-Ser-Cys was specified as the initial point, and the ligand binding energy within the tunnel was calculated, with 2-methylglutaronitrile as the ligand.

3. The method for bidirectional synthesis of chiral lactams via a dual-enzyme cascade-chemical catalysis according to claim 2, characterized in that, This was achieved by configuring the following parameters: minimum probe radius set to 0.9 Å, shell depth to 4 Å, shell radius to 3 Å, clustering threshold to 3.5 Å, maximum distance to 3 Å, and target radius to 5 Å. The residue analysis map generated by the software provided information on how residues around the channel affect its channel profile. A total of 13 residues that may affect the channel properties were identified. Based on the distance between the residues and the active center, F41 and V52 sites were selected for full-saturation mutation.

4. The method for bidirectional synthesis of chiral lactams via a dual-enzyme cascade-chemical catalysis according to claim 1, characterized in that, Step 3 is as follows: First, take out the competent cells stored at -80℃ or in liquid nitrogen and thaw them immediately on ice. Then add the plasmid to the cell culture. Gently tap the bottom of the centrifuge tube to ensure that the cell-plasmid mixture is evenly mixed, and then incubate it in an ice bath for 20-30 minutes. The samples were subjected to heat shock treatment in a 42℃ water bath for 45-90s. Immediately after treatment, they were transferred to an ice bath to cool for 3min before being added to LB medium. After thorough mixing, the samples were cultured at 37℃ with shaking at 200 rpm for 60-100min to promote cell recovery. Finally, 100-200μl of bacterial suspension was evenly spread on LB solid medium containing kanamycin resistance gene, and the culture plate was inverted and cultured overnight at 37℃.

5. The method for bidirectional synthesis of chiral lactams via a dual-enzyme cascade-chemical catalysis according to claim 1, characterized in that, In step 4, the specific steps for preparing the nitrile hydratase mutant strain into competent cells are as follows: Weigh BT medium and dissolve it in ultrapure water, then autoclave. Simultaneously, aseptically pick the *E. coli* strain producing the nitrile hydratase mutant using a sterile inoculation loop, inoculate it into a sterile test tube, and incubate it in a shaker at 37°C and 250 rpm for 12-16 h to allow it to reach the logarithmic growth phase. All operations during the preparation of competent cells must be performed under sterile conditions. The bacterial culture medium must be cooled in an ice bath, and vigorous shaking should be avoided during operation. Initially, transfer an appropriate amount of pre-cultured bacterial solution to sterilized BT medium. Then, incubate in a shaker at 37°C and 250 rpm until OD (digestive growth) is reached. 600 The bacterial concentration reached 0.5-0.6; the bacterial suspension was placed in an ice bath for 5-10 min; then centrifuged at 4℃ and 5000 rpm to collect the bacterial pellet; after centrifugation, the pellet was completely resuspended in BT Buffer A; the bacterial pellet was collected again by centrifugation at 4℃ and 5000 rpm; finally, the pellet was resuspended in BT Buffer B to obtain competent cells, which were aliquoted into centrifuge tubes and stored at -80℃ or in liquid nitrogen for later use.

6. The method for bidirectional synthesis of chiral lactams via a dual-enzyme cascade-chemical catalysis according to claim 1, characterized in that, In step 4, the recombinant plasmid encoding amidase is introduced into the host organism using transformed competent cells to achieve co-expression of nitrile hydratase and amidase in E. coli. The steps are as follows: First, competent cells stored at -80℃ or in liquid nitrogen are taken out and immediately thawed on ice. Then, the recombinant plasmid encoding amidase is added to the cells. The cell-plasmid mixture is gently tapped at the bottom of the centrifuge tube to ensure uniform mixing and incubated in an ice bath for 20-30 min. The sample is placed in a 42℃ water bath for 45-90 s heat shock treatment, and then quickly transferred to an ice bath to cool for 3 min. Then, LB medium preheated to 37℃ is added. After thorough mixing, the cells are cultured at 37℃ and 200 rpm for 60-100 min to promote cell recovery. Finally, 100-200 μl of bacterial suspension is evenly spread on LB solid medium containing kanamycin and ampicillin resistance genes, and the culture plate is inverted and cultured overnight at 37℃.

7. The method for bidirectional synthesis of chiral lactams via a dual-enzyme cascade-chemical catalysis according to claim 1, characterized in that, In step 5, the aqueous whole-cell reaction solution is pretreated: the aqueous whole-cell reaction solution is heated at 100℃ for 10-20 min to denature and inactivate the proteins, and then filtered through a 0.22 μm membrane to remove macromolecular fragments. Subsequently, ammonia gas was introduced under ice bath conditions to convert the free carboxylic acid into an ammonium salt complex.

8. The method for bidirectional synthesis of chiral lactams via a dual-enzyme cascade-chemical catalysis according to claim 1, characterized in that, In step 5, the amount of Raney nickel catalyst used is 10-20% wt.

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