An L-pantolactone dehydrogenase, its mutants, encoding gene, and applications

Abstract

This invention relates to an L-pantolactone dehydrogenase and its mutant, encoding gene, a vector containing the encoding gene, genetically engineered bacteria, and its application in the microbial catalytic preparation of ketopantolactone / D-pantolactone. The L-pantolactone dehydrogenase is derived from *Rhodococcus hoagii*, and its amino acid sequence is shown in SEQ ID NO.4. Its codon-optimized nucleotide sequence is shown in SEQ ID NO.1. The main advantages of this invention are: the three enzymes required for transformation can be simultaneously and efficiently expressed in a single recombinant bacterium; the recombinant bacterium can be cultured in large quantities without cell disruption, freeze-drying, or other processing steps, resulting in lower costs and simpler operation; and a highly efficient, low-cost, and easily industrially scaled-up bioenzymatic synthesis process can be established. This invention utilizes a coenzyme regeneration system in the conversion of the intermediate product ketopantolactone (KPL) to DPL, by using NADP... + The conversion to NADPH ensures a relatively stable NADPH concentration in the system, enabling efficient conversion. This method offers advantages such as high specificity, high product optical purity, and effective control of KPL hydrolysis. Using this method to prepare DPL, with the addition of 200 mM LPL, a conversion rate of 96.4% was achieved after 36 hours without KPL hydrolysis.

Description

Technical Field

[0001] This invention relates to an L-indo-pantolactone dehydrogenase and its mutants, the encoding gene, a vector containing the encoding gene, genetically engineered bacteria, and its application in the microbial catalytic preparation of keto-indo-pantolactone / D-indo-pantolactone. Background Technology

[0002] D-Calcium pantothenate, also known as vitamin B5, is a component of coenzyme A and is widely used in the food, feed, pharmaceutical, chemical, and cosmetic industries. D-(−)-Pantothenic acid lactone, also known as ( RDL-pantothenic acid lactone, with the chemical structure D-(−)-pantothenic acid γ-lactone, is a key chiral intermediate in the synthesis of D-(+)-pantothenic acid. Currently, the industrial synthesis of D-pantothenic acid lactone employs a combined chemical and enzymatic resolution approach. Starting from isobutyraldehyde and formaldehyde, the chemical method synthesizes DL-pantothenic acid lactone. The D-pantothenic acid lactone is stereoselectively hydrolyzed by D-pantothenic acid lactone hydrolase to generate D-pantothenic acid, which is then lactonized to form D-pantothenic acid lactone. The remaining L-pantothenic acid lactone is then chemically racemized to form DL-pantothenic acid lactone for recyclization. The resolution of DL-pantothenic acid lactone is a crucial step in the synthesis of D-pantothenic acid lactone. The enzymatic chiral resolution process requires the racemization of L-pantothenic acid lactone, the separation of D-pantothenic acid and L-pantothenic acid lactone, and the acidification and cyclization of D-pantothenic acid to form D-pantothenic acid lactone. While the enzymatically catalyzed chiral resolution method is a mature technology, it still suffers from problems such as complex processes, high energy and material consumption, and the need for large amounts of acids and bases. Therefore, developing a more direct, efficient, and environmentally friendly asymmetric synthesis method for D-pantolactone to replace existing chiral resolution technologies would have significant application value. D-Pantothenic acid lactone can be synthesized asymmetrically via a redox method. This method can be achieved through two different pathways. The first pathway involves L-pantothenic acid lactone dehydrogenase catalyzing the dehydrogenation of L-pantothenic acid lactone to generate ketopantothenic acid lactone. Then, ketopantothenic acid lactone spontaneously hydrolyzes to form ketopantothenic acid, which is then converted to D-pantothenic acid under the action of D-ketopantothenic acid reductase. Finally, D-pantothenic acid undergoes cyclization under acidic conditions to form D-pantothenic acid lactone. The simpler second pathway uses racemic DL-pantothenic acid lactone as a substrate and utilizes stereoselective L-pantothenic acid lactone dehydrogenase to catalyze the dehydrogenation of L-pantothenic acid lactone to generate ketopantothenic acid lactone. Then, ketopantothenic acid lactone undergoes asymmetric synthesis of D-pantothenic acid lactone under the catalysis of D-ketopantothenic acid reductase. Compared to existing hydrolytic enzyme pathways, the second pathway is simpler, directly yielding optically pure products from the racemic substrate via biocatalysis, eliminating the need for racemization and separation of lactones and acids. Therefore, the oxidoreductase-based asymmetric synthesis of D-pantolactone via the second pathway is a promising alternative to biohydrolytic enzyme methods. The dehydrogenation of L-pantolactone is a key step in this pathway, catalyzed by L-pantolactone dehydrogenase. The asymmetric reduction of ketopantolactone in this pathway is catalyzed by NADPH-dependent conjugated polyton reductase. Since the coenzyme NADPH is expensive, industrial production often requires a coenzyme regeneration system to convert NAD(P)+ to NAD(P)H, ensuring stable regeneration of NAD(P)H and efficient conversion. Multi-enzyme catalytic conversion is a highly selective reaction, enabling directional conversion. However, multi-enzyme conversion processes often face challenges such as low enzyme activity, low enzyme stability, low coenzyme recycling efficiency, complex multi-enzyme addition, and difficulties in scale-up.While the transformation of recombinant bacteria with multiple enzymes involving the whole cell can solve the above problems to some extent, there are still issues such as long transformation time and poor transformation effect. Summary of the Invention

[0003] The purpose of this invention is to overcome the problems in current production technology and provide an efficient, simple, low-cost, and easily industrially produced L-pantolactone dehydrogenase and its mutants, encoding gene, vector containing the encoding gene, genetically engineered bacteria, and its application in the microbial catalytic preparation of ketopantolactone / D-pantolactone.

[0004] The technical solution adopted in this invention is:

[0005] Origin Rhodococcus hoagii The amino acid sequence of L-pantolactone dehydrogenase is shown in SEQ ID NO.4. Its codon-optimized nucleotide sequence is shown in SEQ ID NO.1.

[0006] The present invention also relates to an L-pantolactone dehydrogenase mutant, which is obtained by single mutation or multi-point combined mutation at positions 156, 224, 241 and 254 of the amino acid sequence shown in SEQ ID NO.4.

[0007] Preferably, the mutation is one or a combination of two or more of the following: (1) isoleucine at position 156 is mutated to leucine; (2) phenylalanine at position 224 is mutated to histidine; (3) valine at position 241 is mutated to isoleucine; (4) leucine at position 254 is mutated to isoleucine.

[0008] More preferably, the amino acid sequence of the mutant is shown in one of SEQ ID NO.5~7.

[0009] Proteins derived from the amino acid sequence described in this invention, which have at least 95% identity with transaminase activity after substitution, deletion, or addition of one or more amino acid residues, are all within the scope of protection of this invention.

[0010] This invention also relates to the gene encoding the aforementioned L-pantolactone dehydrogenase and its mutants. The L-pantolactone dehydrogenase... Rho The LPLDH and mutant coding gene sequences are both 1206 bp in length, from the first base to the 1206th base, with the start codon being ATG and the stop codon being TAA.

[0011] The present invention also relates to a recombinant vector containing the L-pantolactone dehydrogenase and the mutant encoding gene described above. Typically, the vector plasmid is pET28b(+).

[0012] Preferably, the vector is a dual expression vector, specifically pETDuet-1 plasmid, pCDFDuet-1 plasmid, or pACYCDuet-1 plasmid, and the conjugated polyketide reductase gene (GenBank NO. CAG61069.1) is derived from Candida glabrata. Candida glabrata The nucleotide sequence after codon optimization is shown in SEQ ID NO.2. The glucose dehydrogenase gene (GenBank NO. KM817194.1) is from... Exiguobacterium sibirium DSM 17290, after codon optimization, has the nucleotide sequence shown in SEQ ID NO.3. The method for constructing the co-expression vector is to... Cgl The CPR and GDH genes were inserted into different multiple cloning sites on the dual expression vector.

[0013] This invention also relates to genetically engineered bacteria containing the L-pantolactone dehydrogenase and its mutant encoding gene. The recombinant bacteria are expressed as Escherichia coli BL21(DE3).

[0014] Preferably, the genetically engineered bacteria is L-pantolactone dehydrogenase or a mutant of conjugated polyketide reductase. Cgl The engineered bacteria co-expressing CPR, glucose dehydrogenase, and GDH were constructed as follows: a recombinant plasmid containing the gene encoding L-pantolactone dehydrogenase or a mutant of pET28b(+) was coupled with a double-linked... Cgl The recombinant plasmids of CPR and GDH were transformed into Escherichia coli BL21(DE3) competent cells, and engineered bacteria with double resistance and three enzymes co-expression were screened; the nucleotide sequence of the conjugated polyketide reductase gene is shown in SEQ ID NO.2, and the nucleotide sequence of the glucose dehydrogenase gene is shown in SEQ ID NO.3.

[0015] This invention also relates to the application of the L-indohydrin dehydrogenase and its mutant in the microbial catalysis of L-indohydrin to prepare ketoindohydrin.

[0016] Furthermore, this invention also relates to the application of the L-pantolactone dehydrogenase and its mutant in the microbial catalytic preparation of D-pantolactone. Specifically, the application involves using the aforementioned multi-enzyme co-expression recombinant bacteria to catalyze the substrate L-pantolactone and coupling it with a coenzyme regeneration system. The coenzyme regeneration system uses glucose as a substrate, and glucose dehydrogenase converts NADP... + The enzyme was converted to NADPH. Using the recombinant bacteria with the above multi-enzyme co-expression, 200 mM L-pantolactone was catalyzed to produce D-pantolactone. After 36 h, the DPL conversion yield reached 96.4% and there was no hydrolysis of the intermediate product KPL.

[0017] Furthermore, the application is as follows: using the wet cells obtained by inducing the co-expressed engineered bacteria as a catalyst, L-pantolactone as a substrate, glucose as a co-substrate, and a pH 7.0, 50 mM PB buffer (0.05 M Na2HPO4, 0.05 M NaH2PO4) as the reaction medium to form a transformation system, the reaction is carried out at 30-40 ℃ and 600-800 rpm (preferably 30 ℃ and 800 rpm) to obtain D-pantolactone in the fermentation broth.

[0018] In the transformation system, the substrate is added to a final concentration of 10-400 mM (preferably 100-200 mM), the glucose is added to a final concentration of 15-600 mM (preferably 150-300 mM), and the catalyst dosage is 10-100 g WCW / L (WCW cell wet weight) based on the wet weight of the cells.

[0019] The wet bacterial cells can be prepared by the following method: [The following text appears to be incomplete and requires further context:] ...containing... Rho LPLDH or mutants and Cgl The engineered bacteria co-expressing CPR and GDH enzymes were inoculated into LB liquid medium containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol, or 50 μg / mL ampicillin, or 50 μg / mL streptomycin sulfate, and cultured at 37 ℃ for 10 h to obtain seed culture. The seed culture was then inoculated at a volume concentration of 1.0% into fresh LB liquid medium containing 50 μg / mL kanamycin and 25 μg / mL chloramphenicol, or 50 μg / mL ampicillin, or 50 μg / mL streptomycin sulfate, and cultured at 37 ℃ and 180 rpm for 2 h (OD). 600 =0.6-0.8), add isopropyl thiogalactoside (Isopropyl) to the culture medium to a final concentration of 0.1 mM. β -D-thiogalactoside (IPTG) was cultured at 28 ℃ for 12 h, followed by centrifugation at 4 ℃ and 8000 rpm for 10 min to obtain the expression. Rho LPLDH or mutant protein and Cgl Wet bacterial cells containing CPR and GDH proteins.

[0020] The present invention relates to the inoculation, transfer, induction, and cell recovery of genetically engineered bacteria. The culture medium can be any medium in the art that can promote bacterial growth and produce the present invention, preferably LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, dissolved in distilled water, and adjusted to pH 7.0. There are no special limitations on the culture method and conditions, which can be optimized according to factors such as host type and culture method.

[0021] The beneficial effects of this invention are mainly reflected in the following: the three enzymes required for the transformation can be simultaneously and efficiently expressed in a single recombinant bacterium. This recombinant bacterium can be cultured in large quantities without the need for cell disruption, freeze-drying, or other processing steps, resulting in lower costs and simpler operation. This allows for the establishment of a highly efficient, low-cost, and easily industrially scaled-up bioenzymatic synthesis process. In the process of converting the intermediate product ketopanolactone (KPL) to DPL, this invention utilizes a coenzyme regeneration system, through the use of NADP... + The conversion to NADPH ensures a relatively stable NADPH concentration in the system, enabling efficient conversion. This method offers advantages such as high specificity, high product optical purity, and effective control of KPL hydrolysis. Using this method to prepare DPL, with the addition of 200 mM LPL, a conversion rate of 96.4% was achieved after 36 hours without KPL hydrolysis. Attached Figure Description

[0022] Figure 1 L-Pantolytic acid lactone dehydrogenase Rho LPLDH, conjugated polyketide reductase Cgl CPR and glucose dehydrogenase Es A schematic diagram of the reaction catalyzed by GDH three-enzyme coupling to induce configuration reversal of L-pantolactone to prepare D-pantolactone.

[0023] Figure 2 This is a schematic diagram of recombinant bacteria with different combinations of three enzymes co-expressed.

[0024] Figure 3 A comparison of the catalytic performance of recombinant bacteria co-expressing three enzymes.

[0025] Figure 4 The reaction process for preparing D-pantolactone by configuration reversal of 200 mM L-pantolactone catalyzed by recombinant bacteria with three co-expression enzymes is shown in the figure; 1 is 50 g / L Strain2, 2 is 50 g / L Strain1, and 3 is 100 g / L Strain2.

[0026] Figure 5 shows the reaction process of preparing D-pantolactone by configuration reversal of different concentrations of L-pantolactone catalyzed by the three-enzyme co-expression recombinant bacteria; 1 is 200 mM substrate, 2 is 250 mM substrate, 3 is 300 mM substrate, and 4 is 400 mM substrate. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to the following embodiments:

[0028] Example 1: Preparation of L-pantolactone dehydrogenase or mutant competent cells

[0029] 1. Starting strain:

[0030] Preservation of engineered bacteria in the laboratory E.coli BL21(DE3) / pET28b- Rho LPLDH, E.coli BL21(DE3) / pET28b- Rho LPLDH L254I / V241I , E.coli BL21(DE3) / pET28b- Rho LPLDH L254I / V241I / I156L , E.coli BL21(DE3) / pET28b- Rho LPLDH L254I / V241I / V308L , E.coli BL21(DE3) / pET28b- Rho LPLDH L254I / V241I / I156L / F224H (mutant) Rho LPLDH L254I / V241I / I156L The amino acid sequence is shown in SEQ ID NO.5, mutant. Rho LPLDH L254I / V241I / I156L / V308L The amino acid sequence is shown in SEQ ID NO.6, mutant. Rho LPLDH L254I / V241I / I156L / F224H The amino acid sequence is shown in SEQ ID NO.7, mutant. Rho

[0031] LPLDH L254I / V241I The amino acid sequence is shown in SEQ ID NO.8. The construction method refers to CN 113564136A) as the original strain, which was activated and chemically competent cells were prepared.

[0032] Example 2: Cgl Construction and transformation of CPR and GDH co-expression plasmids

[0033] 1. Cgl Construction of CPR single expression plasmid

[0034] Using the vector pACYCDuet1-mcs2-CglCPR from strain E. coli BL21(DE3) / pACYCDuet1-mcs2-CglCPR as a template, primers for amplifying the target gene fragment expressing CglCPR were designed, and polymerase chain reaction (PCR) was performed. Using pACYCDuet1, pCDFDuet1, and pETDuet1 plasmids as templates, vector amplification primers for multiple cloning site 1 (mcs1) and multiple cloning site 2 (mcs2) were designed, and PCR was performed.

[0035] PCR reaction system (50 µL): 1 µL forward primer (100 μM), 1 µL reverse primer (100 μM), 25 µL 2×Phanta buffer, 1 µL dNTP mixture (10 mM each), 1 µL plasmid template, 1 µL DNA polymerase Phanta (Novizan, China) and 20 µL ultrapure water.

[0036] The PCR program set according to the Phanta Super-Fidelity DNA polymerase manual is as follows: pre-denaturation at 95 ℃ for 5 min, followed by 30 cycles (denaturation at 95 ℃ for 30 s, annealing at 55-60 ℃ for 30 s, extension at 72 ℃ for 1-4 min), final extension at 72 ℃ for 10 min, and incubation at 16 ℃.

[0037] PCR product digestion: Add 2 µL to the PCR system Dpn I. Digest 5µL CutSmart at 37 ℃ for 1 h.

[0038] PCR product cleaning and recovery: Refer to the AxyPrep cleaning kit. Determine the nucleic acid concentration of the cleaned PCR products using NanoDrop.

[0039] One-step cloning and recombination: Refer to the Novizan ClonExpress II One Step Cloning Kit.

[0040] The recombinant products were transformed into BL21(DE3) competent cells via heat shock, and screened in LB solid medium containing 25 μg / mL chloramphenicol, 50 μg / mL streptomycin sulfate, or 50 μg / mL ampicillin resistance. Cgl CPR-ligated positive clones were obtained. DNA sequencing was used for verification. Ligation at different multiple cloning sites (pACYCDuet1, pCDFDuet1, and pETDuet1) was achieved. Cgl CPR recombinant plasmid.

[0041] 2. Cgl Construction of CPR and GDH dual expression plasmid

[0042] strain E.coli Using the vector pET28b-GDH in BL21(DE3) / pET28b-GDH as a template, primers for amplifying the target gene fragment expressing GDH were designed, and polymerase chain reaction (PCR) was performed. Using pACYCDuet1- Cgl CPR, pCDFDuet1- Cgl CPR and pETDuet1- CglUsing the CPR plasmid as a template, primers for vector amplification with another empty multiple cloning site were designed for PCR. After digestion, cleaning, and recovery of the PCR product, the GDH target gene fragment was cloned and recombined into pACYCDuet1- using the Novizan ClonExpress II One Step Cloning Kit in one step. Cgl CPR, pCDFDuet1- Cgl CPR and pETDuet1- Cgl In the CPR vector, construct Cgl CPR and GDH dual-expression recombinant plasmid.

[0043] 3. Construction of recombinant bacteria expressing three enzymes

[0044] The two-enzyme expression recombinant plasmids successfully constructed in step 2 were transformed into [the following]. E.coli BL21(DE3) / pET28b- Rho LPLDH and its mutant competent cells were plated on 10 mL LB agar plates of the corresponding antibiotics and incubated at 37 °C for 12–16 h. Colony PCR was used to verify expression. Rho LPLDH and its mutants, Cgl The target genes for both CPR and GDH are present in the recombinant bacteria. See the successfully constructed recombinant expression engineered bacteria. Figure 2 .

[0045] Example 3: Inducible expression of L-pantolactone dehydrogenase and its mutants, conjugated polyketide reductase and glucose dehydrogenase

[0046] The recombinant expression engineered bacteria successfully constructed in Example 2, for example E. coli BL21(DE3) / pET28b- Rho LPLDH / pACYCDuet1-m1- Cgl CPR-m2-GDH was inoculated into 10 mL LB liquid medium containing dual antibiotics, such as kanamycin at a final concentration of 50 μg / mL and chloramphenicol at a final concentration of 25 μg / mL, and cultured at 37 ℃ and 180 rpm for 10 h to obtain seed culture. The seed culture was then inoculated at a volume concentration of 1.0% (v / v) into fresh 100 mL LB liquid medium shake flasks containing dual antibiotics, such as kanamycin at a final concentration of 50 μg / mL and chloramphenicol at a final concentration of 25 μg / mL, and cultured at 37 ℃ and 180 rpm until OD (dose retardation). 600 The concentration was set between 0.6 and 0.8. Then, IPTG was added to the culture medium to a final concentration of 0.1 mM. The strain was cultured at 28°C for 12 h, and then centrifuged at 4°C and 8000 rpm for 10 min to obtain the corresponding wet bacterial cells.

[0047] The cells obtained above produce corresponding proteins, which can be used to prepare pure protein enzyme solutions, or to catalyze the configuration reversal of L-indophosphoprolone to prepare D-indophosphoprolone using crude enzyme solutions or whole cells.

[0048] Example 4: Comparison of catalytic abilities of engineered bacteria expressing three recombinant enzymes

[0049] Using wet cells of L-indoglutamate dehydrogenase, conjugated polytonol reductase, and glucose dehydrogenase proteins induced in Example 3 as catalysts, and L-indoglutamate as substrate, the ability of each recombinant strain to catalyze the conversion of LPL to DPL and the accumulation of the intermediate product KPL were compared. The reaction system consisted of 1 mL of the sample, with a catalyst concentration of 20 g / L (wet weight of cells), a final substrate concentration of 100 mM, a final glucose concentration of 150 mM, and a pH 7.5, 50 mM PB buffer as the reaction medium. The reaction was carried out at 30 ℃ and 1200 rpm for 1 h. 200 μL of the reaction solution was then added to 50 μL of 6 M hydrochloric acid (acidified), followed by extraction three times with 200 μL of ethyl acetate. The ethyl acetate phases were combined. The concentrations of L-indoglutamate, ketopantoglutamate, and D-indoglutamate in the ethyl acetate sample were determined by GC, and the percentage concentrations of the three components were calculated. After multiple catalytic validations, the dominant recombinant strains were finally determined to be... E. coli BL21(DE3) / pET28b- Rho DH L241I / L254I / L156L / pACYCDuet1-m1- Cgl CPR-m2-GDH, E. coli BL21(DE3) / pET28b- Rop DH / pACYCDuet-m1-GDH-m2- Cgl CPR E. coli

[0050] BL21(DE3) / pET28b- Rho DH L241I / L254I / V308L / pACYCDuet-m1- Cgl CPR-m2-GDH, E. coli BL21(DE3) / pET28b- Rho DH L241I / L254I / L156L / F224H / pACYCDuet-m1-GDH-m2- Cgl CPR. In the catalytic comparison of these four recombinant bacteria, no intermediate product KPL hydrolysis was observed. E. coli BL21(DE3) / pET28b- Rho DH L241I / L254I / L156L / pACYCDuet1-m1- Cgl CPR-m2-GDH and E. coli BL21(DE3) / pET28b- Rho DH L241I / L254I / L156L / F224H / pACYCDuet-m1-GDH-m2- Cgl The two recombinant bacterial substrates of CPR had the lowest remaining LPL, while the resulting product had the highest DPL. Figure 3 .

[0051] Example 5: Preparation of D-pantolactone by configurational inversion of 200 mM L-pantolactone catalyzed by recombinant bacteria with three-enzyme co-expression

[0052] Prepared by the method of Example 3 E. coli BL21(DE3) / pET28b- Rho DH L241I / L254I / L156L / pACYCDuet1-m1- Cgl CPR-m2-GDH(Strain1) and E. coli BL21(DE3) / pET28b- Rho DH L241I / L254I / L156L / F224H / pACYCDuet-m1-GDH-m2- Cgl The CPR (Strain2) wet cell catalyst was used in a one-pot method to catalyze the configuration inversion of L-pantolactone to D-pantolactone.

[0053] In a 5 mL reaction system, the bacterial cell concentration was 50 or 100 g / L, the substrate L-pantolactone was 200 mM, and the glucose concentration was 300 mM. A 50 mM PB buffer solution at pH 7.0 was used as the reaction medium to construct the transformation system. The reaction was carried out at 30°C and 800 rpm, with the pH adjusted by 1 M NaOH to maintain a pH of 7.0-7.5. The catalytic results were detected using the method described in Example 5. ​ When 50 g / L Strain1 and Strain2 catalyzed 200 mM LPL, Strain1 completely catalyzed the substrate LPL within 10 h, but KPL did not react in time, resulting in hydrolysis and a DPL yield of 81.8% at 36 h. Strain2 catalyzed the LPL reaction at a slightly slower rate than Strain1, and KPL hydrolysis was more pronounced, resulting in a DPL yield of 68.1% at 36 h. However, when 100 g / L Strain2 catalyzed 200 mM LPL, the LPL reaction rate was slower than with the 50 g / L catalyst, and there was no hydrolysis of the intermediate KPL; the DPL yield reached 99.3% at 24 h.

[0054] Example 6: Preparation of D-pantolactone from L-pantolactone via configuration inversion catalyzed by recombinant bacteria co-expressing three enzymes.

[0055] Prepared by the method of Example 3 ​ BL21(DE3) / pET28b- ​ DH L241I / L254I / L156L / F224H / pACYCDuet-m1-GDH-m2- ​ CPR (Strain2) wet bacterial cells were used as catalysts in a one-pot process to catalyze the configuration inversion of L-indophosphoprolactone at a concentration of 200-400 mM to prepare D-indophosphoprolactone.

[0056] In a 5 mL reaction system, the bacterial cell concentration was 100 g / L, the substrate L-pantolactone dosage was 200, 250, 300, and 400 mM, and the glucose concentration was 1.5 times that of the substrate. The transformation system was constructed using a 50 mM PB buffer solution at pH 7.0 as the reaction medium. The reaction was carried out at 30 °C and 800 rpm, with the pH adjusted by 1 M NaOH to maintain a range of 7.0-7.5. The catalytic results were detected using the method described in Example 5. ​ When Strain2 catalyzes 200 mM LPL, the reaction rate decreases compared to Example 5, with 4.0% LPL remaining after 36 h, no KPL hydrolysis, and a DPL yield of 96.4%. With increasing substrate concentration, the overall catalytic activity weakens, the remaining LPL increases, KPL hydrolysis increases, and the final product DPL yield decreases. Overall, Strain2 catalyzes conformational inversion of 200 mM LPL to obtain the highest DPL yield.

[0057] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. An L-pantolactone dehydrogenase mutant, characterized in that, The L-pantolactone dehydrogenase mutant was obtained by multiple combined mutations at positions 156, 224, 241, and 254 of the amino acid sequence shown in SEQ ID NO.4, and its amino acid sequence is shown in SEQ ID NO.

7.

2. A gene encoding an L-pantolactone dehydrogenase mutant, characterized in that, Encodes the L-indohydrin dehydrogenase mutant of claim 1.

3. A recombinant vector, characterized in that, It contains the gene encoding the L-indohydrin dehydrogenase mutant as described in claim 2.

4. A genetically engineered bacterium, characterized in that, It contains the gene encoding the L-indohydrin dehydrogenase mutant as described in claim 2.

5. The genetically engineered bacteria as described in claim 4, characterized in that... The genetically engineered bacteria are an L-pantolactone dehydrogenase mutant and a conjugated polyketide reductase. Cgl The engineered bacteria co-expressing CPR and glucose dehydrogenase (GDH) were constructed as follows: a recombinant plasmid containing the gene encoding the L-pantolactone dehydrogenase mutant pET28b(+) and a recombinant plasmid containing the genes of the double-linked conjugated polyketide reductase gene and glucose dehydrogenase gene were transformed into Escherichia coli BL21(DE3) competent cells, and double-resistant recombinant co-expressing engineered bacteria were screened; the nucleotide sequence of the conjugated polyketide reductase gene is shown in SEQ ID NO.2, and the nucleotide sequence of the glucose dehydrogenase gene is shown in SEQ ID NO.

3.

6. The application of the genetically engineered bacteria according to claim 4 in the microbial catalytic preparation of D-pantolactone, characterized in that, The application involves transforming a recombinant plasmid containing the pET28b(+) gene encoding an L-pantolactone dehydrogenase mutant, along with a recombinant plasmid containing a double-linked conjugated polyketide reductase gene and a glucose dehydrogenase gene, into *E. coli* BL21(DE3) competent cells. Double-resistant, three-enzyme recombinant co-expressing engineered bacteria are screened. The wet cells obtained from the induced culture of the co-expressing engineered bacteria are used as a catalyst, L-pantolactone as a substrate, glucose as a co-substrate, and a pH 7.0, 50 mM PB buffer as the reaction medium to construct the transformation system. The reaction is carried out at 30–40 °C and 600–800 rpm to obtain D-pantolactone in the fermentation broth. The nucleotide sequence of the conjugated polyketide reductase gene is shown in SEQ ID NO.2, and the nucleotide sequence of the glucose dehydrogenase gene is shown in SEQ ID NO.3.

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